[Protein S3 in the human 80S ribosome adjoins mRNA from 3'-side of the A-site codon].

PubMed

Molotkov, M V; Graĭfer, D M; Popugaeva, E A; Bulygin, K N; Meshchaninova, M I; Ven'iaminova, A G; Karpova, G G

2007-01-01

The protein environment of mRNA 3' of the A-site codon (the decoding site) in the human 80S ribosome was studied using a set of oligoribonucleotide derivatives bearing a UUU triplet at the 5'-end and a perfluoroarylazide group at one of the nucleotide residues at the 3'-end of this triplet. Analogues of mRNA were phased into the ribosome using binding at the tRNAPhe P-site, which recognizes the UUU codon. Mild UV irradiation of ribosome complexes with tRNAPhe and mRNA analogues resulted in the predominant crosslinking of the analogues with the 40S subunit components, mainly with proteins and, to a lesser extent, with rRNA. Among the 40S subunit ribosomal proteins, the S3 protein was the main target for modification in all cases. In addition, minor crosslinking with the S2 protein was observed. The crosslinking with the S3 and S2 proteins occurred both in triple complexes and in the absence of tRNA. Within triple complexes, crosslinking with S15 protein was also found, its efficiency considerably falling when the modified nucleotide was moved from positions +5 to +12 relative to the first codon nucleotide in the P-site. In some cases, crosslinking with the S30 protein was observed, it was most efficient for the derivative containing a photoreactive group at the +7 adenosine residue. The results indicate that the S3 protein in the human ribosome plays a key role in the formation of the mRNA binding site 3' of the codon in the decoding site.

Sensitive, Site-Specific, and Stable Vibrational Probe of Local Protein Environments: 4-Azidomethyl-L-Phenylalanine

PubMed Central

Bazewicz, Christopher G.; Liskov, Melanie T.; Hines, Kevin J.; Brewer, Scott H.

2013-01-01

We have synthesized the unnatural amino acid (UAA), 4-azidomethyl-Lphenylalanine (pN3CH2Phe), to serve as an effective vibrational reporter of local protein environments. The position, extinction coefficient, and sensitivity to local environment of the azide asymmetric stretch vibration of pN3CH2Phe are compared to the vibrational reporters: 4-cyano-L-phenylalanine (pCNPhe) and 4-azido-L-phenylalanine (pN3Phe). This UAA was genetically incorporated in a site-specific manner utilizing an engineered, orthogonal aminoacyl-tRNA synthetase in response to an amber codon with high efficiency and fidelity into two distinct sites in superfolder green fluorescent protein (sfGFP). This allowed for the dependence of the azide asymmetric stretch vibration of pN3CH2Phe to different protein environments to be measured. The photo-stability of pN3CH2Phe was also measured relative to the photoreactive UAA, pN3Phe. PMID:23865850

Efficient initiation of mammalian mRNA translation at a CUG codon.

PubMed Central

Dasso, M C; Jackson, R J

1989-01-01

Nucleotide substitutions were made at the initiation codon of an influenza virus NS cDNA clone in a vector carrying the bacteriophage T7 promoter. When capped mRNA transcripts of these constructs were translated in the rabbit reticulocyte lysate, a change in the initiation codon from...AUAAUGG...to...AUACUGG...reduced the in vitro translational efficiency by only 50-60%, and resulted in only a small increase in the yield of short products presumed to be initiated at downstream sites. Synthesis of the full-length product was initiated exclusively at the mutated codon, with negligible use either of in-frame upstream CUG or GUG codons, or of an in-frame downstream GUG codon. We conclude that CUG has the potential to function as an efficient initiation codon in mammalian systems, at least in certain contexts. Images PMID:2780285

Preferences of AAA/AAG codon recognition by modified nucleosides, τm5s2U34 and t6A37 present in tRNALys.

PubMed

Sonawane, Kailas D; Kamble, Asmita S; Fandilolu, Prayagraj M

2017-12-27

Deficiency of 5-taurinomethyl-2-thiouridine, τm 5 s 2 U at the 34th 'wobble' position in tRNA Lys causes MERRF (Myoclonic Epilepsy with Ragged Red Fibers), a neuromuscular disease. This modified nucleoside of mt tRNA Lys , recognizes AAA/AAG codons during protein biosynthesis process. Its preference to identify cognate codons has not been studied at the atomic level. Hence, multiple MD simulations of various molecular models of anticodon stem loop (ASL) of mt tRNA Lys in presence and absence of τm 5 s 2 U 34 and N 6 -threonylcarbamoyl adenosine (t 6 A 37 ) along with AAA and AAG codons have been accomplished. Additional four MD simulations of multiple ASL mt tRNA Lys models in the context of ribosomal A-site residues have also been performed to investigate the role of A-site in recognition of AAA/AAG codons. MD simulation results show that, ASL models in presence of τm 5 s 2 U 34 and t 6 A 37 with codons AAA/AAG are more stable than the ASL lacking these modified bases. MD trajectories suggest that τm 5 s 2 U recognizes the codons initially by 'wobble' hydrogen bonding interactions, and then tRNA Lys might leave the explicit codon by a novel 'single' hydrogen bonding interaction in order to run the protein biosynthesis process smoothly. We propose this model as the 'Foot-Step Model' for codon recognition, in which the single hydrogen bond plays a crucial role. MD simulation results suggest that, tRNA Lys with τm 5 s 2 U and t 6 A recognizes AAA codon more preferably than AAG. Thus, these results reveal the consequences of τm 5 s 2 U and t 6 A in recognition of AAA/AAG codons in mitochondrial disease, MERRF.

CodonLogo: a sequence logo-based viewer for codon patterns.

PubMed

Sharma, Virag; Murphy, David P; Provan, Gregory; Baranov, Pavel V

2012-07-15

Conserved patterns across a multiple sequence alignment can be visualized by generating sequence logos. Sequence logos show each column in the alignment as stacks of symbol(s) where the height of a stack is proportional to its informational content, whereas the height of each symbol within the stack is proportional to its frequency in the column. Sequence logos use symbols of either nucleotide or amino acid alphabets. However, certain regulatory signals in messenger RNA (mRNA) act as combinations of codons. Yet no tool is available for visualization of conserved codon patterns. We present the first application which allows visualization of conserved regions in a multiple sequence alignment in the context of codons. CodonLogo is based on WebLogo3 and uses the same heuristics but treats codons as inseparable units of a 64-letter alphabet. CodonLogo can discriminate patterns of codon conservation from patterns of nucleotide conservation that appear indistinguishable in standard sequence logos. The CodonLogo source code and its implementation (in a local version of the Galaxy Browser) are available at http://recode.ucc.ie/CodonLogo and through the Galaxy Tool Shed at http://toolshed.g2.bx.psu.edu/.

The highly conserved codon following the slippery sequence supports -1 frameshift efficiency at the HIV-1 frameshift site.

PubMed

Mathew, Suneeth F; Crowe-McAuliffe, Caillan; Graves, Ryan; Cardno, Tony S; McKinney, Cushla; Poole, Elizabeth S; Tate, Warren P

2015-01-01

HIV-1 utilises -1 programmed ribosomal frameshifting to translate structural and enzymatic domains in a defined proportion required for replication. A slippery sequence, U UUU UUA, and a stem-loop are well-defined RNA features modulating -1 frameshifting in HIV-1. The GGG glycine codon immediately following the slippery sequence (the 'intercodon') contributes structurally to the start of the stem-loop but has no defined role in current models of the frameshift mechanism, as slippage is inferred to occur before the intercodon has reached the ribosomal decoding site. This GGG codon is highly conserved in natural isolates of HIV. When the natural intercodon was replaced with a stop codon two different decoding molecules-eRF1 protein or a cognate suppressor tRNA-were able to access and decode the intercodon prior to -1 frameshifting. This implies significant slippage occurs when the intercodon is in the (perhaps distorted) ribosomal A site. We accommodate the influence of the intercodon in a model of frame maintenance versus frameshifting in HIV-1.

Synonymous codon usage patterns in different parasitic platyhelminth mitochondrial genomes.

PubMed

Chen, L; Yang, D Y; Liu, T F; Nong, X; Huang, X; Xie, Y; Fu, Y; Zheng, W P; Zhang, R H; Wu, X H; Gu, X B; Wang, S X; Peng, X R; Yang, G Y

2013-02-27

We analyzed synonymous codon usage patterns of the mitochondrial genomes of 43 parasitic platyhelminth species. The relative synonymous codon usage, the effective number of codons (NC) and the frequency of G+C at the third synonymously variable coding position were calculated. Correspondence analysis was used to determine the major variation trends shaping the codon usage patterns. Among the mitochondrial genomes of 19 trematode species, the GC content of third codon positions varied from 0.151 to 0.592, with a mean of 0.295 ± 0.116. In cestodes, the mean GC content of third codon positions was 0.254 ± 0.044. A comparison of the nucleotide composition at 4-fold synonymous sites revealed that, on average, there was a greater abundance of codons ending on U (51.9%) or A (22.7%) than on C (6.3%) or G (19.14%). Twenty-two codons, including UUU, UUA and UUG, were frequently used. In the NC-plot, most of points were distributed well below or around the expected NC curve. In addition to compositional constraints, the degree of hydrophobicity and the aromatic amino acids also influenced codon usage in the mitochondrial genomes of these 43 parasitic platyhelminth species.

The role of modifications in codon discrimination by tRNA(Lys)UUU.

PubMed

Murphy, Frank V; Ramakrishnan, Venki; Malkiewicz, Andrzej; Agris, Paul F

2004-12-01

The natural modification of specific nucleosides in many tRNAs is essential during decoding of mRNA by the ribosome. For example, tRNA(Lys)(UUU) requires the modification N6-threonylcarbamoyladenosine at position 37 (t(6)A37), adjacent and 3' to the anticodon, to bind AAA in the A site of the ribosomal 30S subunit. Moreover, it can only bind both AAA and AAG lysine codons when doubly modified with t(6)A37 and either 5-methylaminomethyluridine or 2-thiouridine at the wobble position (mnm(5)U34 or s(2)U34). Here we report crystal structures of modified tRNA anticodon stem-loops bound to the 30S ribosomal subunit with lysine codons in the A site. These structures allow the rationalization of how modifications in the anticodon loop enable decoding of both lysine codons AAA and AAG.

Detecting site-specific physicochemical selective pressures: applications to the Class I HLA of the human major histocompatibility complex and the SRK of the plant sporophytic self-incompatibility system.

PubMed

Sainudiin, Raazesh; Wong, Wendy Shuk Wan; Yogeeswaran, Krithika; Nasrallah, June B; Yang, Ziheng; Nielsen, Rasmus

2005-03-01

Models of codon substitution are developed that incorporate physicochemical properties of amino acids. When amino acid sites are inferred to be under positive selection, these models suggest the nature and extent of the physicochemical properties under selection. This is accomplished by first partitioning the codons on the basis of some property of the encoded amino acids. This partition is used to parametrize the rates of property-conserving and property-altering base substitutions at the codon level by means of finite mixtures of Markov models that also account for codon and transition:transversion biases. Here, we apply this method to two positively selected receptors involved in ligand-recognition: the class I alleles of the human major histocompatibility complex (MHC) of known structure and the S-locus receptor kinase (SRK) of the sporophytic self-incompatibility system (SSI) in cruciferous plants (Brassicaceae), whose structure is unknown. Through likelihood ratio tests we demonstrate that at some sites, the positively selected MHC and SRK proteins are under physicochemical selective pressures to alter polarity, volume, polarity and/or volume, and charge to various extents. An empirical Bayes approach is used to identify sites that may be important for ligand recognition in these proteins.

Insights into factorless translational initiation by the tRNA-like pseudoknot domain of a viral IRES.

PubMed

Au, Hilda H T; Jan, Eric

2012-01-01

The intergenic region internal ribosome entry site (IGR IRES) of the Dicistroviridae family adopts an overlapping triple pseudoknot structure to directly recruit the 80S ribosome in the absence of initiation factors. The pseudoknot I (PKI) domain of the IRES mimics a tRNA-like codon:anticodon interaction in the ribosomal P site to direct translation initiation from a non-AUG initiation codon in the A site. In this study, we have performed a comprehensive mutational analysis of this region to delineate the molecular parameters that drive IRES translation. We demonstrate that IRES-mediated translation can initiate at an alternate adjacent and overlapping start site, provided that basepairing interactions within PKI remain intact. Consistent with this, IGR IRES translation tolerates increases in the variable loop region that connects the anticodon- and codon-like elements within the PKI domain, as IRES activity remains relatively robust up to a 4-nucleotide insertion in this region. Finally, elements from an authentic tRNA anticodon stem-loop can functionally supplant corresponding regions within PKI. These results verify the importance of the codon:anticodon interaction of the PKI domain and further define the specific elements within the tRNA-like domain that contribute to optimal initiator Met-tRNA(i)-independent IRES translation.

Insights into Factorless Translational Initiation by the tRNA-Like Pseudoknot Domain of a Viral IRES

PubMed Central

Au, Hilda H. T.; Jan, Eric

2012-01-01

The intergenic region internal ribosome entry site (IGR IRES) of the Dicistroviridae family adopts an overlapping triple pseudoknot structure to directly recruit the 80S ribosome in the absence of initiation factors. The pseudoknot I (PKI) domain of the IRES mimics a tRNA-like codon:anticodon interaction in the ribosomal P site to direct translation initiation from a non-AUG initiation codon in the A site. In this study, we have performed a comprehensive mutational analysis of this region to delineate the molecular parameters that drive IRES translation. We demonstrate that IRES-mediated translation can initiate at an alternate adjacent and overlapping start site, provided that basepairing interactions within PKI remain intact. Consistent with this, IGR IRES translation tolerates increases in the variable loop region that connects the anticodon- and codon-like elements within the PKI domain, as IRES activity remains relatively robust up to a 4-nucleotide insertion in this region. Finally, elements from an authentic tRNA anticodon stem-loop can functionally supplant corresponding regions within PKI. These results verify the importance of the codon:anticodon interaction of the PKI domain and further define the specific elements within the tRNA-like domain that contribute to optimal initiator Met-tRNAi-independent IRES translation. PMID:23236506

A Stem-Loop Structure in Potato Leafroll Virus Open Reading Frame 5 (ORF5) Is Essential for Readthrough Translation of the Coat Protein ORF Stop Codon 700 Bases Upstream.

PubMed

Xu, Yi; Ju, Ho-Jong; DeBlasio, Stacy; Carino, Elizabeth J; Johnson, Richard; MacCoss, Michael J; Heck, Michelle; Miller, W Allen; Gray, Stewart M

2018-06-01

Translational readthrough of the stop codon of the capsid protein (CP) open reading frame (ORF) is used by members of the Luteoviridae to produce their minor capsid protein as a readthrough protein (RTP). The elements regulating RTP expression are not well understood, but they involve long-distance interactions between RNA domains. Using high-resolution mass spectrometry, glutamine and tyrosine were identified as the primary amino acids inserted at the stop codon of Potato leafroll virus (PLRV) CP ORF. We characterized the contributions of a cytidine-rich domain immediately downstream and a branched stem-loop structure 600 to 700 nucleotides downstream of the CP stop codon. Mutations predicted to disrupt and restore the base of the distal stem-loop structure prevented and restored stop codon readthrough. Motifs in the downstream readthrough element (DRTE) are predicted to base pair to a site within 27 nucleotides (nt) of the CP ORF stop codon. Consistent with a requirement for this base pairing, the DRTE of Cereal yellow dwarf virus was not compatible with the stop codon-proximal element of PLRV in facilitating readthrough. Moreover, deletion of the complementary tract of bases from the stop codon-proximal region or the DRTE of PLRV prevented readthrough. In contrast, the distance and sequence composition between the two domains was flexible. Mutants deficient in RTP translation moved long distances in plants, but fewer infection foci developed in systemically infected leaves. Selective 2'-hydroxyl acylation and primer extension (SHAPE) probing to determine the secondary structure of the mutant DRTEs revealed that the functional mutants were more likely to have bases accessible for long-distance base pairing than the nonfunctional mutants. This study reveals a heretofore unknown combination of RNA structure and sequence that reduces stop codon efficiency, allowing translation of a key viral protein. IMPORTANCE Programmed stop codon readthrough is used by many animal and plant viruses to produce key viral proteins. Moreover, such "leaky" stop codons are used in host mRNAs or can arise from mutations that cause genetic disease. Thus, it is important to understand the mechanism(s) of stop codon readthrough. Here, we shed light on the mechanism of readthrough of the stop codon of the coat protein ORFs of viruses in the Luteoviridae by identifying the amino acids inserted at the stop codon and RNA structures that facilitate this "leakiness" of the stop codon. Members of the Luteoviridae encode a C-terminal extension to the capsid protein known as the readthrough protein (RTP). We characterized two RNA domains in Potato leafroll virus (PLRV), located 600 to 700 nucleotides apart, that are essential for efficient RTP translation. We further determined that the PLRV readthrough process involves both local structures and long-range RNA-RNA interactions. Genetic manipulation of the RNA structure altered the ability of PLRV to translate RTP and systemically infect the plant. This demonstrates that plant virus RNA contains multiple layers of information beyond the primary sequence and extends our understanding of stop codon readthrough. Strategic targets that can be exploited to disrupt the virus life cycle and reduce its ability to move within and between plant hosts were revealed. Copyright © 2018 American Society for Microbiology.

Near-cognate suppression of amber, opal and quadruplet codons competes with aminoacyl-tRNAPyl for genetic code expansion

PubMed Central

O’Donoghue, Patrick; Prat, Laure; Heinemann, Ilka U.; Ling, Jiqiang; Odoi, Keturah; Liu, Wenshe R.; Söll, Dieter

2012-01-01

Over 300 amino acids are found in proteins in nature, yet typically only 20 are genetically encoded. Reassigning stop codons and use of quadruplet codons emerged as the main avenues for genetically encoding non-canonical amino acids (NCAAs). Canonical aminoacyl-tRNAs with near-cognate anticodons also read these codons to some extent. This background suppression leads to ‘statistical protein’ that contains some natural amino acid(s) at a site intended for NCAA. We characterize near-cognate suppression of amber, opal and a quadruplet codon in common Escherichia coli laboratory strains and find that the PylRS/tRNAPyl orthogonal pair cannot completely outcompete contamination by natural amino acids. PMID:23036644

Two alternative ways of start site selection in human norovirus reinitiation of translation.

PubMed

Luttermann, Christine; Meyers, Gregor

2014-04-25

The calicivirus minor capsid protein VP2 is expressed via termination/reinitiation. This process depends on an upstream sequence element denoted termination upstream ribosomal binding site (TURBS). We have shown for feline calicivirus and rabbit hemorrhagic disease virus that the TURBS contains three sequence motifs essential for reinitiation. Motif 1 is conserved among caliciviruses and is complementary to a sequence in the 18 S rRNA leading to the model that hybridization between motif 1 and 18 S rRNA tethers the post-termination ribosome to the mRNA. Motif 2 and motif 2* are proposed to establish a secondary structure positioning the ribosome relative to the start site of the terminal ORF. Here, we analyzed human norovirus (huNV) sequences for the presence and importance of these motifs. The three motifs were identified by sequence analyses in the region upstream of the VP2 start site, and we showed that these motifs are essential for reinitiation of huNV VP2 translation. More detailed analyses revealed that the site of reinitiation is not fixed to a single codon and does not need to be an AUG, even though this codon is clearly preferred. Interestingly, we were able to show that reinitiation can occur at AUG codons downstream of the canonical start/stop site in huNV and feline calicivirus but not in rabbit hemorrhagic disease virus. Although reinitiation at the original start site is independent of the Kozak context, downstream initiation exhibits requirements for start site sequence context known for linear scanning. These analyses on start codon recognition give a more detailed insight into this fascinating mechanism of gene expression.

Evolutionary conservation of codon optimality reveals hidden signatures of cotranslational folding.

PubMed

Pechmann, Sebastian; Frydman, Judith

2013-02-01

The choice of codons can influence local translation kinetics during protein synthesis. Whether codon preference is linked to cotranslational regulation of polypeptide folding remains unclear. Here, we derive a revised translational efficiency scale that incorporates the competition between tRNA supply and demand. Applying this scale to ten closely related yeast species, we uncover the evolutionary conservation of codon optimality in eukaryotes. This analysis reveals universal patterns of conserved optimal and nonoptimal codons, often in clusters, which associate with the secondary structure of the translated polypeptides independent of the levels of expression. Our analysis suggests an evolved function for codon optimality in regulating the rhythm of elongation to facilitate cotranslational polypeptide folding, beyond its previously proposed role of adapting to the cost of expression. These findings establish how mRNA sequences are generally under selection to optimize the cotranslational folding of corresponding polypeptides.

Identification of the initiation site of poliovirus polyprotein synthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dorner, A.J.; Dorner, L.F.; Larsen, G.R.

1982-06-01

The complete nucleotide sequence of poliovirus RNA has a long open reading frame capable of encoding the precursor polyprotein NCVPOO. The first AUG codon in this reading frame is located 743 nucleotides from the 5' end of the RNA and is preceded by eight AUG codons in all three reading frames. Because all proteins that map at the amino terminus of the polyprotein (P1-1a, VPO, and VP4) are blocked at their amino termini and previous studies of ribosome binding have been inconclusive, direct identification of the initiation site of protein synthesis was difficult. We separated and identified all of themore » tryptic peptides of capsid protein VP4 and correlated these peptides with the amino acid sequence predicted to follow the AUG codon at nucleotide 743. Our data indicate that VP4 begins with a blocked glycine that is encoded immediately after the AUG codon at nucleotide 743. An S1 nuclease analysis of poliovirus mRNA failed to reveal a splice in the 5' region. We concluded that synthesis of poliovirus polyprotein is initiated at nucleotide 743, the first AUG codon in the long open reading frame.« less

Visualization of glucagon secretion from pancreatic α cells by bioluminescence video microscopy: Identification of secretion sites in the intercellular contact regions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yokawa, Satoru; School of Pharmacy, Aichi Gakuin University, Nagoya 464-8650; Suzuki, Takahiro

We have firstly visualized glucagon secretion using a method of video-rate bioluminescence imaging. The fusion protein of proglucagon and Gaussia luciferase (PGCG-GLase) was used as a reporter to detect glucagon secretion and was efficiently expressed in mouse pancreatic α cells (αTC1.6) using a preferred human codon-optimized gene. In the culture medium of the cells expressing PGCG-GLase, luminescence activity determined with a luminometer was increased with low glucose stimulation and KCl-induced depolarization, as observed for glucagon secretion. From immunochemical analyses, PGCG-GLase stably expressed in clonal αTC1.6 cells was correctly processed and released by secretory granules. Luminescence signals of the secreted PGCG-GLase frommore » the stable cells were visualized by video-rate bioluminescence microscopy. The video images showed an increase in glucagon secretion from clustered cells in response to stimulation by KCl. The secretory events were observed frequently at the intercellular contact regions. Thus, the localization and frequency of glucagon secretion might be regulated by cell-cell adhesion. - Highlights: • The fused protein of proglucagon to Gaussia luciferase was used as a reporter. • The fusion protein was highly expressed using a preferred human-codon optimized gene. • Glucagon secretion stimulated by depolarization was determined by luminescence. • Glucagon secretion in α cells was visualized by bioluminescence imaging. • Glucagon secretion sites were localized in the intercellular contact regions.« less

[Protein S3 fragments neighboring mRNA during elongation and translation termination on the human ribosome].

PubMed

Khaĭrulina, Iu S; Molotkov, M V; Bulygin, K N; Graĭfer, D M; Ven'yaminova, A G; Frolova, L Iu; Stahl, J; Karpova, G G

2008-01-01

Protein S3 fragments were determined that crosslink to modified mRNA analogues in positions +5 to +12 relative to the first nucleotide in the P-site binding codon in model complexes mimicking states of ribosomes at the elongation and translation termination steps. The mRNA analogues contained a Phe codon UUU/UUC at the 5'-termini that could predetermine the position of the tRNA(Phe) on the ribosome by the location of P-site binding and perfluorophenylazidobenzoyl group at a nucleotide in various positions 3' of the UUU/UUC codon. The crosslinked S3 protein was isolated from 80S ribosomal complexes irradiated with mild UV light and subjected to cyanogen bromide-induced cleavage at methionine residues with subsequent identification of the crosslinked oligopeptides. An analysis of the positions of modified oligopeptides resulting from the cleavage showed that, in dependence on the positions of modified nucleotides in the mRNA analogue, the crosslinking sites were found in the N-terminal half of the protein (fragment 2-127) and/or in the C-terminal fragment 190-236; the latter reflects a new peculiarity in the structure of the mRNA binding center in the ribosome, unknown to date. The results of crosslinking did not depend on the type of A-site codon or on the presence of translation termination factor eRF1.

Codes in the codons: construction of a codon/amino acid periodic table and a study of the nature of specific nucleic acid-protein interactions.

PubMed

Benyo, B; Biro, J C; Benyo, Z

2004-01-01

The theory of "codon-amino acid coevolution" was first proposed by Woese in 1967. It suggests that there is a stereochemical matching - that is, affinity - between amino acids and certain of the base triplet sequences that code for those amino acids. We have constructed a common periodic table of codons and amino acids, where the nucleic acid table showed perfect axial symmetry for codons and the corresponding amino acid table also displayed periodicity regarding the biochemical properties (charge and hydrophobicity) of the 20 amino acids and the position of the stop signals. The table indicates that the middle (2/sup nd/) amino acid in the codon has a prominent role in determining some of the structural features of the amino acids. The possibility that physical contact between codons and amino acids might exist was tested on restriction enzymes. Many recognition site-like sequences were found in the coding sequences of these enzymes and as many as 73 examples of codon-amino acid co-location were observed in the 7 known 3D structures (December 2003) of endonuclease-nucleic acid complexes. These results indicate that the smallest possible units of specific nucleic acid-protein interaction are indeed the stereochemically compatible codons and amino acids.

TTA codons in some genes prevent their expression in a class of developmental, antibiotic-negative, Streptomyces mutants.

PubMed Central

Leskiw, B K; Lawlor, E J; Fernandez-Abalos, J M; Chater, K F

1991-01-01

In Streptomyces coelicolor A3(2) and the related species Streptomyces lividans 66, aerial mycelium formation and antibiotic production are blocked by mutations in bldA, which specifies a tRNA(Leu)-like gene product which would recognize the UUA codon. Here we show that phenotypic expression of three disparate genes (carB, lacZ, and ampC) containing TTA codons depends strongly on bldA. Site-directed mutagenesis of carB, changing its two TTA codons to CTC (leucine) codons, resulted in bldA-independent expression; hence the bldA product is the principal tRNA for the UUA codon. Two other genes (hyg and aad) containing TTA codons show a medium-dependent reduction in phenotypic expression (hygromycin resistance and spectinomycin resistance, respectively) in bldA mutants. For hyg, evidence is presented that the UUA codon is probably being translated by a tRNA with an imperfectly matched anticodon, giving very low levels of gene product but relatively high resistance to hygromycin. It is proposed that TTA codons may be generally absent from genes expressed during vegetative growth and from the structural genes for differentiation and antibiotic production but present in some regulatory and resistance genes associated with the latter processes. The codon may therefore play a role in developmental regulation. Images PMID:1826053

Translation, modification and cellular distribution of two AC4 variants of African cassava mosaic virus in yeast and their pathogenic potential in plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hipp, Katharina, E-mail: katharina.hipp@bio.uni-st

Plant infecting geminiviruses encode a small (A)C4 protein within the open reading frame of the replication-initiator protein. In African cassava mosaic virus, two in-frame start codons may be used for the translation of a longer and a shorter AC4 variant. Both were fused to green fluorescent protein or glutathione-S-transferase genes and expressed in fission yeast. The longer variant accumulated in discrete spots in the cytoplasm, whereas the shorter variant localized to the plasma membrane. A similar expression pattern was found in plants. A myristoylation motif may promote a targeting of the shorter variant to the plasma membrane. Mass spectrometry analysismore » of the yeast-expressed shorter variant detected the corresponding myristoylation. The biological relevance of the second start codon was confirmed using mutated infectious clones. Whereas mutating the first start codon had no effect on the infectivity in Nicotiana benthamiana plants, the second start codon proved to be essential. -- Highlights: •The ACMV AC4 may be translated from one or the other in-frame start codon. •Both AC4 variants are translated in fission yeast. •The long AC4 protein localizes to the cytoplasm, the short to the plasma membrane. •The short variant is myristoylated in yeast and may promote membrane localization. •Only the shorter AC4 variant has an impact on viral infections in plants.« less

A Simple Combinatorial Codon Mutagenesis Method for Targeted Protein Engineering.

PubMed

Belsare, Ketaki D; Andorfer, Mary C; Cardenas, Frida S; Chael, Julia R; Park, Hyun June; Lewis, Jared C

2017-03-17

Directed evolution is a powerful tool for optimizing enzymes, and mutagenesis methods that improve enzyme library quality can significantly expedite the evolution process. Here, we report a simple method for targeted combinatorial codon mutagenesis (CCM). To demonstrate the utility of this method for protein engineering, CCM libraries were constructed for cytochrome P450 BM3 , pfu prolyl oligopeptidase, and the flavin-dependent halogenase RebH; 10-26 sites were targeted for codon mutagenesis in each of these enzymes, and libraries with a tunable average of 1-7 codon mutations per gene were generated. Each of these libraries provided improved enzymes for their respective transformations, which highlights the generality, simplicity, and tunability of CCM for targeted protein engineering.

Theoretical foundations for quantitative paleogenetics. III - The molecular divergence of nucleic acids and proteins for the case of genetic events of unequal probability

NASA Technical Reports Server (NTRS)

Holmquist, R.; Pearl, D.

1980-01-01

Theoretical equations are derived for molecular divergence with respect to gene and protein structure in the presence of genetic events with unequal probabilities: amino acid and base compositions, the frequencies of nucleotide replacements, the usage of degenerate codons, the distribution of fixed base replacements within codons and the distribution of fixed base replacements among codons. Results are presented in the form of tables relating the probabilities of given numbers of codon base changes with respect to the original codon for the alpha hemoglobin, beta hemoglobin, myoglobin, cytochrome c and parvalbumin group gene families. Application of the calculations to the rabbit alpha and beta hemoglobin mRNAs and proteins indicates that the genes are separated by about 425 fixed based replacements distributed over 114 codon sites, which is a factor of two greater than previous estimates. The theoretical results also suggest that many more base replacements are required to effect a given gene or protein structural change than previously believed.

mRNA 3' of the A site bound codon is located close to protein S3 on the human 80S ribosome.

PubMed

Molotkov, Maxim V; Graifer, Dmitri M; Popugaeva, Elena A; Bulygin, Konstantin N; Meschaninova, Maria I; Ven'yaminova, Aliya G; Karpova, Galina G

2006-07-01

Ribosomal proteins neighboring the mRNA downstream of the codon bound at the decoding site of human 80S ribosomes were identified using three sets of mRNA analogues that contained a UUU triplet at the 5' terminus and a perfluorophenylazide cross-linker at guanosine, adenosine or uridine residues placed at various locations 3' of this triplet. The positions of modified mRNA nucleotides on the ribosome were governed by tRNA(Phe) cognate to the UUU triplet targeted to the P site. Upon mild UV-irradiation, the mRNA analogues cross-linked preferentially to the 40S subunit, to the proteins and to a lesser extent to the 18S rRNA. Cross-linked nucleotides of 18S rRNA were identified previously. In the present study, it is shown that among the proteins the main target for cross-linking with all the mRNA analogues tested was protein S3 (homologous to prokaryotic S3, S3p); minor cross-linking to protein S2 (S5p) was also detected. Both proteins cross-linked to mRNA analogues in the ternary complexes as well as in the binary complexes (without tRNA). In the ternary complexes protein S15 (S19p) also cross-linked, the yield of the cross-link decreased significantly when the modified nucleotide moved from position +5 to position +12 with respect to the first nucleotide of the P site bound codon. In several ternary complexes minor cross-linking to protein S30 was likewise detected. The results of this study indicate that S3 is a key protein at the mRNA binding site neighboring mRNA downstream of the codon at the decoding site in the human ribosome.

Confirmation of translatability and functionality certifies the dual endothelin1/VEGFsp receptor (DEspR) protein.

PubMed

Herrera, Victoria L M; Steffen, Martin; Moran, Ann Marie; Tan, Glaiza A; Pasion, Khristine A; Rivera, Keith; Pappin, Darryl J; Ruiz-Opazo, Nelson

2016-06-14

In contrast to rat and mouse databases, the NCBI gene database lists the human dual-endothelin1/VEGFsp receptor (DEspR, formerly Dear) as a unitary transcribed pseudogene due to a stop [TGA]-codon at codon#14 in automated DNA and RNA sequences. However, re-analysis is needed given prior single gene studies detected a tryptophan [TGG]-codon#14 by manual Sanger sequencing, demonstrated DEspR translatability and functionality, and since the demonstration of actual non-translatability through expression studies, the standard-of-excellence for pseudogene designation, has not been performed. Re-analysis must meet UNIPROT criteria for demonstration of a protein's existence at the highest (protein) level, which a priori, would override DNA- or RNA-based deductions. To dissect the nucleotide sequence discrepancy, we performed Maxam-Gilbert sequencing and reviewed 727 RNA-seq entries. To comply with the highest level multiple UNIPROT criteria for determining DEspR's existence, we performed various experiments using multiple anti-DEspR monoclonal antibodies (mAbs) targeting distinct DEspR epitopes with one spanning the contested tryptophan [TGG]-codon#14, assessing: (a) DEspR protein expression, (b) predicted full-length protein size, (c) sequence-predicted protein-specific properties beyond codon#14: receptor glycosylation and internalization, (d) protein-partner interactions, and (e) DEspR functionality via DEspR-inhibition effects. Maxam-Gilbert sequencing and some RNA-seq entries demonstrate two guanines, hence a tryptophan [TGG]-codon#14 within a compression site spanning an error-prone compression sequence motif. Western blot analysis using anti-DEspR mAbs targeting distinct DEspR epitopes detect the identical glycosylated 17.5 kDa pull-down protein. Decrease in DEspR-protein size after PNGase-F digest demonstrates post-translational glycosylation, concordant with the consensus-glycosylation site beyond codon#14. Like other small single-transmembrane proteins, mass spectrometry analysis of anti-DEspR mAb pull-down proteins do not detect DEspR, but detect DEspR-protein interactions with proteins implicated in intracellular trafficking and cancer. FACS analyses also detect DEspR-protein in different human cancer stem-like cells (CSCs). DEspR-inhibition studies identify DEspR-roles in CSC survival and growth. Live cell imaging detects fluorescently-labeled anti-DEspR mAb targeted-receptor internalization, concordant with the single internalization-recognition sequence also located beyond codon#14. Data confirm translatability of DEspR, the full-length DEspR protein beyond codon#14, and elucidate DEspR-specific functionality. Along with detection of the tryptophan [TGG]-codon#14 within an error-prone compression site, cumulative data demonstrating DEspR protein existence fulfill multiple UNIPROT criteria, thus refuting its pseudogene designation.

A novel nuclear genetic code alteration in yeasts and the evolution of codon reassignment in eukaryotes

PubMed Central

Mühlhausen, Stefanie; Findeisen, Peggy; Plessmann, Uwe; Urlaub, Henning; Kollmar, Martin

2016-01-01

The genetic code is the cellular translation table for the conversion of nucleotide sequences into amino acid sequences. Changes to the meaning of sense codons would introduce errors into almost every translated message and are expected to be highly detrimental. However, reassignment of single or multiple codons in mitochondria and nuclear genomes, although extremely rare, demonstrates that the code can evolve. Several models for the mechanism of alteration of nuclear genetic codes have been proposed (including “codon capture,” “genome streamlining,” and “ambiguous intermediate” theories), but with little resolution. Here, we report a novel sense codon reassignment in Pachysolen tannophilus, a yeast related to the Pichiaceae. By generating proteomics data and using tRNA sequence comparisons, we show that Pachysolen translates CUG codons as alanine and not as the more usual leucine. The Pachysolen tRNACAG is an anticodon-mutated tRNAAla containing all major alanine tRNA recognition sites. The polyphyly of the CUG-decoding tRNAs in yeasts is best explained by a tRNA loss driven codon reassignment mechanism. Loss of the CUG-tRNA in the ancient yeast is followed by gradual decrease of respective codons and subsequent codon capture by tRNAs whose anticodon is not part of the aminoacyl-tRNA synthetase recognition region. Our hypothesis applies to all nuclear genetic code alterations and provides several testable predictions. We anticipate more codon reassignments to be uncovered in existing and upcoming genome projects. PMID:27197221

Methods for selecting fixed-effect models for heterogeneous codon evolution, with comments on their application to gene and genome data.

PubMed

Bao, Le; Gu, Hong; Dunn, Katherine A; Bielawski, Joseph P

2007-02-08

Models of codon evolution have proven useful for investigating the strength and direction of natural selection. In some cases, a priori biological knowledge has been used successfully to model heterogeneous evolutionary dynamics among codon sites. These are called fixed-effect models, and they require that all codon sites are assigned to one of several partitions which are permitted to have independent parameters for selection pressure, evolutionary rate, transition to transversion ratio or codon frequencies. For single gene analysis, partitions might be defined according to protein tertiary structure, and for multiple gene analysis partitions might be defined according to a gene's functional category. Given a set of related fixed-effect models, the task of selecting the model that best fits the data is not trivial. In this study, we implement a set of fixed-effect codon models which allow for different levels of heterogeneity among partitions in the substitution process. We describe strategies for selecting among these models by a backward elimination procedure, Akaike information criterion (AIC) or a corrected Akaike information criterion (AICc). We evaluate the performance of these model selection methods via a simulation study, and make several recommendations for real data analysis. Our simulation study indicates that the backward elimination procedure can provide a reliable method for model selection in this setting. We also demonstrate the utility of these models by application to a single-gene dataset partitioned according to tertiary structure (abalone sperm lysin), and a multi-gene dataset partitioned according to the functional category of the gene (flagellar-related proteins of Listeria). Fixed-effect models have advantages and disadvantages. Fixed-effect models are desirable when data partitions are known to exhibit significant heterogeneity or when a statistical test of such heterogeneity is desired. They have the disadvantage of requiring a priori knowledge for partitioning sites. We recommend: (i) selection of models by using backward elimination rather than AIC or AICc, (ii) use a stringent cut-off, e.g., p = 0.0001, and (iii) conduct sensitivity analysis of results. With thoughtful application, fixed-effect codon models should provide a useful tool for large scale multi-gene analyses.

Evaluating Sense Codon Reassignment with a Simple Fluorescence Screen.

PubMed

Biddle, Wil; Schmitt, Margaret A; Fisk, John D

2015-12-22

Understanding the interactions that drive the fidelity of the genetic code and the limits to which modifications can be made without breaking the translational system has practical implications for understanding the molecular mechanisms of evolution as well as expanding the set of encodable amino acids, particularly those with chemistries not provided by Nature. Because 61 sense codons encode 20 amino acids, reassigning the meaning of sense codons provides an avenue for biosynthetic modification of proteins, furthering both fundamental and applied biochemical research. We developed a simple screen that exploits the absolute requirement for fluorescence of an active site tyrosine in green fluorescent protein (GFP) to probe the pliability of the degeneracy of the genetic code. Our screen monitors the restoration of the fluorophore of GFP by incorporation of a tyrosine in response to a sense codon typically assigned another meaning in the genetic code. We evaluated sense codon reassignment at four of the 21 sense codons read through wobble interactions in Escherichia coli using the Methanocaldococcus jannaschii orthogonal tRNA/aminoacyl tRNA synthetase pair originally developed and commonly used for amber stop codon suppression. By changing only the anticodon of the orthogonal tRNA, we achieved sense codon reassignment efficiencies between 1% (Phe UUU) and 6% (Lys AAG). Each of the orthogonal tRNAs preferentially decoded the codon traditionally read via a wobble interaction in E. coli with the exception of the orthogonal tRNA with an AUG anticodon, which incorporated tyrosine in response to both the His CAU and His CAC codons with approximately equal frequencies. We applied our screen in a high-throughput manner to evaluate a 10(9)-member combined tRNA/aminoacyl tRNA synthetase library to identify improved sense codon reassigning variants for the Lys AAG codon. A single rapid screen with the ability to broadly evaluate reassignable codons will facilitate identification and improvement of the combinations of sense codons and orthogonal pairs that display efficient reassignment.

Efficient Coproduction of Mannanase and Cellulase by the Transformation of a Codon-Optimized Endomannanase Gene from Aspergillus niger into Trichoderma reesei.

PubMed

Sun, Xianhua; Xue, Xianli; Li, Mengzhu; Gao, Fei; Hao, Zhenzhen; Huang, Huoqing; Luo, Huiying; Qin, Lina; Yao, Bin; Su, Xiaoyun

2017-12-20

Cellulase and mannanase are both important enzyme additives in animal feeds. Expressing the two enzymes simultaneously within one microbial host could potentially lead to cost reductions in the feeding of animals. For this purpose, we codon-optimized the Aspergillus niger Man5A gene to the codon-usage bias of Trichoderma reesei. By comparing the free energies and the local structures of the nucleotide sequences, one optimized sequence was finally selected and transformed into the T. reesei pyridine-auxotrophic strain TU-6. The codon-optimized gene was expressed to a higher level than the original one. Further expressing the codon-optimized gene in a mutated T. reesei strain through fed-batch cultivation resulted in coproduction of cellulase and mannanase up to 1376 U·mL -1 and 1204 U·mL -1 , respectively.

Identification of single-nucleotide polymorphisms of the prion protein gene in sika deer (Cervus nippon laiouanus)

PubMed Central

Jeong, Hyun-Jeong; Lee, Joong-Bok; Park, Seung-Yong; Song, Chang-Seon; Kim, Bo-Sook; Rho, Jung-Rae; Yoo, Mi-Hyun; Jeong, Byung-Hoon; Kim, Yong-Sun

2007-01-01

Polymorphisms of the prion protein gene (PRNP) have been detected in several cervid species. In order to confirm the genetic variations, this study examined the DNA sequences of the PRNP obtained from 33 captive sika deer (Cervus nippon laiouanus) in Korea. A total of three single-nucleotide polymorphisms (SNPs) at codons 100, 136 and 226 in the PRNP of the sika deer were identified. The polymorphic site located at codon 100 has not been reported. The SNPs detected at codons 100 and 226 induced amino acid substitutions. The SNP at codon 136 was a silent mutation that does not induce any amino acid change. The genotype and allele frequencies were determined for each of the SNPs. PMID:17679779

Limits of variation, specific infectivity, and genome packaging of massively recoded poliovirus genomes.

PubMed

Song, Yutong; Gorbatsevych, Oleksandr; Liu, Ying; Mugavero, JoAnn; Shen, Sam H; Ward, Charles B; Asare, Emmanuel; Jiang, Ping; Paul, Aniko V; Mueller, Steffen; Wimmer, Eckard

2017-10-10

Computer design and chemical synthesis generated viable variants of poliovirus type 1 (PV1), whose ORF (6,189 nucleotides) carried up to 1,297 "Max" mutations (excess of overrepresented synonymous codon pairs) or up to 2,104 "SD" mutations (randomly scrambled synonymous codons). "Min" variants (excess of underrepresented synonymous codon pairs) are nonviable except for P2 Min , a variant temperature-sensitive at 33 and 39.5 °C. Compared with WT PV1, P2 Min displayed a vastly reduced specific infectivity (si) (WT, 1 PFU/118 particles vs. P2 Min , 1 PFU/35,000 particles), a phenotype that will be discussed broadly. Si of haploid PV presents cellular infectivity of a single genotype. We performed a comprehensive analysis of sequence and structures of the PV genome to determine if evolutionary conserved cis-acting packaging signal(s) were preserved after recoding. We showed that conserved synonymous sites and/or local secondary structures that might play a role in determining packaging specificity do not survive codon pair recoding. This makes it unlikely that numerous "cryptic, sequence-degenerate, dispersed RNA packaging signals mapping along the entire viral genome" [Patel N, et al. (2017) Nat Microbiol 2:17098] play the critical role in poliovirus packaging specificity. Considering all available evidence, we propose a two-step assembly strategy for +ssRNA viruses: step I, acquisition of packaging specificity, either ( a ) by specific recognition between capsid protein(s) and replication proteins (poliovirus), or ( b ) by the high affinity interaction of a single RNA packaging signal (PS) with capsid protein(s) (most +ssRNA viruses so far studied); step II, cocondensation of genome/capsid precursors in which an array of hairpin structures plays a role in virion formation.

High-level tetracycline resistance mediated by efflux pumps Tet(A) and Tet(A)-1 with two start codons.

PubMed

Wang, Weixia; Guo, Qinglan; Xu, Xiaogang; Sheng, Zi-ke; Ye, Xinyu; Wang, Minggui

2014-11-01

Efflux is the most common mechanism of tetracycline resistance. Class A tetracycline efflux pumps, which often have high prevalence in Enterobacteriaceae, are encoded by tet(A) and tet(A)-1 genes. These genes have two potential start codons, GTG and ATG, located upstream of the genes. The purpose of this study was to determine the start codon(s) of the class A tetracycline resistance (tet) determinants tet(A) and tet(A)-1, and the tetracycline resistance level they mediated. Conjugation, transformation and cloning experiments were performed and the genetic environment of tet(A)-1 was analysed. The start codons in class A tet determinants were investigated by site-directed mutagenesis of ATG and GTG, the putative translation initiation codons. High-level tetracycline resistance was transferred from the clinical strain of Klebsiella pneumoniae 10-148 containing tet(A)-1 plasmid pHS27 to Escherichia coli J53 by conjugation. The transformants harbouring recombinant plasmids that carried tet(A) or tet(A)-1 exhibited tetracycline MICs of 256-512 µg ml(-1), with or without tetR(A). Once the ATG was mutated to a non-start codon, the tetracycline MICs were not changed, while the tetracycline MICs decreased from 512 to 64 µg ml(-1) following GTG mutation, and to ≤4 µg ml(-1) following mutation of both GTG and ATG. It was presumed that class A tet determinants had two start codons, which are the primary start codon GTG and secondary start codon ATG. Accordingly, two putative promoters were predicted. In conclusion, class A tet determinants can confer high-level tetracycline resistance and have two start codons. © 2014 The Authors.

A novel nuclear genetic code alteration in yeasts and the evolution of codon reassignment in eukaryotes.

PubMed

Mühlhausen, Stefanie; Findeisen, Peggy; Plessmann, Uwe; Urlaub, Henning; Kollmar, Martin

2016-07-01

The genetic code is the cellular translation table for the conversion of nucleotide sequences into amino acid sequences. Changes to the meaning of sense codons would introduce errors into almost every translated message and are expected to be highly detrimental. However, reassignment of single or multiple codons in mitochondria and nuclear genomes, although extremely rare, demonstrates that the code can evolve. Several models for the mechanism of alteration of nuclear genetic codes have been proposed (including "codon capture," "genome streamlining," and "ambiguous intermediate" theories), but with little resolution. Here, we report a novel sense codon reassignment in Pachysolen tannophilus, a yeast related to the Pichiaceae. By generating proteomics data and using tRNA sequence comparisons, we show that Pachysolen translates CUG codons as alanine and not as the more usual leucine. The Pachysolen tRNACAG is an anticodon-mutated tRNA(Ala) containing all major alanine tRNA recognition sites. The polyphyly of the CUG-decoding tRNAs in yeasts is best explained by a tRNA loss driven codon reassignment mechanism. Loss of the CUG-tRNA in the ancient yeast is followed by gradual decrease of respective codons and subsequent codon capture by tRNAs whose anticodon is not part of the aminoacyl-tRNA synthetase recognition region. Our hypothesis applies to all nuclear genetic code alterations and provides several testable predictions. We anticipate more codon reassignments to be uncovered in existing and upcoming genome projects. © 2016 Mühlhausen et al.; Published by Cold Spring Harbor Laboratory Press.

RNA editing makes mistakes in plant mitochondria: editing loses sense in transcripts of a rps19 pseudogene and in creating stop codons in coxI and rps3 mRNAs of Oenothera.

PubMed Central

Schuster, W; Brennicke, A

1991-01-01

An intact gene for the ribosomal protein S19 (rps19) is absent from Oenothera mitochondria. The conserved rps19 reading frame found in the mitochondrial genome is interrupted by a termination codon. This rps19 pseudogene is cotranscribed with the downstream rps3 gene and is edited on both sides of the translational stop. Editing, however, changes the amino acid sequence at positions that were well conserved before editing. Other strange editings create translational stops in open reading frames coding for functional proteins. In coxI and rps3 mRNAs CGA codons are edited to UGA stop codons only five and three codons, respectively, downstream to the initiation codon. These aberrant editings in essential open reading frames and in the rps19 pseudogene appear to have been shifted to these positions from other editing sites. These observations suggest a requirement for a continuous evolutionary constraint on the editing specificities in plant mitochondria. Images PMID:1762921

High-resolution melting analysis of gyrA codon 84 and grlA codon 80 mutations conferring resistance to fluoroquinolones in Staphylococcus pseudintermedius isolates from canine clinical samples.

PubMed

Loiacono, Monica; Martino, Piera A; Albonico, Francesca; Dell'Orco, Francesca; Ferretti, Manuela; Zanzani, Sergio; Mortarino, Michele

2017-09-01

Staphylococcus pseudintermedius is an opportunistic pathogen of dogs and cats. A high-resolution melting analysis (HRMA) protocol was designed and tested on 42 clinical isolates with known fluoroquinolone (FQ) susceptibility and gyrA codon 84 and grlA codon 80 mutation status. The HRMA approach was able to discriminate between FQ-sensitive and FQ-resistant strains and confirmed previous reports that the main mutation site associated with FQ resistance in S. pseudintermedius is located at position 251 (Ser84Leu) of gyrA. Routine, HRMA-based FQ susceptibility profiles may be a valuable tool to guide therapy. The FQ resistance-predictive power of the assay should be tested in a significantly larger number of isolates.

uORFs with unusual translational start codons autoregulate expression of eukaryotic ornithine decarboxylase homologs

PubMed Central

Ivanov, Ivaylo P.; Loughran, Gary; Atkins, John F.

2008-01-01

In a minority of eukaryotic mRNAs, a small functional upstream ORF (uORF), often performing a regulatory role, precedes the translation start site for the main product(s). Here, conserved uORFs in numerous ornithine decarboxylase homologs are identified from yeast to mammals. Most have noncanonical evolutionarily conserved start codons, the main one being AUU, which has not been known as an initiator for eukaryotic chromosomal genes. The AUG-less uORF present in mouse antizyme inhibitor, one of the ornithine decarboxylase homologs in mammals, mediates polyamine-induced repression of the downstream main ORF. This repression is part of an autoregulatory circuit, and one of its sensors is the AUU codon, which suggests that translation initiation codon identity is likely used for regulation in eukaryotes. PMID:18626014

Translation of vph mRNA in Streptomyces lividans and Escherichia coli after removal of the 5' untranslated leader.

PubMed

Wu, C J; Janssen, G R

1996-10-01

The Streptomyces vinaceus viomycin phosphotransferase (vph) mRNA contains an untranslated leader with a conventional Shine-Dalgarno homology. The vph leader was removed by ligation of the vph coding sequence to the transcriptional start site of a Streptomyces or an Escherichia coli promoter, such that transcription would initiate at the first position of the vph start codon. Analysis of mRNA demonstrated that transcription initiated primarily at the A of the vph AUG translational start codon in both Streptomyces lividans and E. coli; cells expressing the unleadered vph mRNA were resistant to viomycin indicating that the Shine-Dalgarno sequence, or other features contained within the leader, was not necessary for vph translation. Addition of four nucleotides (5'-AUGC-3') onto the 5' end of the unleadered vph mRNA resulted in translation initiation from the vph start codon and the AUG triplet contained within the added sequence. Translational fusions of vph sequence to a Tn5 neo reporter gene indicated that the first 16 codons of vph coding sequence were sufficient to specify the translational start site and reading frame for expression of neomycin resistance in both E. coli and S. lividans.

The Enterococcus faecalis EbpA Pilus Protein: Attenuation of Expression, Biofilm Formation, and Adherence to Fibrinogen Start with the Rare Initiation Codon ATT

PubMed Central

Montealegre, Maria Camila; La Rosa, Sabina Leanti; Roh, Jung Hyeob; Harvey, Barrett R.

2015-01-01

ABSTRACT The endocarditis and biofilm-associated pili (Ebp) are important in Enterococcus faecalis pathogenesis, and the pilus tip, EbpA, has been shown to play a major role in pilus biogenesis, biofilm formation, and experimental infections. Based on in silico analyses, we previously predicted that ATT is the EbpA translational start codon, not the ATG codon, 120 bp downstream of ATT, which is annotated as the translational start. ATT is rarely used to initiate protein synthesis, leading to our hypothesis that this codon participates in translational regulation of Ebp production. To investigate this possibility, site-directed mutagenesis was used to introduce consecutive stop codons in place of two lysines at positions 5 and 6 from the ATT, to replace the ATT codon in situ with ATG, and then to revert this ATG to ATT; translational fusions of ebpA to lacZ were also constructed to investigate the effect of these start codons on translation. Our results showed that the annotated ATG does not start translation of EbpA, implicating ATT as the start codon; moreover, the presence of ATT, compared to the engineered ATG, resulted in significantly decreased EbpA surface display, attenuated biofilm, and reduced adherence to fibrinogen. Corroborating these findings, the translational fusion with the native ATT as the initiation codon showed significantly decreased expression of β-galactosidase compared to the construct with ATG in place of ATT. Thus, these results demonstrate that the rare initiation codon of EbpA negatively regulates EbpA surface display and negatively affects Ebp-associated functions, including biofilm and adherence to fibrinogen. PMID:26015496

PreTIS: A Tool to Predict Non-canonical 5’ UTR Translational Initiation Sites in Human and Mouse

PubMed Central

Reuter, Kerstin; Helms, Volkhard

2016-01-01

Translation of mRNA sequences into proteins typically starts at an AUG triplet. In rare cases, translation may also start at alternative non–AUG codons located in the annotated 5’ UTR which leads to an increased regulatory complexity. Since ribosome profiling detects translational start sites at the nucleotide level, the properties of these start sites can then be used for the statistical evaluation of functional open reading frames. We developed a linear regression approach to predict in–frame and out–of–frame translational start sites within the 5’ UTR from mRNA sequence information together with their translation initiation confidence. Predicted start codons comprise AUG as well as near–cognate codons. The underlying datasets are based on published translational start sites for human HEK293 and mouse embryonic stem cells that were derived by the original authors from ribosome profiling data. The average prediction accuracy of true vs. false start sites for HEK293 cells was 80%. When applied to mouse mRNA sequences, the same model predicted translation initiation sites observed in mouse ES cells with an accuracy of 76%. Moreover, we illustrate the effect of in silico mutations in the flanking sequence context of a start site on the predicted initiation confidence. Our new webservice PreTIS visualizes alternative start sites and their respective ORFs and predicts their ability to initiate translation. Solely, the mRNA sequence is required as input. PreTIS is accessible at http://service.bioinformatik.uni-saarland.de/pretis. PMID:27768687

Model for Codon Position Bias in RNA Editing

NASA Astrophysics Data System (ADS)

Liu, Tsunglin; Bundschuh, Ralf

2005-08-01

RNA editing can be crucial for the expression of genetic information via inserting, deleting, or substituting a few nucleotides at specific positions in an RNA sequence. Within coding regions in an RNA sequence, editing usually occurs with a certain bias in choosing the positions of the editing sites. In the mitochondrial genes of Physarum polycephalum, many more editing events have been observed at the third codon position than at the first and second, while in some plant mitochondria the second codon position dominates. Here we propose an evolutionary model that explains this bias as the basis of selection at the protein level. The model predicts a distribution of the three positions rather close to the experimental observation in Physarum. This suggests that the codon position bias in Physarum is mainly a consequence of selection at the protein level.

A model for codon position bias in RNA editing

NASA Astrophysics Data System (ADS)

Bundschuh, Ralf; Liu, Tsunglin

2006-03-01

RNA editing can be crucial for the expression of genetic information via inserting, deleting, or substituting a few nucleotides at specific positions in an RNA sequence. Within coding regions in an RNA sequence, editing usually occurs with a certain bias in choosing the positions of the editing sites. In the mitochondrial genes of Physarum polycephalum, many more editing events have been observed at the third codon position than at the first and second, while in some plant mitochondria the second codon position dominates. Here we propose an evolutionary model that explains this bias as the basis of selection at the protein level. The model predicts a distribution of the three positions rather close to the experimental observation in Physarum. This suggests that the codon position bias in Physarum is mainly a consequence of selection at the protein level.

Expanding the utility of 4-cyano-L-phenylalanine as a vibrational reporter of protein environments.

PubMed

Bazewicz, Christopher G; Lipkin, Jacob S; Smith, Emily E; Liskov, Melanie T; Brewer, Scott H

2012-09-06

The ability to genetically incorporate amino acids modified with spectroscopic reporters site-specifically into proteins with high efficiency and fidelity has greatly enhanced the ability to probe local protein structure and dynamics. Here, we have synthesized the unnatural amino acid (UAA), 4-cyano-L-phenylalanine (pCNPhe), containing the nitrile vibrational reporter and three isotopomers ((15)N, (13)C, (13)C(15)N) of this UAA to enhance the ability of pCNPhe to study local protein environments. Each pCNPhe isotopic variant was genetically incorporated in an efficient, site-specific manner into superfolder green fluorescent protein (sfGFP) in response to an amber codon with high fidelity utilizing an engineered, orthogonal aminoacyl-tRNA synthetase. The isotopomers of 4-cyano-L-phenylalanine permitted the nitrile symmetric stretch vibration of these UAAs to be unambiguously assigned utilizing the magnitude and direction of the isotopic shift of this vibration. The sensitivity of the nitrile symmetric stretching frequency of each isotopic variant to the local environment was measured by individually incorporating the probes into two distinct local environments of sfGFP. The UAAs were also utilized in concert to probe multiple local environments in sfGFP simultaneously to increase the utility of 4-cyano-L-phenylalanine.

Mucopolysaccharidosis IVA: Four new exonic mutations in patients with N-acetylgalactosamine-6-sulfate sulfatase deficiency

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tomatsu, Shunji; Fukuda, Seiji; Yamagishi, Atsushi

1996-05-01

We report four new mutations in Japanese patients with mucopolysaccharidosis IVA (MPSIVA) who were heterozygous for a common double gene deletion. A nonsense mutation of CAG to TAG at codon 148 in exon 4 was identified, resulting in a change of Q to a stop codon and three missense mutations: V (GTC) to A (GCC) at codon 138 in exon 4, P (CCC) to S (TCC) at codon 151 in exon 5, and P (CCC) to L (CTC) at codon 151 in exon 5. Introduction of these mutations into the normal GALNS cDNA and transient expression in cultured fibroblasts resultedmore » in a significant decrease in the enzyme activity. V138A and Q148X mutations result in changes of restriction site, which were analyzed by restriction-enzyme assay. P151S and P151L mutations that did not alter the restriction site were detected by direct sequencing or allele specific oligohybridization. Detection of the double gene deletion was initially done using Southern blots and was confirmed by PCR. Haplotypes were determined using seven polymorphisms to the GALNS locus in families with the double gene deletion. Haplotype analysis showed that the common double gene deletion occurred on a single haplotype, except for some variation in a VNTR-like polymorphism. This finding is consistent with a common founder for all individuals with this mutation. 48 refs., 5 figs., 1 tab.« less

Cytochrome oxidase subunit II gene in mitochondria of Oenothera has no intron

PubMed Central

Hiesel, Rudolf; Brennicke, Axel

1983-01-01

The cytochrome oxidase subunit II gene has been localized in the mitochondrial genome of Oenothera berteriana and the nucleotide sequence has been determined. The coding sequence contains 777 bp and, unlike the corresponding gene in Zea mays, is not interrupted by an intron. No TGA codon is found within the open reading frame. The codon CGG, as in the maize gene, is used in place of tryptophan codons of corresponding genes in other organisms. At position 742 in the Oenothera sequence the TGG of maize is changed into a CGG codon, where Trp is conserved as the amino acid in other organisms. Homologous sequences occur more than once in the mitochondrial genome as several mitochondrial DNA species hybridize with DNA probes of the cytochrome oxidase subunit II gene. ImagesFig. 5. PMID:16453484

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ohshima, Kazusato, E-mail: ohshimak@cc.saga-u.ac.jp; The United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima; Matsumoto, Kosuke

Cucumber mosaic virus (CMV) is a damaging pathogen of over 200 mono- and dicotyledonous crop species worldwide. It has the broadest known host range of any virus, but the timescale of its evolution is unknown. To investigate the evolutionary history of this virus, we obtained the genomic sequences of 40 CMV isolates from brassicas sampled in Iran, Turkey and Japan, and combined them with published sequences. Our synonymous ('silent') site analyses revealed that the present CMV population is the progeny of a single ancestor existing 1550–2600 years ago, but that the population mostly radiated 295–545 years ago. We found thatmore » the major CMV lineages are not phylogeographically confined, but that recombination and reassortment is restricted to local populations and that no reassortant lineage is more than 251 years old. Our results highlight the different evolutionary patterns seen among viral pathogens of brassica crops across the world. - Highlights: • Present-day CMV lineages had a most recent common ancestor 1550–2600 years ago. • The CMV population mostly radiated less than 295–545 years ago. • No reassortant found in the present populations is more than 251 years old. • The open-reading frames evolve at around 2.3–4.7×10{sup −4} substitutions/site/year. • Synonymous codons of CMV seem to have a more precise temporal signal than all codons.« less

EvoDB: a database of evolutionary rate profiles, associated protein domains and phylogenetic trees for PFAM-A

PubMed Central

Ndhlovu, Andrew; Durand, Pierre M.; Hazelhurst, Scott

2015-01-01

The evolutionary rate at codon sites across protein-coding nucleotide sequences represents a valuable tier of information for aligning sequences, inferring homology and constructing phylogenetic profiles. However, a comprehensive resource for cataloguing the evolutionary rate at codon sites and their corresponding nucleotide and protein domain sequence alignments has not been developed. To address this gap in knowledge, EvoDB (an Evolutionary rates DataBase) was compiled. Nucleotide sequences and their corresponding protein domain data including the associated seed alignments from the PFAM-A (protein family) database were used to estimate evolutionary rate (ω = dN/dS) profiles at codon sites for each entry. EvoDB contains 98.83% of the gapped nucleotide sequence alignments and 97.1% of the evolutionary rate profiles for the corresponding information in PFAM-A. As the identification of codon sites under positive selection and their position in a sequence profile is usually the most sought after information for molecular evolutionary biologists, evolutionary rate profiles were determined under the M2a model using the CODEML algorithm in the PAML (Phylogenetic Analysis by Maximum Likelihood) suite of software. Validation of nucleotide sequences against amino acid data was implemented to ensure high data quality. EvoDB is a catalogue of the evolutionary rate profiles and provides the corresponding phylogenetic trees, PFAM-A alignments and annotated accession identifier data. In addition, the database can be explored and queried using known evolutionary rate profiles to identify domains under similar evolutionary constraints and pressures. EvoDB is a resource for evolutionary, phylogenetic studies and presents a tier of information untapped by current databases. Database URL: http://www.bioinf.wits.ac.za/software/fire/evodb PMID:26140928

EvoDB: a database of evolutionary rate profiles, associated protein domains and phylogenetic trees for PFAM-A.

PubMed

Ndhlovu, Andrew; Durand, Pierre M; Hazelhurst, Scott

2015-01-01

The evolutionary rate at codon sites across protein-coding nucleotide sequences represents a valuable tier of information for aligning sequences, inferring homology and constructing phylogenetic profiles. However, a comprehensive resource for cataloguing the evolutionary rate at codon sites and their corresponding nucleotide and protein domain sequence alignments has not been developed. To address this gap in knowledge, EvoDB (an Evolutionary rates DataBase) was compiled. Nucleotide sequences and their corresponding protein domain data including the associated seed alignments from the PFAM-A (protein family) database were used to estimate evolutionary rate (ω = dN/dS) profiles at codon sites for each entry. EvoDB contains 98.83% of the gapped nucleotide sequence alignments and 97.1% of the evolutionary rate profiles for the corresponding information in PFAM-A. As the identification of codon sites under positive selection and their position in a sequence profile is usually the most sought after information for molecular evolutionary biologists, evolutionary rate profiles were determined under the M2a model using the CODEML algorithm in the PAML (Phylogenetic Analysis by Maximum Likelihood) suite of software. Validation of nucleotide sequences against amino acid data was implemented to ensure high data quality. EvoDB is a catalogue of the evolutionary rate profiles and provides the corresponding phylogenetic trees, PFAM-A alignments and annotated accession identifier data. In addition, the database can be explored and queried using known evolutionary rate profiles to identify domains under similar evolutionary constraints and pressures. EvoDB is a resource for evolutionary, phylogenetic studies and presents a tier of information untapped by current databases. © The Author(s) 2015. Published by Oxford University Press.

Large-scale analyses of synonymous substitution rates can be sensitive to assumptions about the process of mutation.

PubMed

Aris-Brosou, Stéphane; Bielawski, Joseph P

2006-08-15

A popular approach to examine the roles of mutation and selection in the evolution of genomes has been to consider the relationship between codon bias and synonymous rates of molecular evolution. A significant relationship between these two quantities is taken to indicate the action of weak selection on substitutions among synonymous codons. The neutral theory predicts that the rate of evolution is inversely related to the level of functional constraint. Therefore, selection against the use of non-preferred codons among those coding for the same amino acid should result in lower rates of synonymous substitution as compared with sites not subject to such selection pressures. However, reliably measuring the extent of such a relationship is problematic, as estimates of synonymous rates are sensitive to our assumptions about the process of molecular evolution. Previous studies showed the importance of accounting for unequal codon frequencies, in particular when synonymous codon usage is highly biased. Yet, unequal codon frequencies can be modeled in different ways, making different assumptions about the mutation process. Here we conduct a simulation study to evaluate two different ways of modeling uneven codon frequencies and show that both model parameterizations can have a dramatic impact on rate estimates and affect biological conclusions about genome evolution. We reanalyze three large data sets to demonstrate the relevance of our results to empirical data analysis.

A mutated hygromycin resistance gene is functional in the n-alkane-assimilating yeast Candida tropicalis.

PubMed

Hara, A; Ueda, M; Misawa, S; Matsui, T; Furuhashi, K; Tanaka, A

2000-03-01

Development of a transformation system in the n-alkane-assimilating diploid yeast Candida tropicalis requires an antibiotic resistance gene in order to establish a selectable marker. The resistance gene for hygromycin B has often been used as a selectable marker in yeast transformation. However, C. tropicalis harboring the hygromycin resistance gene (HYG) was as sensitive to hygromycin B as the wild-type strain. Nine CTG codons were found in the ORF of the HYG gene. This codon has been reported to be translated as serine rather than leucine in Candida species. Analysis of the tRNA gene in C. tropicalis with the anticodon CAG [tRNA(CAG) gene], which is complementary to the codon CTG, showed that the sequence was highly similar to that of the C. maltosa tRNA(CAG) gene. In C. maltosa, the codon CTG is read as serine and not leucine. These results suggested that the HYG gene was not functional due to the nonuniversal usage of the CTG codon. Each of the nine CTG codons in the ORF of the HYG gene was changed to a CTC codon, which is read as leucine, by site-directed mutagenesis. When a plasmid containing the mutated HYG gene (HYG#) was constructed and introduced into C. tropicalis, hygromycin-resistant transformants were successfully obtained. This mutated hygromycin resistance gene may be useful for direct selection of C. tropicalis transformants.

Adaptive molecular evolution of the Major Histocompatibility Complex genes, DRA and DQA, in the genus Equus

PubMed Central

2011-01-01

Background Major Histocompatibility Complex (MHC) genes are central to vertebrate immune response and are believed to be under balancing selection by pathogens. This hypothesis has been supported by observations of extremely high polymorphism, elevated nonsynonymous to synonymous base pair substitution rates and trans-species polymorphisms at these loci. In equids, the organization and variability of this gene family has been described, however the full extent of diversity and selection is unknown. As selection is not expected to act uniformly on a functional gene, maximum likelihood codon-based models of selection that allow heterogeneity in selection across codon positions can be valuable for examining MHC gene evolution and the molecular basis for species adaptations. Results We investigated the evolution of two class II MHC genes of the Equine Lymphocyte Antigen (ELA), DRA and DQA, in the genus Equus with the addition of novel alleles identified in plains zebra (E. quagga, formerly E. burchelli). We found that both genes exhibited a high degree of polymorphism and inter-specific sharing of allele lineages. To our knowledge, DRA allelic diversity was discovered to be higher than has ever been observed in vertebrates. Evidence was also found to support a duplication of the DQA locus. Selection analyses, evaluated in terms of relative rates of nonsynonymous to synonymous mutations (dN/dS) averaged over the gene region, indicated that the majority of codon sites were conserved and under purifying selection (dN

Conserved nonsense-prone CpG sites in apoptosis-regulatory genes: conditional stop signs on the road to cell death.

PubMed

Zhao, Yongzhong; Epstein, Richard J

2013-01-01

Methylation-prone CpG dinucleotides are strongly conserved in the germline, yet are also predisposed to somatic mutation. Here we quantify the relationship between germline codon mutability and somatic carcinogenesis by comparing usage of the nonsense-prone CGA (→TGA) codons in gene groups that differ in apoptotic function; to this end, suppressor genes were subclassified as either apoptotic (gatekeepers) or repair (caretakers). Mutations affecting CGA codons in sporadic tumors proved to be highly asymmetric. Moreover, nonsense mutations were 3-fold more likely to affect gatekeepers than caretakers. In addition, intragenic CGA clustering nonrandomly affected functionally critical regions of gatekeepers. We conclude that human gatekeeper suppressor genes are enriched for nonsense-prone codons, and submit that this germline vulnerability to tumors could reflect in utero selection for a methylation-dependent capability to short-circuit environmental insults that otherwise trigger apoptosis and fetal loss.

Idiosyncratic recognition of UUG/UUA codons by modified nucleoside 5-taurinomethyluridine, τm5U present at 'wobble' position in anticodon loop of tRNALeu: A molecular modeling approach.

PubMed

Kamble, Asmita S; Fandilolu, Prayagraj M; Sambhare, Susmit B; Sonawane, Kailas D

2017-01-01

Lack of naturally occurring modified nucleoside 5-taurinomethyluridine (τm5U) at the 'wobble' 34th position in tRNALeu causes mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS). The τm5U34 specifically recognizes UUG and UUA codons. Structural consequences of τm5U34 to read cognate codons have not been studied so far in detail at the atomic level. Hence, 50ns multiple molecular dynamics (MD) simulations of various anticodon stem loop (ASL) models of tRNALeu in presence and absence of τm5U34 along with UUG and UUA codons were performed to explore the dynamic behaviour of τm5U34 during codon recognition process. The MD simulation results revealed that τm5U34 recognizes G/A ending codons by 'wobble' as well as a novel 'single' hydrogen bonding interactions. RMSD and RMSF values indicate the comparative stability of the ASL models containing τm5U34 modification over the other models, lacking τm5U34. Another MD simulation study of 55S mammalian mitochondrial rRNA with tRNALeu showed crucial interactions between the A-site residues, A918, A919, G256 and codon-anticodon bases. Thus, these results could improve our understanding about the decoding efficiency of human mt tRNALeu with τm5U34 to recognize UUG and UUA codons.

Idiosyncratic recognition of UUG/UUA codons by modified nucleoside 5-taurinomethyluridine, τm5U present at ‘wobble’ position in anticodon loop of tRNALeu: A molecular modeling approach

PubMed Central

Kamble, Asmita S.; Fandilolu, Prayagraj M.; Sambhare, Susmit B.; Sonawane, Kailas D.

2017-01-01

Lack of naturally occurring modified nucleoside 5-taurinomethyluridine (τm5U) at the ‘wobble’ 34th position in tRNALeu causes mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS). The τm5U34 specifically recognizes UUG and UUA codons. Structural consequences of τm5U34 to read cognate codons have not been studied so far in detail at the atomic level. Hence, 50ns multiple molecular dynamics (MD) simulations of various anticodon stem loop (ASL) models of tRNALeu in presence and absence of τm5U34 along with UUG and UUA codons were performed to explore the dynamic behaviour of τm5U34 during codon recognition process. The MD simulation results revealed that τm5U34 recognizes G/A ending codons by ‘wobble’ as well as a novel ‘single’ hydrogen bonding interactions. RMSD and RMSF values indicate the comparative stability of the ASL models containing τm5U34 modification over the other models, lacking τm5U34. Another MD simulation study of 55S mammalian mitochondrial rRNA with tRNALeu showed crucial interactions between the A-site residues, A918, A919, G256 and codon-anticodon bases. Thus, these results could improve our understanding about the decoding efficiency of human mt tRNALeu with τm5U34 to recognize UUG and UUA codons. PMID:28453549

Alterations of the three short open reading frames in the Rous sarcoma virus leader RNA modulate viral replication and gene expression.

PubMed Central

Moustakas, A; Sonstegard, T S; Hackett, P B

1993-01-01

The Rous sarcoma virus (RSV) leader RNA has three short open reading frames (ORF1 to ORF3) which are conserved in all avian sarcoma-leukosis retroviruses. Effects on virus propagation were determined following three types of alterations in the ORFs: (i) replacement of AUG initiation codons in order to prohibit ORF translation, (ii) alterations of the codon context around the AUG initiation codon to enhance translation of the normally silent ORF3, and (iii) elongation of the ORF coding sequences. Mutagenesis of the AUG codons for ORF1 and ORF2 (AUG1 and AUG2) singly or together delayed the onset of viral replication and cell transformation. In contrast, mutagenesis of AUG3 almost completely suppressed these viral activities. Mutagenesis of ORF3 to enhance its translation inhibited viral propagation. When the mutant ORF3 included an additional frameshift mutation which extended the ORF beyond the initiation site for the gag, gag-pol, and env proteins, host cells were initially transformed but died soon thereafter. Elongation of ORF1 from 7 to 62 codons led to the accumulation of transformation-defective virus with a delayed onset of replication. In contrast, viruses with elongation of ORF1 from 7 to 30 codons, ORF2 from 16 to 48 codons, or ORF3 from 9 to 64 codons, without any alterations in the AUG context, exhibited wild-type phenotypes. These results are consistent with a model that translation of the ORFs is necessary to facilitate virus production. Images PMID:7685415

Effect of the nucleotides surrounding the start codon on the translation of foot-and-mouth disease virus RNA.

PubMed

Ma, X X; Feng, Y P; Gu, Y X; Zhou, J H; Ma, Z R

2016-06-01

As for the alternative AUGs in foot-and-mouth disease virus (FMDV), nucleotide bias of the context flanking the AUG(2nd) could be used as a strong signal to initiate translation. To determine the role of the specific nucleotide context, dicistronic reporter constructs were engineered to contain different versions of nucleotide context linking between internal ribosome entry site (IRES) and downstream gene. The results indicate that under FMDV IRES-dependent mechanism, the nucleotide contexts flanking start codon can influence the translation initiation efficiencies. The most optimal sequences for both start codons have proved to be UUU AUG(1st) AAC and AAG AUG(2nd) GAA.

rpoB gene mutations among Mycobacterium tuberculosis isolates from extrapulmonary sites.

PubMed

Khosravi, Azar Dokht; Meghdadi, Hossein; Ghadiri, Ata A; Alami, Ameneh; Sina, Amir Hossein; Mirsaeidi, Mehdi

2018-03-01

The aim of this study was to analyze mutations occurring in the rpoB gene of Mycobacterium tuberculosis (MTB) isolates from clinical samples of extrapulmonary tuberculosis (EPTB). Seventy formalin-fixed, paraffin-embedded samples and fresh tissue samples from confirmed EPTB cases were analyzed. Nested PCR based on the rpoB gene was performed on the extracted DNAs, combined with cloning and subsequent sequencing. Sixty-seven (95.7%) samples were positive for nester PCR. Sequence analysis of the 81 bp region of the rpoB gene demonstrated mutations in 41 (61.2%) of 67 sequenced samples. Several point mutations including deletion mutations at codons 510, 512, 513 and 515, with 45% and 51% of the mutations in codons 512 and 513 respectively were seen, along with 26% replacement mutations at codons 509, 513, 514, 518, 520, 524 and 531. The most common alteration was Gln → His, at codon 513, presented in 30 (75.6%) isolates. This study demonstrated sequence alterations in codon 513 of the 81 bp region of the rpoB gene as the most common mutation occurred in 75.6% of molecularly confirmed rifampin-resistant strains. In addition, simultaneous mutation at codons 512 and 513 was demonstrated in 34.3% of the isolates. © 2018 APMIS. Published by John Wiley & Sons Ltd.

Detecting Adaptation in Protein-Coding Genes Using a Bayesian Site-Heterogeneous Mutation-Selection Codon Substitution Model.

PubMed

Rodrigue, Nicolas; Lartillot, Nicolas

2017-01-01

Codon substitution models have traditionally attempted to uncover signatures of adaptation within protein-coding genes by contrasting the rates of synonymous and non-synonymous substitutions. Another modeling approach, known as the mutation-selection framework, attempts to explicitly account for selective patterns at the amino acid level, with some approaches allowing for heterogeneity in these patterns across codon sites. Under such a model, substitutions at a given position occur at the neutral or nearly neutral rate when they are synonymous, or when they correspond to replacements between amino acids of similar fitness; substitutions from high to low (low to high) fitness amino acids have comparatively low (high) rates. Here, we study the use of such a mutation-selection framework as a null model for the detection of adaptation. Following previous works in this direction, we include a deviation parameter that has the effect of capturing the surplus, or deficit, in non-synonymous rates, relative to what would be expected under a mutation-selection modeling framework that includes a Dirichlet process approach to account for across-codon-site variation in amino acid fitness profiles. We use simulations, along with a few real data sets, to study the behavior of the approach, and find it to have good power with a low false-positive rate. Altogether, we emphasize the potential of recent mutation-selection models in the detection of adaptation, calling for further model refinements as well as large-scale applications. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

How the Sequence of a Gene Specifies Structural Symmetry in Proteins

PubMed Central

Shen, Xiaojuan; Huang, Tongcheng; Wang, Guanyu; Li, Guanglin

2015-01-01

Internal symmetry is commonly observed in the majority of fundamental protein folds. Meanwhile, sufficient evidence suggests that nascent polypeptide chains of proteins have the potential to start the co-translational folding process and this process allows mRNA to contain additional information on protein structure. In this paper, we study the relationship between gene sequences and protein structures from the viewpoint of symmetry to explore how gene sequences code for structural symmetry in proteins. We found that, for a set of two-fold symmetric proteins from left-handed beta-helix fold, intragenic symmetry always exists in their corresponding gene sequences. Meanwhile, codon usage bias and local mRNA structure might be involved in modulating translation speed for the formation of structural symmetry: a major decrease of local codon usage bias in the middle of the codon sequence can be identified as a common feature; and major or consecutive decreases in local mRNA folding energy near the boundaries of the symmetric substructures can also be observed. The results suggest that gene duplication and fusion may be an evolutionarily conserved process for this protein fold. In addition, the usage of rare codons and the formation of higher order of secondary structure near the boundaries of symmetric substructures might have coevolved as conserved mechanisms to slow down translation elongation and to facilitate effective folding of symmetric substructures. These findings provide valuable insights into our understanding of the mechanisms of translation and its evolution, as well as the design of proteins via symmetric modules. PMID:26641668

Pathways of Metabolite-related Damage to A Synthetic p53 Gene Exon 7 Oligonucleotide using Magnetic Enzyme Bioreactor Beads and LC-MS/MS Sequencing.

PubMed

Malla, Spundana; Kadimisetty, Karteek; Jiang, Di; Choudhary, Dharamainder; Rusling, James F

2018-05-11

Reactive metabolites of environmental chemicals and drugs can cause site-specific damage to p53 tumor suppressor gene in a major pathway for genotoxicity. We report here a high throughput, cell-free, 96-well plate magnetic bead-enzyme system interfaced with LC-MS/MS sequencing to bioactivate test chemicals and identify resulting adduction sites on genes. Bioactivated aflatoxin B1 was reacted with a 32 bp exon 7 fragment of the p53 gene using 8 microsomal cyt P450 enzymes from different organs coated on magnetic beads. All cyt P450s converted aflatoxin B1 to aflatoxin B1-8,9-epoxide that adducts guanine (G) in codon 249, with subsequent depurination to give abasic sites, then strand breaks. This is the first demonstration in a cell-free medium that aflatoxin B1 metabolite selectively causes abasic site formation and strand breaks at codon 249 of the p53 probe, corresponding to the chemical pathway and mutations of p53 in human liver cells and tumors. Molecular modeling supports the view that binding of aflatoxin B1-8,9-epoxide to G in codon 249 precedes the SN2 adduction reaction. Among a range of metabolic enzymes characteristic of different organs, human liver microsomes and cyt P450 3A5 supersomes showed the highest bioactivation rate for p53 exon 7 damage. This method to identify metabolite-related gene damage sites may facilitate predictions of organ-specific cancers for test chemicals via correlations with mutation sites.

Global analysis of translation termination in E. coli.

PubMed

Baggett, Natalie E; Zhang, Yan; Gross, Carol A

2017-03-01

Terminating protein translation accurately and efficiently is critical for both protein fidelity and ribosome recycling for continued translation. The three bacterial release factors (RFs) play key roles: RF1 and 2 recognize stop codons and terminate translation; and RF3 promotes disassociation of bound release factors. Probing release factors mutations with reporter constructs containing programmed frameshifting sequences or premature stop codons had revealed a propensity for readthrough or frameshifting at these specific sites, but their effects on translation genome-wide have not been examined. We performed ribosome profiling on a set of isogenic strains with well-characterized release factor mutations to determine how they alter translation globally. Consistent with their known defects, strains with increasingly severe release factor defects exhibit increasingly severe accumulation of ribosomes over stop codons, indicative of an increased duration of the termination/release phase of translation. Release factor mutant strains also exhibit increased occupancy in the region following the stop codon at a significant number of genes. Our global analysis revealed that, as expected, translation termination is generally efficient and accurate, but that at a significant number of genes (≥ 50) the ribosome signature after the stop codon is suggestive of translation past the stop codon. Even native E. coli K-12 exhibits the ribosome signature suggestive of protein extension, especially at UGA codons, which rely exclusively on the reduced function RF2 variant of the K-12 strain for termination. Deletion of RF3 increases the severity of the defect. We unambiguously demonstrate readthrough and frameshifting protein extensions and their further accumulation in mutant strains for a few select cases. In addition to enhancing recoding, ribosome accumulation over stop codons disrupts attenuation control of biosynthetic operons, and may alter expression of some overlapping genes. Together, these functional alterations may either augment the protein repertoire or produce deleterious proteins.

Somatic mutations in cancer: Stochastic versus predictable.

PubMed

Gold, Barry

2017-02-01

The origins of human cancers remain unclear except for a limited number of potent environmental mutagens, such as tobacco and UV light, and in rare cases, familial germ line mutations that affect tumor suppressor genes or oncogenes. A significant component of cancer etiology has been deemed stochastic and correlated with the number of stem cells in a tissue, the number of times the stem cells divide and a low incidence of random DNA polymerase errors that occur during each cell division. While somatic mutations occur during each round of DNA replication, mutations in cancer driver genes are not stochastic. Out of a total of 2843 codons, 1031 can be changed to stop codons by a single base substitution in the tumor suppressor APC gene, which is mutated in 76% of colorectal cancers (CRC). However, the nonsense mutations, which comprise 65% of all the APC driver mutations in CRC, are not random: 43% occur at Arg CGA codons, although they represent <3% of the codons. In TP53, CGA codons comprise <3% of the total 393 codons but they account for 72% and 39% of the mutations in CRC and ovarian cancer OVC, respectively. This mutation pattern is consistent with the kinetically slow, but not stochastic, hydrolytic deamination of 5-methylcytosine residues at specific methylated CpG sites to afford T·G mismatches that lead to C→T transitions and stop codons at CGA. Analysis of nonsense mutations in CRC, OVC and a number of other cancers indicates the need to expand the predictable risk factors for cancer to include, in addition to random polymerase errors, the methylation status of gene body CGA codons in tumor suppressor genes. Copyright © 2017. Published by Elsevier B.V.

The Acheta domesticus Densovirus, Isolated from the European House Cricket, Has Evolved an Expression Strategy Unique among Parvoviruses▿†

PubMed Central

Liu, Kaiyu; Li, Yi; Jousset, Françoise-Xavière; Zadori, Zoltan; Szelei, Jozsef; Yu, Qian; Pham, Hanh Thi; Lépine, François; Bergoin, Max; Tijssen, Peter

2011-01-01

The Acheta domesticus densovirus (AdDNV), isolated from crickets, has been endemic in Europe for at least 35 years. Severe epizootics have also been observed in American commercial rearings since 2009 and 2010. The AdDNV genome was cloned and sequenced for this study. The transcription map showed that splicing occurred in both the nonstructural (NS) and capsid protein (VP) multicistronic RNAs. The splicing pattern of NS mRNA predicted 3 nonstructural proteins (NS1 [576 codons], NS2 [286 codons], and NS3 [213 codons]). The VP gene cassette contained two VP open reading frames (ORFs), of 597 (ORF-A) and 268 (ORF-B) codons. The VP2 sequence was shown by N-terminal Edman degradation and mass spectrometry to correspond with ORF-A. Mass spectrometry, sequencing, and Western blotting of baculovirus-expressed VPs versus native structural proteins demonstrated that the VP1 structural protein was generated by joining ORF-A and -B via splicing (splice II), eliminating the N terminus of VP2. This splice resulted in a nested set of VP1 (816 codons), VP3 (467 codons), and VP4 (429 codons) structural proteins. In contrast, the two splices within ORF-B (Ia and Ib) removed the donor site of intron II and resulted in VP2, VP3, and VP4 expression. ORF-B may also code for several nonstructural proteins, of 268, 233, and 158 codons. The small ORF-B contains the coding sequence for a phospholipase A2 motif found in VP1, which was shown previously to be critical for cellular uptake of the virus. These splicing features are unique among parvoviruses and define a new genus of ambisense densoviruses. PMID:21775445

Unraveling patterns of site-to-site synonymous rates variation and associated gene properties of protein domains and families.

PubMed

Dimitrieva, Slavica; Anisimova, Maria

2014-01-01

In protein-coding genes, synonymous mutations are often thought not to affect fitness and therefore are not subject to natural selection. Yet increasingly, cases of non-neutral evolution at certain synonymous sites were reported over the last decade. To evaluate the extent and the nature of site-specific selection on synonymous codons, we computed the site-to-site synonymous rate variation (SRV) and identified gene properties that make SRV more likely in a large database of protein-coding gene families and protein domains. To our knowledge, this is the first study that explores the determinants and patterns of the SRV in real data. We show that the SRV is widespread in the evolution of protein-coding sequences, putting in doubt the validity of the synonymous rate as a standard neutral proxy. While protein domains rarely undergo adaptive evolution, the SRV appears to play important role in optimizing the domain function at the level of DNA. In contrast, protein families are more likely to evolve by positive selection, but are less likely to exhibit SRV. Stronger SRV was detected in genes with stronger codon bias and tRNA reusage, those coding for proteins with larger number of interactions or forming larger number of structures, located in intracellular components and those involved in typically conserved complex processes and functions. Genes with extreme SRV show higher expression levels in nearly all tissues. This indicates that codon bias in a gene, which often correlates with gene expression, may often be a site-specific phenomenon regulating the speed of translation along the sequence, consistent with the co-translational folding hypothesis. Strikingly, genes with SRV were strongly overrepresented for metabolic pathways and those associated with several genetic diseases, particularly cancers and diabetes.

Two Isoforms of Geobacter sulfurreducens PilA Have Distinct Roles in Pilus Biogenesis, Cytochrome Localization, Extracellular Electron Transfer, and Biofilm Formation

PubMed Central

Richter, Lubna V.; Sandler, Steven J.

2012-01-01

Type IV pili of Geobacter sulfurreducens are composed of PilA monomers and are essential for long-range extracellular electron transfer to insoluble Fe(III) oxides and graphite anodes. A previous analysis of pilA expression indicated that transcription was initiated at two positions, with two predicted ribosome-binding sites and translation start codons, potentially producing two PilA preprotein isoforms. The present study supports the existence of two functional translation start codons for pilA and identifies two isoforms (short and long) of the PilA preprotein. The short PilA isoform is found predominantly in an intracellular fraction. It seems to stabilize the long isoform and to influence the secretion of several outer-surface c-type cytochromes. The long PilA isoform is required for secretion of PilA to the outer cell surface, a process that requires coexpression of pilA with nine downstream genes. The long isoform was determined to be essential for biofilm formation on certain surfaces, for optimum current production in microbial fuel cells, and for growth on insoluble Fe(III) oxides. PMID:22408162

Cloning and sequencing of the pheP gene, which encodes the phenylalanine-specific transport system of Escherichia coli.

PubMed Central

Pi, J; Wookey, P J; Pittard, A J

1991-01-01

The phenylalanine-specific permease gene (pheP) of Escherichia coli has been cloned and sequenced. The gene was isolated on a 6-kb Sau3AI fragment from a chromosomal library, and its presence was verified by complementation of a mutant lacking the functional phenylalanine-specific permease. Subcloning from this fragment localized the pheP gene on a 2.7-kb HindIII-HindII fragment. The nucleotide sequence of this 2.7-kb region was determined. An open reading frame was identified which extends from a putative start point of translation (GTG at position 636) to a termination signal (TAA at position 2010). The assignment of the GTG as the initiation codon was verified by site-directed mutagenesis of the initiation codon and by introducing a chain termination mutation into the pheP-lacZ fusion construct. A single initiation site of transcription 30 bp upstream of the start point of translation was identified by the primer extension analysis. The pheP structural gene consists of 1,374 nucleotides specifying a protein of 458 amino acid residues. The PheP protein is very hydrophobic (71% nonpolar residues). A topological model predicted from the sequence analysis defines 12 transmembrane segments. This protein is highly homologous with the AroP (general aromatic transport) system of E. coli (59.6% identity) and to a lesser extent with the yeast permeases CAN1 (arginine), PUT4 (proline), and HIP1 (histidine) of Saccharomyces cerevisiae. Images PMID:1711024

Site-specific incorporation of 4-iodo-L-phenylalanine through opal suppression.

PubMed

Kodama, Koichiro; Nakayama, Hiroshi; Sakamoto, Kensaku; Fukuzawa, Seketsu; Kigawa, Takanori; Yabuki, Takashi; Kitabatake, Makoto; Takio, Koji; Yokoyama, Shigeyuki

2010-08-01

A variety of unique codons have been employed to expand the genetic code. The use of the opal (UGA) codon is promising, but insufficient information is available about the UGA suppression approach, which facilitates the incorporation of non-natural amino acids through suppression of the UGA codon. In this study, the UGA codon was used to incorporate 4-iodo-l-phenylalanine into position 32 of the Ras protein in an Escherichia coli cell-free translation system. The undesired incorporation of tryptophan in response to the UGA codon was completely repressed by the addition of indolmycin. The minor amount (3%) of contaminating 4-bromo-l-phenylalanine in the building block 4-iodo-l-phenylalanine led to the significant incorporation of 4-bromo-l-phenylalanine (21%), and this problem was solved by using a purified 4-iodo-l-phenylalanine sample. Optimization of the incubation time was also important, since the undesired incorporation of free phenylalanine increased during the cell-free translation reaction. The 4-iodo-l-phenylalanine residue can be used for the chemoselective modification of proteins. This method will contribute to advancements in protein engineering studies with non-natural amino acid substitutions.

Modulation of c-fms proto-oncogene in an ovarian carcinoma cell line by a hammerhead ribozyme.

PubMed Central

Yokoyama, Y.; Morishita, S.; Takahashi, Y.; Hashimoto, M.; Tamaya, T.

1997-01-01

Co-expression of macrophage colony-stimulating factor (M-CSF) and its receptor (c-fms) is often found in ovarian epithelial carcinoma, suggesting the existence of autocrine regulation of cell growth by M-CSF. To block this autocrine loop, we have developed hammerhead ribozymes against c-fms mRNA. As target sites of the ribozyme, we chose the GUC sequence in codon 18 and codon 27 of c-fms mRNA. Two kinds of ribozymes were able to cleave an artificial c-fms RNA substrate in a cell-free system, although the ribozyme against codon 18 was much more efficient than that against codon 27. We next constructed an expression vector carrying a ribozyme sequence that targeted the GUC sequence in codon 18 of c-fms mRNA. It was introduced into TYK-nu cells that expressed M-CSF and its receptor. Its transfectant showed a reduced growth potential. The expression levels of c-fms protein and mRNA in the transfectant were clearly decreased with the expression of ribozyme RNA compared with that of an untransfected control or a transfectant with the vector without the ribozyme sequence. These results suggest that the ribozyme against GUC in codon 18 of c-fms mRNA is a promising tool for blocking the autocrine loop of M-CSF in ovarian epithelial carcinoma. Images Figure 2 Figure 3 Figure 5 Figure 6 PMID:9376277

Methylation of class I translation termination factors: structural and functional aspects.

PubMed

Graille, Marc; Figaro, Sabine; Kervestin, Stéphanie; Buckingham, Richard H; Liger, Dominique; Heurgué-Hamard, Valérie

2012-07-01

During protein synthesis, release of polypeptide from the ribosome occurs when an in frame termination codon is encountered. Contrary to sense codons, which are decoded by tRNAs, stop codons present in the A-site are recognized by proteins named class I release factors, leading to the release of newly synthesized proteins. Structures of these factors bound to termination ribosomal complexes have recently been obtained, and lead to a better understanding of stop codon recognition and its coordination with peptidyl-tRNA hydrolysis in bacteria. Release factors contain a universally conserved GGQ motif which interacts with the peptidyl-transferase centre to allow peptide release. The Gln side chain from this motif is methylated, a feature conserved from bacteria to man, suggesting an important biological role. However, methylation is catalysed by completely unrelated enzymes. The function of this motif and its post-translational modification will be discussed in the context of recent structural and functional studies. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

A method for multi-codon scanning mutagenesis of proteins based on asymmetric transposons.

PubMed

Liu, Jia; Cropp, T Ashton

2012-02-01

Random mutagenesis followed by selection or screening is a commonly used strategy to improve protein function. Despite many available methods for random mutagenesis, nearly all generate mutations at the nucleotide level. An ideal mutagenesis method would allow for the generation of 'codon mutations' to change protein sequence with defined or mixed amino acids of choice. Herein we report a method that allows for mutations of one, two or three consecutive codons. Key to this method is the development of a Mu transposon variant with asymmetric terminal sequences. As a demonstration of the method, we performed multi-codon scanning on the gene encoding superfolder GFP (sfGFP). Characterization of 50 randomly chosen clones from each library showed that more than 40% of the mutants in these three libraries contained seamless, in-frame mutations with low site preference. By screening only 500 colonies from each library, we successfully identified several spectra-shift mutations, including a S205D variant that was found to bear a single excitation peak in the UV region.

Living colors in the gray mold pathogen Botrytis cinerea: codon-optimized genes encoding green fluorescent protein and mCherry, which exhibit bright fluorescence.

PubMed

Leroch, Michaela; Mernke, Dennis; Koppenhoefer, Dieter; Schneider, Prisca; Mosbach, Andreas; Doehlemann, Gunther; Hahn, Matthias

2011-05-01

The green fluorescent protein (GFP) and its variants have been widely used in modern biology as reporters that allow a variety of live-cell imaging techniques. So far, GFP has rarely been used in the gray mold fungus Botrytis cinerea because of low fluorescence intensity. The codon usage of B. cinerea genes strongly deviates from that of commonly used GFP-encoding genes and reveals a lower GC content than other fungi. In this study, we report the development and use of a codon-optimized version of the B. cinerea enhanced GFP (eGFP)-encoding gene (Bcgfp) for improved expression in B. cinerea. Both the codon optimization and, to a smaller extent, the insertion of an intron resulted in higher mRNA levels and increased fluorescence. Bcgfp was used for localization of nuclei in germinating spores and for visualizing host penetration. We further demonstrate the use of promoter-Bcgfp fusions for quantitative evaluation of various toxic compounds as inducers of the atrB gene encoding an ABC-type drug efflux transporter of B. cinerea. In addition, a codon-optimized mCherry-encoding gene was constructed which yielded bright red fluorescence in B. cinerea.

The ribosome uses two active mechanisms to unwind messenger RNA during translation.

PubMed

Qu, Xiaohui; Wen, Jin-Der; Lancaster, Laura; Noller, Harry F; Bustamante, Carlos; Tinoco, Ignacio

2011-07-06

The ribosome translates the genetic information encoded in messenger RNA into protein. Folded structures in the coding region of an mRNA represent a kinetic barrier that lowers the peptide elongation rate, as the ribosome must disrupt structures it encounters in the mRNA at its entry site to allow translocation to the next codon. Such structures are exploited by the cell to create diverse strategies for translation regulation, such as programmed frameshifting, the modulation of protein expression levels, ribosome localization and co-translational protein folding. Although strand separation activity is inherent to the ribosome, requiring no exogenous helicases, its mechanism is still unknown. Here, using a single-molecule optical tweezers assay on mRNA hairpins, we find that the translation rate of identical codons at the decoding centre is greatly influenced by the GC content of folded structures at the mRNA entry site. Furthermore, force applied to the ends of the hairpin to favour its unfolding significantly speeds translation. Quantitative analysis of the force dependence of its helicase activity reveals that the ribosome, unlike previously studied helicases, uses two distinct active mechanisms to unwind mRNA structure: it destabilizes the helical junction at the mRNA entry site by biasing its thermal fluctuations towards the open state, increasing the probability of the ribosome translocating unhindered; and it mechanically pulls apart the mRNA single strands of the closed junction during the conformational changes that accompany ribosome translocation. The second of these mechanisms ensures a minimal basal rate of translation in the cell; specialized, mechanically stable structures are required to stall the ribosome temporarily. Our results establish a quantitative mechanical basis for understanding the mechanism of regulation of the elongation rate of translation by structured mRNAs. ©2011 Macmillan Publishers Limited. All rights reserved

Evolution of CCL11: genetic characterization in lagomorphs and evidence of positive and purifying selection in mammals.

PubMed

Neves, Fabiana; Abrantes, Joana; Esteves, Pedro J

2016-07-01

The interactions between chemokines and their receptors are crucial for differentiation and activation of inflammatory cells. CC chemokine ligand 11 (CCL11) binds to CCR3 and to CCR5 that in leporids underwent gene conversion with CCR2. Here, we genetically characterized CCL11 in lagomorphs (leporids and pikas). All lagomorphs have a potentially functional CCL11, and the Pygmy rabbit has a mutation in the stop codon that leads to a longer protein. Other mammals also have mutations at the stop codon that result in proteins with different lengths. By employing maximum likelihood methods, we observed that, in mammals, CCL11 exhibits both signatures of purifying and positive selection. Signatures of purifying selection were detected in sites important for receptor binding and activation. Of the three sites detected as under positive selection, two were located close to the stop codon. Our results suggest that CCL11 is functional in all lagomorphs, and that the signatures of purifying and positive selection in mammalian CCL11 probably reflect the protein's biological roles. © The Author(s) 2016.

tRNA tKUUU, tQUUG, and tEUUC wobble position modifications fine-tune protein translation by promoting ribosome A-site binding.

PubMed

Rezgui, Vanessa Anissa Nathalie; Tyagi, Kshitiz; Ranjan, Namit; Konevega, Andrey L; Mittelstaet, Joerg; Rodnina, Marina V; Peter, Matthias; Pedrioli, Patrick G A

2013-07-23

tRNA modifications are crucial to ensure translation efficiency and fidelity. In eukaryotes, the URM1 and ELP pathways increase cellular resistance to various stress conditions, such as nutrient starvation and oxidative agents, by promoting thiolation and methoxycarbonylmethylation, respectively, of the wobble uridine of cytoplasmic (tK(UUU)), (tQ(UUG)), and (tE(UUC)). Although in vitro experiments have implicated these tRNA modifications in modulating wobbling capacity and translation efficiency, their exact in vivo biological roles remain largely unexplored. Using a combination of quantitative proteomics and codon-specific translation reporters, we find that translation of a specific gene subset enriched for AAA, CAA, and GAA codons is impaired in the absence of URM1- and ELP-dependent tRNA modifications. Moreover, in vitro experiments using native tRNAs demonstrate that both modifications enhance binding of tK(UUU) to the ribosomal A-site. Taken together, our data suggest that tRNA thiolation and methoxycarbonylmethylation regulate translation of genes with specific codon content.

Molecular Scanning of β-Thalassemia in the Southern Region of Central Java, Indonesia; a Step Towards a Local Prevention Program.

PubMed

Rujito, Lantip; Basalamah, Muhammad; Mulatsih, Sri; Sofro, Abdul Salam M

2015-08-03

Thalassemia is the most prevalent genetic blood disorder worldwide, and particularly prevalent in Indonesia. The purpose of this study was to determine the spectrum of β-thalassemia (β-thal) mutations found in the southern region of Central Java, Indonesia. The subjects of the study included 209 β-thal Javanese patients from Banyumas Residency, a southwest region of Central Java Province. DNA analysis was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), amplification refractory mutation system (ARMS), and the direct sequencing method. The results showed that 14 alleles were found in the following order: IVS-I-5 (G > C) (HBB: c.92 + 5G > C) 43.5%, codon 26 (Hb E; HBB: c.79G > A) 28.2%, IVS-I-1 (G > A) (HBB: c.92 + 1G > A) 5.0%, codon 15 (TGG > TAG) (HBB: c.47G > A) 3.8%, IVS-I-1 (G > T) (HBB: c.92 + 1G > T) 3.1%, codon 35 (-C) (HBB: c.110delC) 2.4%. The rest, including codons 41/42 (-TTCT) (HBB: c.126_129delCTTT), codons 8/9 (+G) (HBB: c.27_28insG), codon 19 (AAC > AGC) (HBB: c.59A > G), codon 17 (AAG > TAG) (HBB: c.52A > T), IVS-I-2 (T > C) (HBB: c.92 + 2T > C), codons 123/124/125 (-ACCCCACC) (HBB: c.370_378delACCCCACCA), codon 40 (-G) (HBB: c.123delG) and Cap +1 (A > C) (HBB: c.-50A > C), accounted for up to 1.0% each. The most prevalent alleles would be recommended to be used as part of β-thal screening for the Javanese, one of the major ethnic groups in the country.

Molecular Scanning of β-Thalassemia in the Southern Region of Central Java, Indonesia; a Step Towards a Local Prevention Program.

PubMed

Rujito, Lantip; Basalamah, Muhammad; Mulatsih, Sri; Sofro, Abdul Salam M

2015-01-01

Thalassemia is the most prevalent genetic blood disorder worldwide, and particularly prevalent in Indonesia. The purpose of this study was to determine the spectrum of β-thalassemia (β-thal) mutations found in the southern region of Central Java, Indonesia. The subjects of the study included 209 β-thal Javanese patients from Banyumas Residency, a southwest region of Central Java Province. DNA analysis was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), amplification refractory mutation system (ARMS), and the direct sequencing method. The results showed that 14 alleles were found in the following order: IVS-I-5 (G > C) (HBB: c.92 + 5G > C) 43.5%, codon 26 (Hb E; HBB: c.79G > A) 28.2%, IVS-I-1 (G > A) (HBB: c.92 + 1G > A) 5.0%, codon 15 (TGG > TAG) (HBB: c.47G > A) 3.8%, IVS-I-1 (G > T) (HBB: c.92 + 1G > T) 3.1%, codon 35 (-C) (HBB: c.110delC) 2.4%. The rest, including codons 41/42 (-TTCT) (HBB: c.126_129delCTTT), codons 8/9 (+G) (HBB: c.27_28insG), codon 19 (AAC > AGC) (HBB: c.59A > G), codon 17 (AAG > TAG) (HBB: c.52A > T), IVS-I-2 (T > C) (HBB: c.92 + 2T > C), codons 123/124/125 (-ACCCCACC) (HBB: c.370_378delACCCCACCA), codon 40 (-G) (HBB: c.123delG) and Cap +1 (A > C) (HBB: c.-50A > C), accounted for up to 1.0% each. The most prevalent alleles would be recommended to be used as part of β-thal screening for the Javanese, one of the major ethnic groups in the country.

[Positioning of mRNA 3' of the a site bound codon on the human 80S ribosome].

PubMed

Molotkov, M V; Graĭfer, D M; Demeshkina, N A; Repkova, M N; Ven'iaminova, A G; Karpova, G G

2005-01-01

Short mRNA analogues carrying a UUU triplet at the 5'-termini and a perfluorophenylazide group at either the N7 atom of the guanosine or the C5 atom of the uridine 3' of the triplet were applied to study positioning of mRNA 3' of the A site codon. Complexes of 80S ribosomes with the mRNA analogues were obtained in the presence of tRNAPhe that directed UUU codon to the P site and consequently provided placement of the nucleotide with cross-linker in positions +9 or +12 with respect to the first nucleotide of the P site bound codon. Both types mRNA analogues cross-linked to the 18S rRNA and 40S proteins under mild UV-irradiation. Cross-linking patterns in the complexes where modified nucleotides of the mRNA analogues were in position +7 were analyzed for comparison (cross-linking to the 18S rRNA in such complexes has been studied previously). The efficiency of cross-linking to the ribosomal components depended on the nature of the modified nucleotide in the mRNA analogue and its position on the ribosome, extent of cross-linking to the 18S rRNA being decreased drastically when the modified nucleotide was moved from position +7 to position +12. The nucleotides of 18S rRNA cross-linked to mRNA analogues were determined. Modified nucleotides in positions +9 and +12 cross-linked to the invariant dinucleotide A1824/A1825 and to variable A1823 in the 3'-minidomain of 18S rRNA as well as to protein S15. The same ribosomal components have been found earlier to cross-link to modified mRNA nucleotides in positions from +4 to +7. Besides, all mRNA analogues cross-linked to the invariant nucleotide c1698 in the 3'-minidomain and to and the conserved region 605-620 closing helix 18 in the 5'-domain.

Methyl-Cytosine-Driven Structural Changes Enhance Adduction Kinetics of an Exon 7 fragment of the p53 Gene

NASA Astrophysics Data System (ADS)

Malla, Spundana; Kadimisetty, Karteek; Fu, You-Jun; Choudhary, Dharamainder; Schenkman, John B.; Rusling, James F.

2017-01-01

Methylation of cytosine (C) at C-phosphate-guanine (CpG) sites enhances reactivity of DNA towards electrophiles. Mutations at CpG sites on the p53 tumor suppressor gene that can result from these adductions are in turn correlated with specific cancers. Here we describe the first restriction-enzyme-assisted LC-MS/MS sequencing study of the influence of methyl cytosines (MeC) on kinetics of p53 gene adduction by model metabolite benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), using methodology applicable to correlate gene damage sites for drug and pollutant metabolites with mutation sites. This method allows direct kinetic measurements by LC-MS/MS sequencing for oligonucleotides longer than 20 base pairs (bp). We used MeC and non-MeC (C) versions of a 32 bp exon 7 fragment of the p53 gene. Methylation of 19 cytosines increased the rate constant 3-fold for adduction on G at the major reactive CpG in codon 248 vs. the non-MeC fragment. Rate constants for non-CpG codons 244 and 243 were not influenced significantly by MeC. Conformational and hydrophobicity changes in the MeC-p53 exon 7 fragment revealed by CD spectra and molecular modeling increase the BPDE binding constant to G in codon 248 consistent with a pathway in which preceding reactant binding greatly facilitates the rate of covalent SN2 coupling.

Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

PubMed Central

Cozens, Christopher

2018-01-01

Abstract Engineering proteins for designer functions and biotechnological applications almost invariably requires (or at least benefits from) multiple mutations to non-contiguous residues. Several methods for multiple site-directed mutagenesis exist, but there remains a need for fast and simple methods to efficiently introduce such mutations – particularly for generating large, high quality libraries for directed evolution. Here, we present Darwin Assembly, which can deliver high quality libraries of >108 transformants, targeting multiple (>10) distal sites with minimal wild-type contamination (<0.25% of total population) and which takes a single working day from purified plasmid to library transformation. We demonstrate its efficacy with whole gene codon reassignment of chloramphenicol acetyl transferase, mutating 19 codons in a single reaction in KOD DNA polymerase and generating high quality, multiple-site libraries in T7 RNA polymerase and Tgo DNA polymerase. Darwin Assembly uses commercially available enzymes, can be readily automated, and offers a cost-effective route to highly complex and customizable library generation. PMID:29409059

The Role of +4U as an Extended Translation Termination Signal in Bacteria

PubMed Central

Wei, Yulong; Xia, Xuhua

2017-01-01

Termination efficiency of stop codons depends on the first 3′ flanking (+4) base in bacteria and eukaryotes. In both Escherichia coli and Saccharomyces cerevisiae, termination read-through is reduced in the presence of +4U; however, the molecular mechanism underlying +4U function is poorly understood. Here, we perform comparative genomics analysis on 25 bacterial species (covering Actinobacteria, Bacteriodetes, Cyanobacteria, Deinococcus-Thermus, Firmicutes, Proteobacteria, and Spirochaetae) with bioinformatics approaches to examine the influence of +4U in bacterial translation termination by contrasting highly- and lowly-expressed genes (HEGs and LEGs, respectively). We estimated gene expression using the recently formulated Index of Translation Elongation, ITE, and identified stop codon near-cognate transfer RNAs (tRNAs) from well-annotated genomes. We show that +4U was consistently overrepresented in UAA-ending HEGs relative to LEGs. The result is consistent with the interpretation that +4U enhances termination mainly for UAA. Usage of +4U decreases in GC-rich species where most stop codons are UGA and UAG, with few UAA-ending genes, which is expected if UAA usage in HEGs drives up +4U usage. In HEGs, +4U usage increases significantly with abundance of UAA nc_tRNAs (near-cognate tRNAs that decode codons differing from UAA by a single nucleotide), particularly those with a mismatch at the first stop codon site. UAA is always the preferred stop codon in HEGs, and our results suggest that UAAU is the most efficient translation termination signal in bacteria. PMID:27903612

Defining the mRNA recognition signature of a bacterial toxin protein

DOE PAGES

Schureck, Marc A.; Dunkle, Jack A.; Maehigashi, Tatsuya; ...

2015-10-27

Bacteria contain multiple type II toxins that selectively degrade mRNAs bound to the ribosome to regulate translation and growth and facilitate survival during the stringent response. Ribosome-dependent toxins recognize a variety of three-nucleotide codons within the aminoacyl (A) site, but how these endonucleases achieve substrate specificity remains poorly understood. In this paper, we identify the critical features for how the host inhibition of growth B (HigB) toxin recognizes each of the three A-site nucleotides for cleavage. X-ray crystal structures of HigB bound to two different codons on the ribosome illustrate how HigB uses a microbial RNase-like nucleotide recognition loop tomore » recognize either cytosine or adenosine at the second A-site position. Strikingly, a single HigB residue and 16S rRNA residue C1054 form an adenosine-specific pocket at the third A-site nucleotide, in contrast to how tRNAs decode mRNA. Finally, our results demonstrate that the most important determinant for mRNA cleavage by ribosome-dependent toxins is interaction with the third A-site nucleotide.« less

Defining the mRNA recognition signature of a bacterial toxin protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schureck, Marc A.; Dunkle, Jack A.; Maehigashi, Tatsuya

Bacteria contain multiple type II toxins that selectively degrade mRNAs bound to the ribosome to regulate translation and growth and facilitate survival during the stringent response. Ribosome-dependent toxins recognize a variety of three-nucleotide codons within the aminoacyl (A) site, but how these endonucleases achieve substrate specificity remains poorly understood. In this paper, we identify the critical features for how the host inhibition of growth B (HigB) toxin recognizes each of the three A-site nucleotides for cleavage. X-ray crystal structures of HigB bound to two different codons on the ribosome illustrate how HigB uses a microbial RNase-like nucleotide recognition loop tomore » recognize either cytosine or adenosine at the second A-site position. Strikingly, a single HigB residue and 16S rRNA residue C1054 form an adenosine-specific pocket at the third A-site nucleotide, in contrast to how tRNAs decode mRNA. Finally, our results demonstrate that the most important determinant for mRNA cleavage by ribosome-dependent toxins is interaction with the third A-site nucleotide.« less

On origin of genetic code and tRNA before translation

PubMed Central

2011-01-01

Background Synthesis of proteins is based on the genetic code - a nearly universal assignment of codons to amino acids (aas). A major challenge to the understanding of the origins of this assignment is the archetypal "key-lock vs. frozen accident" dilemma. Here we re-examine this dilemma in light of 1) the fundamental veto on "foresight evolution", 2) modular structures of tRNAs and aminoacyl-tRNA synthetases, and 3) the updated library of aa-binding sites in RNA aptamers successfully selected in vitro for eight amino acids. Results The aa-binding sites of arginine, isoleucine and tyrosine contain both their cognate triplets, anticodons and codons. We have noticed that these cases might be associated with palindrome-dinucleotides. For example, one-base shift to the left brings arginine codons CGN, with CG at 1-2 positions, to the respective anticodons NCG, with CG at 2-3 positions. Formally, the concomitant presence of codons and anticodons is also expected in the reverse situation, with codons containing palindrome-dinucleotides at their 2-3 positions, and anticodons exhibiting them at 1-2 positions. A closer analysis reveals that, surprisingly, RNA binding sites for Arg, Ile and Tyr "prefer" (exactly as in the actual genetic code) the anticodon(2-3)/codon(1-2) tetramers to their anticodon(1-2)/codon(2-3) counterparts, despite the seemingly perfect symmetry of the latter. However, since in vitro selection of aa-specific RNA aptamers apparently had nothing to do with translation, this striking preference provides a new strong support to the notion of the genetic code emerging before translation, in response to catalytic (and possibly other) needs of ancient RNA life. Consistently with the pre-translation origin of the code, we propose here a new model of tRNA origin by the gradual, Fibonacci process-like, elongation of a tRNA molecule from a primordial coding triplet and 5'DCCA3' quadruplet (D is a base-determinator) to the eventual 76 base-long cloverleaf-shaped molecule. Conclusion Taken together, our findings necessarily imply that primordial tRNAs, tRNA aminoacylating ribozymes, and (later) the translation machinery in general have been co-evolving to ''fit'' the (likely already defined) genetic code, rather than the opposite way around. Coding triplets in this primal pre-translational code were likely similar to the anticodons, with second and third nucleotides being more important than the less specific first one. Later, when the code was expanding in co-evolution with the translation apparatus, the importance of 2-3 nucleotides of coding triplets "transferred" to the 1-2 nucleotides of their complements, thus distinguishing anticodons from codons. This evolutionary primacy of anticodons in genetic coding makes the hypothesis of primal stereo-chemical affinity between amino acids and cognate triplets, the hypothesis of coding coenzyme handles for amino acids, the hypothesis of tRNA-like genomic 3' tags suggesting that tRNAs originated in replication, and the hypothesis of ancient ribozymes-mediated operational code of tRNA aminoacylation not mutually contradicting but rather co-existing in harmony. Reviewers This article was reviewed by Eugene V. Koonin, Wentao Ma (nominated by Juergen Brosius) and Anthony Poole. PMID:21342520

On the Evolution of the Standard Genetic Code: Vestiges of Critical Scale Invariance from the RNA World in Current Prokaryote Genomes

PubMed Central

José, Marco V.; Govezensky, Tzipe; García, José A.; Bobadilla, Juan R.

2009-01-01

Herein two genetic codes from which the primeval RNA code could have originated the standard genetic code (SGC) are derived. One of them, called extended RNA code type I, consists of all codons of the type RNY (purine-any base-pyrimidine) plus codons obtained by considering the RNA code but in the second (NYR type) and third (YRN type) reading frames. The extended RNA code type II, comprises all codons of the type RNY plus codons that arise from transversions of the RNA code in the first (YNY type) and third (RNR) nucleotide bases. In order to test if putative nucleotide sequences in the RNA World and in both extended RNA codes, share the same scaling and statistical properties to those encountered in current prokaryotes, we used the genomes of four Eubacteria and three Archaeas. For each prokaryote, we obtained their respective genomes obeying the RNA code or the extended RNA codes types I and II. In each case, we estimated the scaling properties of triplet sequences via a renormalization group approach, and we calculated the frequency distributions of distances for each codon. Remarkably, the scaling properties of the distance series of some codons from the RNA code and most codons from both extended RNA codes turned out to be identical or very close to the scaling properties of codons of the SGC. To test for the robustness of these results, we show, via computer simulation experiments, that random mutations of current genomes, at the rates of 10−10 per site per year during three billions of years, were not enough for destroying the observed patterns. Therefore, we conclude that most current prokaryotes may still contain relics of the primeval RNA World and that both extended RNA codes may well represent two plausible evolutionary paths between the RNA code and the current SGC. PMID:19183813

Global analysis of translation termination in E. coli

PubMed Central

Baggett, Natalie E.

2017-01-01

Terminating protein translation accurately and efficiently is critical for both protein fidelity and ribosome recycling for continued translation. The three bacterial release factors (RFs) play key roles: RF1 and 2 recognize stop codons and terminate translation; and RF3 promotes disassociation of bound release factors. Probing release factors mutations with reporter constructs containing programmed frameshifting sequences or premature stop codons had revealed a propensity for readthrough or frameshifting at these specific sites, but their effects on translation genome-wide have not been examined. We performed ribosome profiling on a set of isogenic strains with well-characterized release factor mutations to determine how they alter translation globally. Consistent with their known defects, strains with increasingly severe release factor defects exhibit increasingly severe accumulation of ribosomes over stop codons, indicative of an increased duration of the termination/release phase of translation. Release factor mutant strains also exhibit increased occupancy in the region following the stop codon at a significant number of genes. Our global analysis revealed that, as expected, translation termination is generally efficient and accurate, but that at a significant number of genes (≥ 50) the ribosome signature after the stop codon is suggestive of translation past the stop codon. Even native E. coli K-12 exhibits the ribosome signature suggestive of protein extension, especially at UGA codons, which rely exclusively on the reduced function RF2 variant of the K-12 strain for termination. Deletion of RF3 increases the severity of the defect. We unambiguously demonstrate readthrough and frameshifting protein extensions and their further accumulation in mutant strains for a few select cases. In addition to enhancing recoding, ribosome accumulation over stop codons disrupts attenuation control of biosynthetic operons, and may alter expression of some overlapping genes. Together, these functional alterations may either augment the protein repertoire or produce deleterious proteins. PMID:28301469

Codon 13 KRAS mutation predicts patterns of recurrence in patients undergoing hepatectomy for colorectal liver metastases.

PubMed

Margonis, Georgios A; Kim, Yuhree; Sasaki, Kazunari; Samaha, Mario; Amini, Neda; Pawlik, Timothy M

2016-09-01

Investigations regarding the impact of tumor biology after surgical management of colorectal liver metastasis have focused largely on overall survival. We investigated the impact of codon-specific KRAS mutations on the rates and patterns of recurrence in patients after surgery for colorectal liver metastasis (CRLM). All patients who underwent curative-intent surgery for CRLM between 2002 and 2015 at Johns Hopkins who had available data on KRAS mutation status were identified. Clinico-pathologic data, recurrence patterns, and recurrence-free survival (RFS) were assessed using univariable and multivariable analyses. A total of 512 patients underwent resection only (83.2%) or resection plus radiofrequency ablation (16.8%). Although 5-year overall survival was 64.6%, 284 (55.5%) patients recurred with a median RFS time of 18.1 months. The liver was the initial recurrence site for 181 patients, whereas extrahepatic recurrence was observed in 162 patients. Among patients with an extrahepatic recurrence, 102 (63%) had a lung recurrence. Although overall KRAS mutation was not associated with overall RFS (P = 0.186), it was independently associated with a worse extrahepatic (P = 0.004) and lung RFS (P = 0.007). Among patients with known KRAS codon-specific mutations, patients with codon 13 KRAS mutation had a worse 5-year extrahepatic RFS (P = 0.01), whereas codon 12 mutations were not associated with extrahepatic (P = 0.11) or lung-specific recurrence rate (P = 0.24). On multivariable analysis, only codon 13 mutation independently predicted worse overall extrahepatic RFS (P = 0.004) and lung-specific RFS (P = 0.023). Among patients undergoing resection of CRLM, overall KRAS mutation was not associated with RFS. KRAS codon 13 mutations, but not codon 12 mutations, were associated with a higher risk for overall extrahepatic recurrence and lung-specific recurrence. Cancer 2016. © 2016 American Cancer Society. Cancer 2016;122:2698-2707. © 2016 American Cancer Society. © 2016 American Cancer Society.

Conserved small mRNA with an unique, extended Shine-Dalgarno sequence

PubMed Central

Hahn, Julia; Migur, Anzhela; von Boeselager, Raphael Freiherr; Kubatova, Nina; Kubareva, Elena; Schwalbe, Harald

2017-01-01

ABSTRACT Up to now, very small protein-coding genes have remained unrecognized in sequenced genomes. We identified an mRNA of 165 nucleotides (nt), which is conserved in Bradyrhizobiaceae and encodes a polypeptide with 14 amino acid residues (aa). The small mRNA harboring a unique Shine-Dalgarno sequence (SD) with a length of 17 nt was localized predominantly in the ribosome-containing P100 fraction of Bradyrhizobium japonicum USDA 110. Strong interaction between the mRNA and 30S ribosomal subunits was demonstrated by their co-sedimentation in sucrose density gradient. Using translational fusions with egfp, we detected weak translation and found that it is impeded by both the extended SD and the GTG start codon (instead of ATG). Biophysical characterization (CD- and NMR-spectroscopy) showed that synthesized polypeptide remained unstructured in physiological puffer. Replacement of the start codon by a stop codon increased the stability of the transcript, strongly suggesting additional posttranscriptional regulation at the ribosome. Therefore, the small gene was named rreB (ribosome-regulated expression in Bradyrhizobiaceae). Assuming that the unique ribosome binding site (RBS) is a hallmark of rreB homologs or similarly regulated genes, we looked for similar putative RBS in bacterial genomes and detected regions with at least 16 nt complementarity to the 3′-end of 16S rRNA upstream of sORFs in Caulobacterales, Rhizobiales, Rhodobacterales and Rhodospirillales. In the Rhodobacter/Roseobacter lineage of α-proteobacteria the corresponding gene (rreR) is conserved and encodes an 18 aa protein. This shows how specific RBS features can be used to identify new genes with presumably similar control of expression at the RNA level. PMID:27834614

AUU-to-AUG mutation in the initiator codon of the translation initiation factor IF3 abolishes translational autocontrol of its own gene (infC) in vivo.

PubMed Central

Butler, J S; Springer, M; Grunberg-Manago, M

1987-01-01

We previously showed that Escherichia coli translation initiation factor IF3 regulates the expression of its own gene infC at the translational level in vivo. Here we create two alterations in the infC gene and test their effects on translational autocontrol of infC expression in vivo by measuring beta-galactosidase activity expressed from infC-lacZ gene fusions under conditions of up to 4-fold derepression or 3-fold repression of infC expression. Replacement of the infC promoter with the trp promoter deletes 120 nucleotides of the infC mRNA 5' to the translation initiation site without affecting autogenous translational control. Mutation of the unusual AUU initiator codon of infC to the more common AUG initiator codon abolishes translation initiation factor IF3-dependent repression and derepression of infC expression in vivo. These results establish the AUU initiator codon of infC as an essential cis-acting element in autogenous translational control of translation initiation factor IF3 expression in vivo. PMID:2954162

AUU-to-AUG mutation in the initiator codon of the translation initiation factor IF3 abolishes translational autocontrol of its own gene (infC) in vivo.

PubMed

Butler, J S; Springer, M; Grunberg-Manago, M

1987-06-01

We previously showed that Escherichia coli translation initiation factor IF3 regulates the expression of its own gene infC at the translational level in vivo. Here we create two alterations in the infC gene and test their effects on translational autocontrol of infC expression in vivo by measuring beta-galactosidase activity expressed from infC-lacZ gene fusions under conditions of up to 4-fold derepression or 3-fold repression of infC expression. Replacement of the infC promoter with the trp promoter deletes 120 nucleotides of the infC mRNA 5' to the translation initiation site without affecting autogenous translational control. Mutation of the unusual AUU initiator codon of infC to the more common AUG initiator codon abolishes translation initiation factor IF3-dependent repression and derepression of infC expression in vivo. These results establish the AUU initiator codon of infC as an essential cis-acting element in autogenous translational control of translation initiation factor IF3 expression in vivo.

Novel coding, translation, and gene expression of a replicating covalently closed circular RNA of 220 nt.

PubMed

AbouHaidar, Mounir Georges; Venkataraman, Srividhya; Golshani, Ashkan; Liu, Bolin; Ahmad, Tauqeer

2014-10-07

The highly structured (64% GC) covalently closed circular (CCC) RNA (220 nt) of the virusoid associated with rice yellow mottle virus codes for a 16-kDa highly basic protein using novel modalities for coding, translation, and gene expression. This CCC RNA is the smallest among all known viroids and virusoids and the only one that codes proteins. Its sequence possesses an internal ribosome entry site and is directly translated through two (or three) completely overlapping ORFs (shifting to a new reading frame at the end of each round). The initiation and termination codons overlap UGAUGA (underline highlights the initiation codon AUG within the combined initiation-termination sequence). Termination codons can be ignored to obtain larger read-through proteins. This circular RNA with no noncoding sequences is a unique natural supercompact "nanogenome."

Upstream open reading frames regulate the expression of the nuclear Wnt13 isoforms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tang Tao; Rector, Kyle; Barnett, Corey D.

2008-02-22

Wnt proteins control cell survival and cell fate during development. Although Wnt expression is tightly regulated in a spatio-temporal manner, the mechanisms involved both at the transcriptional and translational levels are poorly defined. We have identified a downstream translation initiation codon, AUG(+74), in Wnt13B and Wnt13C mRNAs responsible for the expression of Wnt13 nuclear forms. In this report, we demonstrate that the expression of the nuclear Wnt13C form is translationally regulated in response to stress and apoptosis. Though the 5'-leaders of both Wnt13C and Wnt13B mRNAs have an inhibitory effect on translation, they did not display an internal ribosome entrymore » site activity as demonstrated by dicistronic reporter assays. However, mutations or deletions of the upstream AUG(-99) and AUG(+1) initiation codons abrogate these translation inhibitory effects, demonstrating that Wnt13C expression is controlled by upstream open reading frames. Since long 5'-untranslated region with short upstream open reading frames characterize other Wnt transcripts, our present data on the translational control of Wnt13 expression open the way to further studies on the translation control of Wnt expression as a modulator of their subcellular localization and activity.« less

A novel Rieske-type protein derived from an apoptosis-inducing factor-like (AIFL) transcript with a retained intron 4 induces change in mitochondrial morphology and growth arrest

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murata, Yasuhiko, E-mail: 97318@ib.k.u-tokyo.ac.jp; Furuyama, Isao; Oda, Shoji

2011-04-01

Highlights: {yields} A novel major transcript, AIFL-I4, is found. {yields} Nuclear localization of AIFL-I4 induces mitochondrial morphology change and suppression of cell proliferation. {yields} AIFL-I4 mutant with a lesion in [2Fe-2S] cluster binding site does not induce these phenotypes. {yields} [2Fe-2S] cluster binding site is essential for these phenotypes. -- Abstract: Apoptosis-inducing factor-like (AIFL) protein contains a Rieske domain and pyridine nucleotide-disulfide oxidoreductase (Pyr{sub r}edox) domain that shows 35% homology to that of apoptosis-inducing factor (AIF) protein. We identified a novel major transcript of the medaka (Oryzias latipes) AIFL gene that retained intron 4 (AIFL-I4) in embryos and tissues frommore » adult fish. The product of this transcript, AIFL-I4 protein, lacked the Pyr{sub r}edox domain because of a nonsense codon in intron 4. Both AIFL-I4 and full-length AIFL (fAIFL) transcripts were highly expressed in the brain and late embryos, and relative fAIFL and AIFL-I4 expression levels differed among tissues. Transient expression of AIFL-I4 and fAIFL tagged with GFP showed that AIFL-I4 localized in the nucleus, while fAIFL localized throughout the cytoplasm. We also found that overexpression of AIFL-I4 induced a change in mitochondrial morphology and suppression of cell proliferation. AIFL-I4 mutant with a lesion in [2Fe-2S] cluster binding site of the Rieske domain did not induce these phenotypes. This report is the first to demonstrate nuclear localization of a Rieske-type protein translated from the AIFL gene. Our data suggested that the [2Fe-2S] cluster binding site was essential for the nuclear localization and involved in mitochondrial morphology and suppression of cell proliferation.« less

Amino acid and nucleotide recurrence in aligned sequences: synonymous substitution patterns in association with global and local base compositions.

PubMed

Nishizawa, M; Nishizawa, K

2000-10-01

The tendency for repetitiveness of nucleotides in DNA sequences has been reported for a variety of organisms. We show that the tendency for repetitive use of amino acids is widespread and is observed even for segments conserved between human and Drosophila melanogaster at the level of >50% amino acid identity. This indicates that repetitiveness influences not only the weakly constrained segments but also those sequence segments conserved among phyla. Not only glutamine (Q) but also many of the 20 amino acids show a comparable level of repetitiveness. Repetitiveness in bases at codon position 3 is stronger for human than for D.melanogaster, whereas local repetitiveness in intron sequences is similar between the two organisms. While genes for immune system-specific proteins, but not ancient human genes (i.e. human homologs of Escherichia coli genes), have repetitiveness at codon bases 1 and 2, repetitiveness at codon base 3 for these groups is similar, suggesting that the human genome has at least two mechanisms generating local repetitiveness. Neither amino acid nor nucleotide repetitiveness is observed beyond the exon boundary, denying the possibility that such repetitiveness could mainly stem from natural selection on mRNA or protein sequences. Analyses of mammalian sequence alignments show that while the 'between gene' GC content heterogeneity, which is linked to 'isochores', is a principal factor associated with the bias in substitution patterns in human, 'within gene' heterogeneity in nucleotide composition is also associated with such bias on a more local scale. The relationship amongst the various types of repetitiveness is discussed.

Amino acid and nucleotide recurrence in aligned sequences: synonymous substitution patterns in association with global and local base compositions

PubMed Central

Nishizawa, Manami; Nishizawa, Kazuhisa

2000-01-01

The tendency for repetitiveness of nucleotides in DNA sequences has been reported for a variety of organisms. We show that the tendency for repetitive use of amino acids is widespread and is observed even for segments conserved between human and Drosophila melanogaster at the level of >50% amino acid identity. This indicates that repetitiveness influences not only the weakly constrained segments but also those sequence segments conserved among phyla. Not only glutamine (Q) but also many of the 20 amino acids show a comparable level of repetitiveness. Repetitiveness in bases at codon position 3 is stronger for human than for D.melanogaster, whereas local repetitiveness in intron sequences is similar between the two organisms. While genes for immune system-specific proteins, but not ancient human genes (i.e. human homologs of Escherichia coli genes), have repetitiveness at codon bases 1 and 2, repetitiveness at codon base 3 for these groups is similar, suggesting that the human genome has at least two mechanisms generating local repetitiveness. Neither amino acid nor nucleotide repetitiveness is observed beyond the exon boundary, denying the possibility that such repetitiveness could mainly stem from natural selection on mRNA or protein sequences. Analyses of mammalian sequence alignments show that while the ‘between gene’ GC content heterogeneity, which is linked to ‘isochores’, is a principal factor associated with the bias in substitution patterns in human, ‘within gene’ heterogeneity in nucleotide composition is also associated with such bias on a more local scale. The relationship amongst the various types of repetitiveness is discussed. PMID:11000273

Selenium. Role of the Essential Metalloid in Health

PubMed Central

Kurokawa, Suguru; Berry, Marla J.

2015-01-01

Selenium is an essential micronutrient in mammals, but is also recognized as toxic in excess. It is a non-metal with properties that are intermediate between the chalcogen elements sulfur and tellurium. Selenium exerts its biological functions through selenoproteins. Selenoproteins contain selenium in the form of the 21st amino acid, selenocysteine (Sec), which is an analog of cysteine with the sulfur-containing side chain replaced by a Se-containing side chain. Sec is encoded by the codon UGA, which is one of three termination codons for mRNA translation in non-selenoprotein genes. Recognition of the UGA codon as a Sec insertion site instead of stop requires a Sec insertion sequence (SECIS) element in selenoprotein mRNAs and a unique selenocysteyl-tRNA, both of which are recognized by specialized protein factors. Unlike the 20 standard amino acids, Sec is biosynthesized from serine on its tRNA. Twenty-five selenoproteins are encoded in the human genome. Most of the selenoprotein genes were discovered by bioinformatics approaches, searching for SECIS elements downstream of in-frame UGA codons. Sec has been described as having stronger nucleophilic and electrophilic properties than cysteine, and Sec is present in the catalytic site of all selenoenzymes. Most selenoproteins, whose functions are known, are involved in redox systems and signaling pathways. However, several selenoproteins are not well characterized in terms of their function. The selenium field has grown dramatically in the last few decades, and research on selenium biology is providing extensive new information regarding its importance for human health. PMID:24470102

Protein encoding genes in an ancient plant: analysis of codon usage, retained genes and splice sites in a moss, Physcomitrella patens

PubMed Central

Rensing, Stefan A; Fritzowsky, Dana; Lang, Daniel; Reski, Ralf

2005-01-01

Background The moss Physcomitrella patens is an emerging plant model system due to its high rate of homologous recombination, haploidy, simple body plan, physiological properties as well as phylogenetic position. Available EST data was clustered and assembled, and provided the basis for a genome-wide analysis of protein encoding genes. Results We have clustered and assembled Physcomitrella patens EST and CDS data in order to represent the transcriptome of this non-seed plant. Clustering of the publicly available data and subsequent prediction resulted in a total of 19,081 non-redundant ORF. Of these putative transcripts, approximately 30% have a homolog in both rice and Arabidopsis transcriptome. More than 130 transcripts are not present in seed plants but can be found in other kingdoms. These potential "retained genes" might have been lost during seed plant evolution. Functional annotation of these genes reveals unequal distribution among taxonomic groups and intriguing putative functions such as cytotoxicity and nucleic acid repair. Whereas introns in the moss are larger on average than in the seed plant Arabidopsis thaliana, position and amount of introns are approximately the same. Contrary to Arabidopsis, where CDS contain on average 44% G/C, in Physcomitrella the average G/C content is 50%. Interestingly, moss orthologs of Arabidopsis genes show a significant drift of codon fraction usage, towards the seed plant. While averaged codon bias is the same in Physcomitrella and Arabidopsis, the distribution pattern is different, with 15% of moss genes being unbiased. Species-specific, sensitive and selective splice site prediction for Physcomitrella has been developed using a dataset of 368 donor and acceptor sites, utilizing a support vector machine. The prediction accuracy is better than those achieved with tools trained on Arabidopsis data. Conclusion Analysis of the moss transcriptome displays differences in gene structure, codon and splice site usage in comparison with the seed plant Arabidopsis. Putative retained genes exhibit possible functions that might explain the peculiar physiological properties of mosses. Both the transcriptome representation (including a BLAST and retrieval service) and splice site prediction have been made available on , setting the basis for assembly and annotation of the Physcomitrella genome, of which draft shotgun sequences will become available in 2005. PMID:15784153

Ribosome A and P sites revealed by length analysis of ribosome profiling data

PubMed Central

Martens, Andrew T.; Taylor, James; Hilser, Vincent J.

2015-01-01

The high-throughput sequencing of nuclease-protected mRNA fragments bound to ribosomes, a technique known as ribosome profiling, quantifies the relative frequencies with which different regions of transcripts are translated. This technique has revealed novel translation initiation sites with unprecedented scope and has furthered investigations into the connections between codon biases and translation rates. Yet the location of the codon being decoded in ribosome footprints is still unknown, and has been complicated by the recent observation of footprints with non-canonical lengths. Here we show how taking into account the variations in ribosome footprint lengths can reveal the ribosome aminoacyl (A) and peptidyl (P) site locations. These location assignments are in agreement with the proposed mechanisms for various ribosome pauses and further enhance the resolution of the profiling data. We also show that GC-rich motifs at the 5′ ends of footprints are found in yeast, calling into question the anti-Shine-Dalgarno effect's role in ribosome pausing. PMID:25805170

Novel coding, translation, and gene expression of a replicating covalently closed circular RNA of 220 nt

PubMed Central

AbouHaidar, Mounir Georges; Venkataraman, Srividhya; Golshani, Ashkan; Liu, Bolin; Ahmad, Tauqeer

2014-01-01

The highly structured (64% GC) covalently closed circular (CCC) RNA (220 nt) of the virusoid associated with rice yellow mottle virus codes for a 16-kDa highly basic protein using novel modalities for coding, translation, and gene expression. This CCC RNA is the smallest among all known viroids and virusoids and the only one that codes proteins. Its sequence possesses an internal ribosome entry site and is directly translated through two (or three) completely overlapping ORFs (shifting to a new reading frame at the end of each round). The initiation and termination codons overlap UGAUGA (underline highlights the initiation codon AUG within the combined initiation-termination sequence). Termination codons can be ignored to obtain larger read-through proteins. This circular RNA with no noncoding sequences is a unique natural supercompact “nanogenome.” PMID:25253891

Ribosomal protein S14 transcripts are edited in Oenothera mitochondria.

PubMed Central

Schuster, W; Unseld, M; Wissinger, B; Brennicke, A

1990-01-01

The gene encoding ribosomal protein S14 (rps14) in Oenothera mitochondria is located upstream of the cytochrome b gene (cob). Sequence analysis of independently derived cDNA clones covering the entire rps14 coding region shows two nucleotides edited from the genomic DNA to the mRNA derived sequences by C to U modifications. A third editing event occurs four nucleotides upstream of the AUG initiation codon and improves a potential ribosome binding site. A CGG codon specifying arginine in a position conserved in evolution between chloroplasts and E. coli as a UGG tryptophan codon is not edited in any of the cDNAs analysed. An inverted repeat 3' of an unidentified open reading frame is located upstream of the rps14 gene. The inverted repeat sequence is highly conserved at analogous regions in other Oenothera mitochondrial loci. Images PMID:2326162

Characterization of the human UDP-galactose:ceramide galactosyltransferase gene promoter.

PubMed

Tencomnao, T; Yu, R K; Kapitonov, D

2001-02-16

UDP-galactose:ceramide galactosyltransferase (CGT, EC 2.4.1.45) is a key enzyme in the biosynthesis of galactocerebroside, the most abundant glycosphingolipid in the myelin sheath. An 8 kb fragment upstream from the transcription initiation site of CGT gene was isolated from a human genomic DNA library. Primer extension analysis revealed a single transcription initiation site 329 bp upstream from the ATG start codon. Neither a consensus TATA nor a CCAAT box was identified in the proximity to the transcription start site; however, this region contains a high GC content and multiple putative regulatory elements. To investigate the transcriptional regulation of CGT, a series of 5' deletion constructs of the 5'-flanking region were generated and cloned upstream from the luciferase reporter gene. By comparing promoter activity in the human oligodendroglioma (HOG) and human neuroblastoma (LAN-5) cell lines, we found that the CGT promoter functions in a cell type-specific manner. Three positive cis-acting regulatory regions were identified, including a proximal region at -292/-256 which contains the potential binding sites for known transcription factors (TFs) such as Ets and SP1 (GC box), a distal region at -747/-688 comprising a number of binding sites such as the ERE half-site, NF1-like, TGGCA-BP, and CRE, and a third positive cis-acting region distally localized at -1325/-1083 consisting of binding sites for TFs such as nitrogen regulatory, TCF-1, TGGCA-BP, NF-IL6, CF1, bHLH, NF1-like, GATA, and gamma-IRE. A negative cis-acting domain localized in a far distal region at -1594/-1326 was also identified. Our results suggest the presence of both positive and negative cis-regulatory regions essential for the cell-specific expression in the TATA-less promoter of the human CGT gene.

Consequences of germline variation disrupting the constitutional translational initiation codon start sites of MLH1 and BRCA2: use of potential alternative start sites and implications for predicting variant pathogenicity

PubMed Central

Parsons, Michael T.; Whiley, Phillip J.; Beesley, Jonathan; Drost, Mark; de Wind, Niels; Thompson, Bryony A.; Marquart, Louise; Hopper, John L.; Jenkins, Mark A.; Brown, Melissa A.; Tucker, Kathy; Warwick, Linda; Buchanan, Daniel D.; Spurdle, Amanda B.

2014-01-01

Variants that disrupt the translation initiation sequences in cancer predisposition genes are generally assumed to be deleterious. However few studies have validated these assumptions with functional and clinical data. Two cancer syndrome gene variants likely to affect native translation initiation were identified by clinical genetic testing: MLH1:c.1A>G p.(Met1?) and BRCA2:c.67+3A>G. In vitro GFP-reporter assays were conducted to assess the consequences of translation initiation disruption on alternative downstream initiation codon usage. Analysis of MLH1:c.1A>G p.(Met1?) showed that translation was mostly initiated at an in-frame position 103 nucleotides downstream, but also at two ATG sequences downstream. The protein product encoded by the in-frame transcript initiating from position c.103 showed loss of in vitro mismatch repair activity comparable to known pathogenic mutations. BRCA2:c.67+3A>G was shown by mRNA analysis to result in an aberrantly spliced transcript deleting exon 2 and the consensus ATG site. In the absence of exon 2, translation initiated mostly at an out-of-frame ATG 323 nucleotides downstream, and to a lesser extent at an in-frame ATG 370 nucleotides downstream. Initiation from any of the downstream alternative sites tested in both genes would lead to loss of protein function, but further clinical data is required to confirm if these variants are associated with a high cancer risk. Importantly, our results highlight the need for caution in interpreting the functional and clinical consequences of variation that leads to disruption of the initiation codon, since translation may not necessarily occur from the first downstream alternative start site, or from a single alternative start site. PMID:24302565

Positive selection in the SLC11A1 gene in the family Equidae.

PubMed

Bayerova, Zuzana; Janova, Eva; Matiasovic, Jan; Orlando, Ludovic; Horin, Petr

2016-05-01

Immunity-related genes are a suitable model for studying effects of selection at the genomic level. Some of them are highly conserved due to functional constraints and purifying selection, while others are variable and change quickly to cope with the variation of pathogens. The SLC11A1 gene encodes a transporter protein mediating antimicrobial activity of macrophages. Little is known about the patterns of selection shaping this gene during evolution. Although it is a typical evolutionarily conserved gene, functionally important polymorphisms associated with various diseases were identified in humans and other species. We analyzed the genomic organization, genetic variation, and evolution of the SLC11A1 gene in the family Equidae to identify patterns of selection within this important gene. Nucleotide SLC11A1 sequences were shown to be highly conserved in ten equid species, with more than 97 % sequence identity across the family. Single nucleotide polymorphisms (SNPs) were found in the coding and noncoding regions of the gene. Seven codon sites were identified to be under strong purifying selection. Codons located in three regions, including the glycosylated extracellular loop, were shown to be under diversifying selection. A 3-bp indel resulting in a deletion of the amino acid 321 in the predicted protein was observed in all horses, while it has been maintained in all other equid species. This codon comprised in an N-glycosylation site was found to be under positive selection. Interspecific variation in the presence of predicted N-glycosylation sites was observed.

The Influence of HIV on the Evolution of Mycobacterium tuberculosis

PubMed Central

Brites, Daniela; Stucki, David; Evans, Joanna C.; Seldon, Ronnett; Heekes, Alexa; Mulder, Nicola; Nicol, Mark; Oni, Tolu; Mizrahi, Valerie; Warner, Digby F.; Parkhill, Julian; Gagneux, Sebastien; Martin, Darren P.; Wilkinson, Robert J.

2017-01-01

Abstract HIV significantly affects the immunological environment during tuberculosis coinfection, and therefore may influence the selective landscape upon which M. tuberculosis evolves. To test this hypothesis whole genome sequences were determined for 169 South African M. tuberculosis strains from HIV-1 coinfected and uninfected individuals and analyzed using two Bayesian codon-model based selection analysis approaches: FUBAR which was used to detect persistent positive and negative selection (selection respectively favoring and disfavoring nonsynonymous substitutions); and MEDS which was used to detect episodic directional selection specifically favoring nonsynonymous substitutions within HIV-1 infected individuals. Among the 25,251 polymorphic codon sites analyzed, FUBAR revealed that 189-fold more were detectably evolving under persistent negative selection than were evolving under persistent positive selection. Three specific codon sites within the genes celA2b, katG, and cyp138 were identified by MEDS as displaying significant evidence of evolving under directional selection influenced by HIV-1 coinfection. All three genes encode proteins that may indirectly interact with human proteins that, in turn, interact functionally with HIV proteins. Unexpectedly, epitope encoding regions were enriched for sites displaying weak evidence of directional selection influenced by HIV-1. Although the low degree of genetic diversity observed in our M. tuberculosis data set means that these results should be interpreted carefully, the effects of HIV-1 on epitope evolution in M. tuberculosis may have implications for the design of M. tuberculosis vaccines that are intended for use in populations with high HIV-1 infection rates. PMID:28369607

Photo-dependent protein biosynthesis using a caged aminoacyl-tRNA.

PubMed

Akahoshi, Akiya; Doi, Yoshio; Sisido, Masahiko; Watanabe, Kazunori; Ohtsuki, Takashi

2014-12-01

Translation systems with four-base codons provide a powerful strategy for protein engineering and protein studies because they enable site-specific incorporation of non-natural amino acids into proteins. In this study, a caged aminoacyl-tRNA with a four-base anticodon was synthesized. The caged aminoacyl-tRNA contains a photocleavable nitroveratryloxycarbonyl (NVOC) group. This study showed that the caged aminoacyl-tRNA was not deacylated, did not bind to EF-Tu, and was activated by light. Photo-dependent translation of an mRNA containing the four-base codon was demonstrated using the caged aminoacyl-tRNA.

Self-organizing approach for meta-genomes.

PubMed

Zhu, Jianfeng; Zheng, Wei-Mou

2014-12-01

We extend the self-organizing approach for annotation of a bacterial genome to analyze the raw sequencing data of the human gut metagenome without sequence assembling. The original approach divides the genomic sequence of a bacterium into non-overlapping segments of equal length and assigns to each segment one of seven 'phases', among which one is for the noncoding regions, three for the direct coding regions to indicate the three possible codon positions of the segment starting site, and three for the reverse coding regions. The noncoding phase and the six coding phases are described by two frequency tables of the 64 triplet types or 'codon usages'. A set of codon usages can be used to update the phase assignment and vice versa. An iteration after an initialization leads to a convergent phase assignment to give an annotation of the genome. In the extension of the approach to a metagenome, we consider a mixture model of a number of categories described by different codon usages. The Illumina Genome Analyzer sequencing data of the total DNA from faecal samples are then examined to understand the diversity of the human gut microbiome. Copyright © 2014 Elsevier Ltd. All rights reserved.

DNA abasic site-directed formation of fluorescent silver nanoclusters for selective nucleobase recognition

NASA Astrophysics Data System (ADS)

Ma, Kun; Cui, Qinghua; Liu, Guiying; Wu, Fei; Xu, Shujuan; Shao, Yong

2011-07-01

DNA single-nucleotide polymorphism (SNP) detection has attracted much attention due to mutation related diseases. Various methods for SNP detection have been proposed and many are already in use. Here, we find that the abasic site (AP site) in the DNA duplex can be developed as a capping scaffold for the generation of fluorescent silver nanoclusters (Ag NCs). As a proof of concept, the DNA sequences from fragments near codon 177 of cancer supression gene p53 were used as a model for SNP detection by in situ formed Ag NCs. The formation of fluorescent Ag NCs in the AP site-containing DNA duplex is highly selective for cytosine facing the AP site and guanines flanking the site and can be employed in situ as readout for SNP detection. The fluorescent signal-on sensing for SNP based on this inorganic fluorophore is substantially advantageous over the previously reported signal-off responses using low-molecular-weight organic ligands. The strong dependence of fluorescent Ag NC formation on the sequences surrounding the AP site was successfully used to identify mutations in codon 177 of cancer supression gene p53. We anticipate that this approach will be employed to develop a practical SNP detection method by locating an AP site toward the midway cytosine in a target strand containing more than three consecutive cytosines.

[C-terminal fragment of ribosomal protein S15 is located at the decoding site of the human ribosome].

PubMed

Khaĭrulina, Iu S; Molotkov, M V; Bulygin, K N; Graĭfer, D M; Ven'iaminova, A G; Karpova, G G

2008-01-01

Protein S15 is a characteristic component of the mammalian 80S ribosome that neighbors mRNA codon at the decoding site and the downstream triplets. In this study we determined S15 protein fragments located close to mRNA positions +4 to +12 with respect to the first nucleotide of the P site codon on the human ribosome. For cross-linking to ribosomal protein S15, a set of mRNA was used that contained triplet UUU/UUC at the 5'-termini and a perfluorophenyl azide-modified uridine in position 3' of this triplet. The locations of mRNA analogues on the ribosome were governed by tRNAPhe cognate to the UUU/UUC triplet targeted to the P site. Cross-linked S15 protein was isolated from the irradiated with mild UV light complexes of 80S ribosomes with tRNAPhe and mRNA analogues with subsequent cleavage with CNBr that splits polypeptide chain after methionines. Analysis of modified oligopeptides resulted from the cleavage revealed that in all cases cross-linking site was located in C-terminal fragment 111-145 of protein S15 indicating that this fragment is involved in formation of decoding site of the eukaryotic ribosome.

Codon optimization of antigen coding sequences improves the immune potential of DNA vaccines against avian influenza virus H5N1 in mice and chickens.

PubMed

Stachyra, Anna; Redkiewicz, Patrycja; Kosson, Piotr; Protasiuk, Anna; Góra-Sochacka, Anna; Kudla, Grzegorz; Sirko, Agnieszka

2016-08-26

Highly pathogenic avian influenza viruses are a serious threat to domestic poultry and can be a source of new human pandemic and annual influenza strains. Vaccination is the main strategy of protection against influenza, thus new generation vaccines, including DNA vaccines, are needed. One promising approach for enhancing the immunogenicity of a DNA vaccine is to maximize its expression in the immunized host. The immunogenicity of three variants of a DNA vaccine encoding hemagglutinin (HA) from the avian influenza virus A/swan/Poland/305-135V08/2006 (H5N1) was compared in two animal models, mice (BALB/c) and chickens (broilers and layers). One variant encoded the wild type HA while the other two encoded HA without proteolytic site between HA1 and HA2 subunits and differed in usage of synonymous codons. One of them was enriched for codons preferentially used in chicken genes, while in the other modified variant the third position of codons was occupied in almost 100 % by G or C nucleotides. The variant of the DNA vaccine containing almost 100 % of the GC content in the third position of codons stimulated strongest immune response in two animal models, mice and chickens. These results indicate that such modification can improve not only gene expression but also immunogenicity of DNA vaccine. Enhancement of the GC content in the third position of the codon might be a good strategy for development of a variant of a DNA vaccine against influenza that could be highly effective in distant hosts, such as birds and mammals, including humans.

New Universal Rules of Eukaryotic Translation Initiation Fidelity

PubMed Central

Zur, Hadas; Tuller, Tamir

2013-01-01

The accepted model of eukaryotic translation initiation begins with the scanning of the transcript by the pre-initiation complex from the 5′end until an ATG codon with a specific nucleotide (nt) context surrounding it is recognized (Kozak rule). According to this model, ATG codons upstream to the beginning of the ORF should affect translation. We perform for the first time, a genome-wide statistical analysis, uncovering a new, more comprehensive and quantitative, set of initiation rules for improving the cost of translation and its efficiency. Analyzing dozens of eukaryotic genomes, we find that in all frames there is a universal trend of selection for low numbers of ATG codons; specifically, 16–27 codons upstream, but also 5–11 codons downstream of the START ATG, include less ATG codons than expected. We further suggest that there is selection for anti optimal ATG contexts in the vicinity of the START ATG. Thus, the efficiency and fidelity of translation initiation is encoded in the 5′UTR as required by the scanning model, but also at the beginning of the ORF. The observed nt patterns suggest that in all the analyzed organisms the pre-initiation complex often misses the START ATG of the ORF, and may start translation from an alternative initiation start-site. Thus, to prevent the translation of undesired proteins, there is selection for nucleotide sequences with low affinity to the pre-initiation complex near the beginning of the ORF. With the new suggested rules we were able to obtain a twice higher correlation with ribosomal density and protein levels in comparison to the Kozak rule alone (e.g. for protein levels r = 0.7 vs. r = 0.31; p<10−12). PMID:23874179

Multiple Origins of Mutations in the mdr1 Gene—A Putative Marker of Chloroquine Resistance in P. vivax

PubMed Central

Schousboe, Mette L.; Ranjitkar, Samir; Rajakaruna, Rupika S.; Amerasinghe, Priyanie H.; Morales, Francisco; Pearce, Richard; Ord, Rosalyn; Leslie, Toby; Rowland, Mark; Gadalla, Nahla B.; Konradsen, Flemming; Bygbjerg, Ib C.; Roper, Cally; Alifrangis, Michael

2015-01-01

Background Chloroquine combined with primaquine has been the recommended antimalarial treatment of Plasmodium vivax malaria infections for six decades but the efficacy of this treatment regimen is threatened by chloroquine resistance (CQR). Single nucleotide polymorphisms (SNPs) in the multidrug resistance gene, Pvmdr1 are putative determinants of CQR but the extent of their emergence at population level remains to be explored. Objective In this study we describe the prevalence of SNPs in the Pvmdr1 among samples collected in seven P. vivax endemic countries and we looked for molecular evidence of drug selection by characterising polymorphism at microsatellite (MS) loci flanking the Pvmdr1 gene. Methods We examined the prevalence of SNPs in the Pvmdr1 gene among 267 samples collected from Pakistan, Afghanistan, Sri Lanka, Nepal, Sudan, São Tomé and Ecuador. We measured and diversity in four microsatellite (MS) markers flanking the Pvmdr1 gene to look evidence of selection on mutant alleles. Results SNP polymorphism in the Pvmdr1 gene was largely confined to codons T958M, Y976F and F1076L. Only 2.4% of samples were wildtype at all three codons (TYF, n = 5), 13.3% (n = 28) of the samples were single mutant MYF, 63.0% of samples (n = 133) were double mutant MYL, and 21.3% (n = 45) were triple mutant MFL. Clear geographic differences in the prevalence of these Pvmdr mutation combinations were observed. Significant linkage disequilibrium (LD) between Pvmdr1 and MS alleles was found in populations sampled in Ecuador, Nepal and Sri Lanka, while significant LD between Pvmdr1 and the combined 4 MS locus haplotype was only seen in Ecuador and Sri Lanka. When combining the 5 loci, high level diversity, measured as expected heterozygosity (He), was seen in the complete sample set (He = 0.99), while He estimates for individual loci ranged from 0.00–0.93. Although Pvmdr1 haplotypes were not consistently associated with specific flanking MS alleles, there was significant differentiation between geographic sites which could indicate directional selection through local drug pressure. Conclusions Our observations suggest that Pvmdr1 mutations emerged independently on multiple occasions even within the same population. In Sri Lanka population analysis at multiple sites showed evidence of local selection and geographical dispersal of Pvmdr1 mutations between sites. PMID:26539821

Molecular analysis of markers associated with chloroquine and sulfadoxine/pyrimethamine resistance in Plasmodium falciparum malaria parasites from southeastern Côte-d'Ivoire by the time of Artemisinin-based Combination Therapy adoption in 2005.

PubMed

Ako, Berenger Aristide; Offianan, André Toure; Johansson, Marnie; Penali, Louis Koné; Nguetta, Simon-Pierre Assanvo; Sibley, Carol Hopkin

2012-01-01

Artemisin-based combination therapies became the recommended therapy in Côte-d'Ivoire in 2005, but both chloroquine (CQ) and sulfadoxine/pyrimethamine (SP) have been heavily used for many decades. Despite this long history, little is known about the geographical distribution of drug resistance-conferring genotypes outside the capital city of Abidjan. In this work, we compared the prevalence of drug-resistant genotypes in Bonoua, an urban area, and Samo, a rural agricultural area, in southeastern Côte-d'Ivoire, about 59 km from Abidjan. Samples were collected from symptomatic patients in both sites during the rainy season in 2005. Genomic DNA was isolated and codons associated with resistance to CQ and SP were analyzed: pfcrt codons Cys-72-Ser, Val-73-Val, Met-74-Ile, Arg-75-Glu, Lys-76-Thr; pfdhfr codons Ala-16-Val, Arg-51-Ile, Cys-59-Arg, Ser-108-Arg/Thr, and Ile-164-Leu; pfdhps codons Ser-436-Ala, Ala-437-Gly, Lys-540-Glu, Ala-581-Gly, and Ala-613-Thr/Ser. A limited number of genotypes were found in Bonoua compared with Samo. In both sites, the triple-mutant allele CVIET of pfcrt predominated: 100% in Bonoua and 86.2% in Samo. The wild-type allele, NCSI of pfdhfr, was common - 50% in Bonoua and 38.7% in Samo - but the triple-mutant IRNI and double-mutant NRNI were also frequent (IRNI, 32.6% in Bonoua and 19.4% in Samo; NRNI, 15.2% in Bonoua and 9.7% in Samo). In Samo, a wide range of different genotypes of Pfdhps was observed, with alleles carrying the Gly-437 codon fixed in Bonoua and comprising 73% of the isolates in Samo. Although these two sites are only 8 km apart, they belonged to very different ecological environments. The overall prevalence of alleles of single-nucleotide polymorphisms associated with resistance to CQ and SP in both locations was among the highest of the region by 2005, although the more rural site showed a more diverse set of alleles and mixed infections. Continued surveillance of these markers will be a useful tool for drug policy, as both CQ and SP are still frequently used years after withdrawal, and SP is recommended by the World Health Organization for intermittent preventive therapy for pregnant women and infants. Data analyzed herein are among the first to be generated during the year of artemisin-based combination-therapy introduction in Côte-d'Ivoire and could be of some interest for malaria policy-makers.

Nucleotide substitutions in dengue virus serotypes from Asian and American countries: insights into intracodon recombination and purifying selection

PubMed Central

2013-01-01

Background Dengue virus (DENV) infection represents a significant public health problem in many subtropical and tropical countries. Although genetically closely related, the four serotypes of DENV differ in antigenicity for which cross protection among serotypes is limited. It is also believed that both multi-serotype infection as well as the evolution of viral antigenicity may have confounding effects in increased dengue epidemics. Numerous studies have been performed that investigated genetic diversity of DENV, but the precise mechanism(s) of dengue virus evolution are not well understood. Results We investigated genome-wide genetic diversity and nucleotide substitution patterns in the four serotypes among samples collected from different countries in Asia and Central and South America and sequenced as part of the Genome Sequencing Center for Infectious Diseases at the Broad Institute. We applied bioinformatics, statistical and coalescent simulation methods to investigate diversity of codon sequences of DENV samples representing the four serotypes. We show that fixation of nucleotide substitutions is more prominent among the inter-continental isolates (Asian and American) of serotypes 1, 2 and 3 compared to serotype 4 isolates (South and Central America) and are distributed in a non-random manner among the genes encoded by the virus. Nearly one third of the negatively selected sites are associated with fixed mutation sites within serotypes. Our results further show that of all the sites showing evidence of recombination, the majority (~84%) correspond to sites under purifying selection in the four serotypes. The analysis further shows that genetic recombination occurs within specific codons, albeit with low frequency (< 5% of all recombination sites) throughout the DENV genome of the four serotypes and reveals significant enrichment (p < 0.05) among sites under purifying selection in the virus. Conclusion The study provides the first evidence for intracodon recombination in DENV and suggests that within codons, genetic recombination has a significant role in maintaining extensive purifying selection of DENV in natural populations. Our study also suggests that fixation of beneficial mutations may lead to virus evolution via translational selection of specific sites in the DENV genome. PMID:23410119

Estimating time of HIV-1 infection from next-generation sequence diversity

PubMed Central

2017-01-01

Estimating the time since infection (TI) in newly diagnosed HIV-1 patients is challenging, but important to understand the epidemiology of the infection. Here we explore the utility of virus diversity estimated by next-generation sequencing (NGS) as novel biomarker by using a recent genome-wide longitudinal dataset obtained from 11 untreated HIV-1-infected patients with known dates of infection. The results were validated on a second dataset from 31 patients. Virus diversity increased linearly with time, particularly at 3rd codon positions, with little inter-patient variation. The precision of the TI estimate improved with increasing sequencing depth, showing that diversity in NGS data yields superior estimates to the number of ambiguous sites in Sanger sequences, which is one of the alternative biomarkers. The full advantage of deep NGS was utilized with continuous diversity measures such as average pairwise distance or site entropy, rather than the fraction of polymorphic sites. The precision depended on the genomic region and codon position and was highest when 3rd codon positions in the entire pol gene were used. For these data, TI estimates had a mean absolute error of around 1 year. The error increased only slightly from around 0.6 years at a TI of 6 months to around 1.1 years at 6 years. Our results show that virus diversity determined by NGS can be used to estimate time since HIV-1 infection many years after the infection, in contrast to most alternative biomarkers. We provide the regression coefficients as well as web tool for TI estimation. PMID:28968389

Functional analysis of the promoter of the molt-inhibiting hormone (mih) gene in mud crab Scylla paramamosain.

PubMed

Zhang, Xin; Huang, Danping; Jia, Xiwei; Zou, Zhihua; Wang, Yilei; Zhang, Ziping

2018-04-01

In this study, the 5'-flanking region of molt-inhibiting hormone (MIH) gene was cloned by Tail-PCR. It is 2024 bp starting from the translation initiation site, and 1818 bp starting from the predicted transcription start site. Forecast analysis results by the bioinformatics software showed that the transcription start site is located at 207 bp upstream of the start codon ATG, and TATA box is located at 240 bp upstream of the start codon ATG. Potential transcription factor binding sites include Sp1, NF-1, Oct-1, Sox-2, RAP1, and so on. There are two CpG islands, located at -25- +183 bp and -1451- -1316 bp respectively. The transfection results of luciferase reporter constructs showed that the core promoter region was located in the fragment -308 bp to -26 bp. NF-kappaB and RAP1 were essential for mih basal transcriptional activity. There are three kinds of polymorphism CA in the 5'-flanking sequence, and they can influence mih promoter activity. These findings provide a genetic foundation of the further research of mih transcription regulation. Copyright © 2017 Elsevier Inc. All rights reserved.

Positive Newborn Screen for Methylmalonic Aciduria Identifies the First Mutation in TCblR/CD320, the Gene for Cellular Uptake of Transcobalamin-bound Vitamin B12

PubMed Central

Quadros, Edward V.; Lai, Shao-Chiang; Nakayama, Yasumi; Sequeira, Jeffrey M.; Hannibal, Luciana; Wang, Sihe; Jacobsen, Donald W.; Fedosov, Sergey; Wright, Erica; Gallagher, Renata C.; Anastasio, Natascia; Watkins, David; Rosenblatt, David S.

2010-01-01

Elevated methylmalonic acid in five asymptomatic newborns whose fibroblasts showed decreased uptake of transcobalamin-bound cobalamin (holo-TC), suggested a defect in the cellular uptake of cobalamin. Analysis of TCblR/CD320, the gene for the receptor for cellular uptake of holo-TC, identified a homozygous single codon deletion, c.262_264GAG (p.E88del), resulting in the loss of a glutamic acid residue in the low-density lipoprotein receptor type A-like domain. Inserting the codon by site-directed mutagenesis fully restored TCblR function. PMID:20524213

Defect in the GTPase activating protein (GAP) function of eIF5 causes repression of GCN4 translation.

PubMed

Antony A, Charles; Alone, Pankaj V

2017-05-13

In eukaryotes, the eIF5 protein plays an important role in translation start site selection by providing the GAP (GTPase activating protein) function. However, in yeast translation initiation fidelity defective eIF5 G31R mutant causes preferential utilization of UUG as initiation codon and is termed as Suppressor of initiation codon (Sui - ) phenotype due to its hyper GTPase activity. The eIF5 G31R mutant dominantly represses GCN4 expression and confers sensitivity to 3-Amino-1,2,4-Trizole (3AT) induced starvation. The down-regulation of the GCN4 expression (Gcn - phenotype) in the eIF5 G31R mutant was not because of leaky scanning defects; rather was due to the utilization of upUUG initiation codons at the 5' regulatory region present between uORF1 and the main GCN4 ORF. Copyright © 2017 Elsevier Inc. All rights reserved.

[Organization and expression of poliovirus genome].

PubMed

Vevcherenko, S G

1984-01-01

In the present paper on the basis of analysis of literary data it is postulated that along with the AUG codon at N743 there exists a second initiation codon in the poliovirus RNA (the AUG codon at N586). The translation initiated at N586 can be transferred to the phase of the major reading frame by removing the small hairpin N732-N744 formed near the first initiation site, or by removing the small region N739-N745. In the first case at the boundary between the hypothetical leader peptide encoded by the 5'-terminus of the long, open reading frame of the spliced poliovirus RNA and the capsid protein VP4 must be the Gln-Gly proteolytic cleavage signal, and in the second case--the Tyr-Gly signal. In both cases the leader peptide can be chipped off by the virus specific proteinase. It is supposed that the exon-intronic structure of the poliovirus genome is needed for coordination of translation and transcription during the poliovirus reproduction cycle.

2'-O-methylation in mRNA disrupts tRNA decoding during translation elongation.

PubMed

Choi, Junhong; Indrisiunaite, Gabriele; DeMirci, Hasan; Ieong, Ka-Weng; Wang, Jinfan; Petrov, Alexey; Prabhakar, Arjun; Rechavi, Gideon; Dominissini, Dan; He, Chuan; Ehrenberg, Måns; Puglisi, Joseph D

2018-03-01

Chemical modifications of mRNA may regulate many aspects of mRNA processing and protein synthesis. Recently, 2'-O-methylation of nucleotides was identified as a frequent modification in translated regions of human mRNA, showing enrichment in codons for certain amino acids. Here, using single-molecule, bulk kinetics and structural methods, we show that 2'-O-methylation within coding regions of mRNA disrupts key steps in codon reading during cognate tRNA selection. Our results suggest that 2'-O-methylation sterically perturbs interactions of ribosomal-monitoring bases (G530, A1492 and A1493) with cognate codon-anticodon helices, thereby inhibiting downstream GTP hydrolysis by elongation factor Tu (EF-Tu) and A-site tRNA accommodation, leading to excessive rejection of cognate aminoacylated tRNAs in initial selection and proofreading. Our current and prior findings highlight how chemical modifications of mRNA tune the dynamics of protein synthesis at different steps of translation elongation.

Ribosome profiling reveals pervasive and regulated stop codon readthrough in Drosophila melanogaster

PubMed Central

Dunn, Joshua G; Foo, Catherine K; Belletier, Nicolette G; Gavis, Elizabeth R; Weissman, Jonathan S

2013-01-01

Ribosomes can read through stop codons in a regulated manner, elongating rather than terminating the nascent peptide. Stop codon readthrough is essential to diverse viruses, and phylogenetically predicted to occur in a few hundred genes in Drosophila melanogaster, but the importance of regulated readthrough in eukaryotes remains largely unexplored. Here, we present a ribosome profiling assay (deep sequencing of ribosome-protected mRNA fragments) for Drosophila melanogaster, and provide the first genome-wide experimental analysis of readthrough. Readthrough is far more pervasive than expected: the vast majority of readthrough events evolved within D. melanogaster and were not predicted phylogenetically. The resulting C-terminal protein extensions show evidence of selection, contain functional subcellular localization signals, and their readthrough is regulated, arguing for their importance. We further demonstrate that readthrough occurs in yeast and humans. Readthrough thus provides general mechanisms both to regulate gene expression and function, and to add plasticity to the proteome during evolution. DOI: http://dx.doi.org/10.7554/eLife.01179.001 PMID:24302569

Multiple Transcript Properties Related to Translation Affect mRNA Degradation Rates in Saccharomyces cerevisiae

PubMed Central

Neymotin, Benjamin; Ettorre, Victoria; Gresham, David

2016-01-01

Degradation of mRNA contributes to variation in transcript abundance. Studies of individual mRNAs have shown that both cis and trans factors affect mRNA degradation rates. However, the factors underlying transcriptome-wide variation in mRNA degradation rates are poorly understood. We investigated the contribution of different transcript properties to transcriptome-wide degradation rate variation in the budding yeast, Saccharomyces cerevisiae, using multiple regression analysis. We find that multiple transcript properties are significantly associated with variation in mRNA degradation rates, and that a model incorporating these properties explains ∼50% of the genome-wide variance. Predictors of mRNA degradation rates include transcript length, ribosome density, biased codon usage, and GC content of the third position in codons. To experimentally validate these factors, we studied individual transcripts expressed from identical promoters. We find that decreasing ribosome density by mutating the first translational start site of a transcript increases its degradation rate. Using coding sequence variants of green fluorescent protein (GFP) that differ only at synonymous sites, we show that increased GC content of the third position of codons results in decreased rates of mRNA degradation. Thus, in steady-state conditions, a large fraction of genome-wide variation in mRNA degradation rates is determined by inherent properties of transcripts, many of which are related to translation, rather than specific regulatory mechanisms. PMID:27633789

Structure of the 5' region of the Hst70 gene transcription unit: presence of an intron and multiple transcription initiation sites.

PubMed Central

Scieglinska, D; Widłak, W; Konopka, W; Poutanen, M; Rahman, N; Huhtaniemi, I; Krawczyk, Z

2001-01-01

The rat Hst70 gene and its mouse counterpart Hsp70.2 belong to the family of Hsp70 heat shock genes and are specifically expressed in male germ cells. Previous studies regarding the structure of the 5' region of the transcription unit of these genes as well as localization of the 'cis' elements conferring their testis-specific expression gave contradictory results [Widlak, Markkula, Krawczyk, Kananen and Huhtaniemi (1995) Biochim. Biophys. Acta 1264, 191-200; Dix, Rosario-Herrle, Gotoh, Mori, Goulding, Barret and Eddy (1996) Dev. Biol. 174, 310-321]. In the present paper we solve these controversies and show that the 5' untranslated region (UTR) of the Hst70 gene contains an intron which is localized similar to that of the mouse Hsp70.2 gene. Reverse transcriptase-mediated PCR, Northern blotting and RNase protection analysis revealed that the transcription initiation of both genes starts at two main distant sites, and one of them is localized within the intron. As a result two populations of Hst70 gene transcripts with similar sizes but different 5' UTR structures can be detected in total testicular RNA. Functional analysis of the Hst70 gene promoter in transgenic mice and transient transfection assays proved that the DNA fragment of approx. 360 bp localized upstream of the ATG transcription start codon is the minimal promoter required for testis-specific expression of the HST70/chloramphenicol acetyltransferase transgene. These experiments also suggest that the expression of the gene may depend on 'cis' regulatory elements localized within exon 1 and the intron sequences. PMID:11563976

Translation initiation at an upstream CUG codon regulates the expression of Hibiscus chlorotic ringspot virus coat protein.

PubMed

Koh, Dora Chin-Yen; Wang, Xiaoxing; Wong, Sek-Man; Liu, D X

2006-12-01

Viruses depend heavily on host cells for replication and exploit the host translation machinery for its gene expression using various unorthodox translation mechanisms. According to the conventional scanning model, only the 5'-proximal gene in the viral RNA is accessible to the ribosomes whereas other genes are silent. In this study, we use a model plant RNA virus, Hibiscus chlorotic ringspot virus (HCRSV), to investigate various translation mechanisms involved in regulation of the expression of internal genes. The 3'-end 1.2kb region of HCRSV genomic and subgenomic RNAs were shown to encode four polypeptides of 38, 27, 25 and 22.5kDa. Mutagenesis studies revealed that a CUG codon ((2570)CUG) is the initiation codon for p27, the longest of the three co-C-terminal products (p27, p25 and p22.5), and translation of p25 and p22.5 was initiated at (2603)AUG and (2666)AUG, respectively. Translation initiation of the p27 expression at the (2570)CUG codon regulates the expression of p38, the viral coat protein through a leaky scanning mechanism and mutational analysis of an upstream open reading frame (ORF) demonstrated that initiation of the p27 expression at this CUG codon (instead of an AUG) may play a role in maintaining the ratio of p27 and p38. In addition, a previously identified internal ribosome entry site was shown to control the expression of p27 and p38 in the subgenomic RNA 2.

Does the Genetic Code Have A Eukaryotic Origin?

PubMed Central

Zhang, Zhang; Yu, Jun

2013-01-01

In the RNA world, RNA is assumed to be the dominant macromolecule performing most, if not all, core “house-keeping” functions. The ribo-cell hypothesis suggests that the genetic code and the translation machinery may both be born of the RNA world, and the introduction of DNA to ribo-cells may take over the informational role of RNA gradually, such as a mature set of genetic code and mechanism enabling stable inheritance of sequence and its variation. In this context, we modeled the genetic code in two content variables—GC and purine contents—of protein-coding sequences and measured the purine content sensitivities for each codon when the sensitivity (% usage) is plotted as a function of GC content variation. The analysis leads to a new pattern—the symmetric pattern—where the sensitivity of purine content variation shows diagonally symmetry in the codon table more significantly in the two GC content invariable quarters in addition to the two existing patterns where the table is divided into either four GC content sensitivity quarters or two amino acid diversity halves. The most insensitive codon sets are GUN (valine) and CAN (CAR for asparagine and CAY for aspartic acid) and the most biased amino acid is valine (always over-estimated) followed by alanine (always under-estimated). The unique position of valine and its codons suggests its key roles in the final recruitment of the complete codon set of the canonical table. The distinct choice may only be attributable to sequence signatures or signals of splice sites for spliceosomal introns shared by all extant eukaryotes. PMID:23402863

Mutations in the glucose-6-phosphatase gene that cause glycogen storage disease type 1a

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chou, J.Y.; Lei, K.J.; Shelly, L.L.

1994-09-01

Glycogen storage disease (GSD) type la (von Gierke disease) is caused by the deficiency of glucose-6-phosphatase (G6Pase), the key enzyme in glucose homeostasis. The disease presents with clinical manifestations of severe hypoglycemia, hepatomegaly, growth retardation, lactic acidemia, hyperlipidemia, and hyperuricemia. We have succeeded in isolating a murine G6Pase cDNA from a normal mouse liver cDNA library by differentially screening method. We then isolated the human G6Pase cDNA and gene. To date, we have characterized the G6Pase genes of twelve GSD type la patients and uncovered a total of six different mutations. The mutations are comprised of R83C (an Arg atmore » codon 83 to a Cys), Q347X (a Gly at codon 347 to a stop codon), 459insTA (a two basepair insertion at nucleotide 459 yielding a truncated G6Pase of 129 residues), R295C (an Arg at codon 295 to a Cys), G222R (a Gly at codon 222 to an Arg) and {delta}F327 (a codon deletion for Phe-327 at nucleotides 1058 to 1060). The relative incidences of these mutations are 37.5% (R83C), 33.3% (Q347X), 16.6% (459insTA), 4.2% (G222R), 4.2% (R295C) and 4.2% ({delta}F327). Site-directed mutagenesis and transient expression assays demonstrated that the R83C, Q347X, R295C, and {delta}F327 mutations abolished whereas the G222R mutation greatly reduced G6Pase activity. We further characterized the structure-function requirements of amino acids 83, 222, and 295 in G6Pase catalysis. The identification of mutations in GSD type la patients has unequivocally established the molecular basis of the type la disorder. Knowledge of the mutations may be applied to prenatal diagnosis and opens the way for developing and evaluating new therapeutic approaches.« less

Molecular Structure and Transformation of the Glucose Dehydrogenase Gene in Drosophila Melanogaster

PubMed Central

Whetten, R.; Organ, E.; Krasney, P.; Cox-Foster, D.; Cavener, D.

1988-01-01

We have precisely mapped and sequenced the three 5' exons of the Drosophila melanogaster Gld gene and have identified the start sites for transcription and translation. The first exon is composed of 335 nucleotides and does not contain any putative translation start codons. The second exon is separated from the first exon by 8 kb and contains the Gld translation start codon. The inferred amino acid sequence of the amino terminus contains two unusual features: three tandem repeats of serine-alanine, and a relatively high density of cysteine residues. P element-mediated transformation experiments demonstrated that a 17.5-kb genomic fragment contains the functional and regulatory components of the Gld gene. PMID:3143620

Beyond the Triplet Code: Context Cues Transform Translation.

PubMed

Brar, Gloria A

2016-12-15

The elucidation of the genetic code remains among the most influential discoveries in biology. While innumerable studies have validated the general universality of the code and its value in predicting and analyzing protein coding sequences, established and emerging work has also suggested that full genome decryption may benefit from a greater consideration of a codon's neighborhood within an mRNA than has been broadly applied. This Review examines the evidence for context cues in translation, with a focus on several recent studies that reveal broad roles for mRNA context in programming translation start sites, the rate of translation elongation, and stop codon identity. Copyright © 2016 Elsevier Inc. All rights reserved.

Introduction of translation stop codons into the viral glycoprotein gene in a fish DNA vaccine eliminates induction of protective immunity

USGS Publications Warehouse

Garver, K.A.; Conway, C.M.; Kurath, G.

2006-01-01

A highly efficacious DNA vaccine against a fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV), was mutated to introduce two stop codons to prevent glycoprotein translation while maintaining the plasmid DNA integrity and RNA transcription ability. The mutated plasmid vaccine, denoted pIHNw-G2stop, when injected intramuscularly into fish at high doses, lacked detectable glycoprotein expression in the injection site muscle, and did not provide protection against lethal virus challenge 7 days post-vaccination. These results suggest that the G-protein itself is required to stimulate the early protective antiviral response observed after vaccination with the nonmutated parental DNA vaccine. ?? Springer Science+Business Media, Inc. 2006.

Novel Immune Modulating Cellular Vaccine for Prostate Cancer

DTIC Science & Technology

2014-10-01

restriction sites. Murine PSMA : The cDNA encoding mPSMA was purchased from Sino Biologicals and was cloned into the HindIII and BamHI sites of pSP73-Sph/A64...sequence) and reverse primer 5’-TATATAGAGCTCTCAGATGTTCCGATACACATCTC-3’ Murine PSMA no signal sequence (mPSMA-SS): Murine PSMA minus the signal sequence...contains a HindIII site for cloning and utilizes an ATG that lies downstream of the signal sequence as the start codon in PSMA -SS ( PSMA without signal

Multiple Site-Directed and Saturation Mutagenesis by the Patch Cloning Method.

PubMed

Taniguchi, Naohiro; Murakami, Hiroshi

2017-01-01

Constructing protein-coding genes with desired mutations is a basic step for protein engineering. Herein, we describe a multiple site-directed and saturation mutagenesis method, termed MUPAC. This method has been used to introduce multiple site-directed mutations in the green fluorescent protein gene and in the moloney murine leukemia virus reverse transcriptase gene. Moreover, this method was also successfully used to introduce randomized codons at five desired positions in the green fluorescent protein gene, and for simple DNA assembly for cloning.

Identification of a novel selD homolog from Eukaryotes, Bacteria, and Archaea: Is there an autoregulatory mechanism in selenocysteine metabolism?

PubMed Central

Guimarães, M. Jorge; Peterson, David; Vicari, Alain; Cocks, Benjamin G.; Copeland, Neal G.; Gilbert, Debra J.; Jenkins, Nancy A.; Ferrick, David A.; Kastelein, Robert A.; Bazan, J. Fernando; Zlotnik, Albert

1996-01-01

Escherichia coli selenophosphate synthetase (SPS, the selD gene product) catalyzes the production of monoselenophosphate, the selenium donor compound required for synthesis of selenocysteine (Sec) and seleno-tRNAs. We report the molecular cloning of human and mouse homologs of the selD gene, designated Sps2, which contains an in-frame TGA codon at a site corresponding to the enzyme’s putative active site. These sequences allow the identification of selD gene homologs in the genomes of the bacterium Haemophilus influenzae and the archaeon Methanococcus jannaschii, which had been previously misinterpreted due to their in-frame TGA codon. Sps2 mRNA levels are elevated in organs previously implicated in the synthesis of selenoproteins and in active sites of blood cell development. In addition, we show that Sps2 mRNA is up-regulated upon activation of T lymphocytes and have mapped the Sps2 gene to mouse chromosome 7. Using the mouse gene isolated from the hematopoietic cell line FDCPmixA4, we devised a construct for protein expression that results in the insertion of a FLAG tag sequence at the N terminus of the SPS2 protein. This strategy allowed us to document the readthrough of the in-frame TGA codon and the incorporation of 75Se into SPS2. These results suggest the existence of an autoregulatory mechanism involving the incorporation of Sec into SPS2 that might be relevant to blood cell biology. This mechanism is likely to have been present in ancient life forms and conserved in a variety of living organisms from all domains of life. PMID:8986768

Odilorhabdins, Antibacterial Agents that Cause Miscoding by Binding at a New Ribosomal Site.

PubMed

Pantel, Lucile; Florin, Tanja; Dobosz-Bartoszek, Malgorzata; Racine, Emilie; Sarciaux, Matthieu; Serri, Marine; Houard, Jessica; Campagne, Jean-Marc; de Figueiredo, Renata Marcia; Midrier, Camille; Gaudriault, Sophie; Givaudan, Alain; Lanois, Anne; Forst, Steve; Aumelas, André; Cotteaux-Lautard, Christelle; Bolla, Jean-Michel; Vingsbo Lundberg, Carina; Huseby, Douglas L; Hughes, Diarmaid; Villain-Guillot, Philippe; Mankin, Alexander S; Polikanov, Yury S; Gualtieri, Maxime

2018-04-05

Growing resistance of pathogenic bacteria and shortage of antibiotic discovery platforms challenge the use of antibiotics in the clinic. This threat calls for exploration of unconventional sources of antibiotics and identification of inhibitors able to eradicate resistant bacteria. Here we describe a different class of antibiotics, odilorhabdins (ODLs), produced by the enzymes of the non-ribosomal peptide synthetase gene cluster of the nematode-symbiotic bacterium Xenorhabdus nematophila. ODLs show activity against Gram-positive and Gram-negative pathogens, including carbapenem-resistant Enterobacteriaceae, and can eradicate infections in animal models. We demonstrate that the bactericidal ODLs interfere with protein synthesis. Genetic and structural analyses reveal that ODLs bind to the small ribosomal subunit at a site not exploited by current antibiotics. ODLs induce miscoding and promote hungry codon readthrough, amino acid misincorporation, and premature stop codon bypass. We propose that ODLs' miscoding activity reflects their ability to increase the affinity of non-cognate aminoacyl-tRNAs to the ribosome. Copyright © 2018 Elsevier Inc. All rights reserved.

Markov-modulated Markov chains and the covarion process of molecular evolution.

PubMed

Galtier, N; Jean-Marie, A

2004-01-01

The covarion (or site specific rate variation, SSRV) process of biological sequence evolution is a process by which the evolutionary rate of a nucleotide/amino acid/codon position can change in time. In this paper, we introduce time-continuous, space-discrete, Markov-modulated Markov chains as a model for representing SSRV processes, generalizing existing theory to any model of rate change. We propose a fast algorithm for diagonalizing the generator matrix of relevant Markov-modulated Markov processes. This algorithm makes phylogeny likelihood calculation tractable even for a large number of rate classes and a large number of states, so that SSRV models become applicable to amino acid or codon sequence datasets. Using this algorithm, we investigate the accuracy of the discrete approximation to the Gamma distribution of evolutionary rates, widely used in molecular phylogeny. We show that a relatively large number of classes is required to achieve accurate approximation of the exact likelihood when the number of analyzed sequences exceeds 20, both under the SSRV and among site rate variation (ASRV) models.

Analysis of synonymous codon usage patterns in the genus Rhizobium.

PubMed

Wang, Xinxin; Wu, Liang; Zhou, Ping; Zhu, Shengfeng; An, Wei; Chen, Yu; Zhao, Lin

2013-11-01

The codon usage patterns of rhizobia have received increasing attention. However, little information is available regarding the conserved features of the codon usage patterns in a typical rhizobial genus. The codon usage patterns of six completely sequenced strains belonging to the genus Rhizobium were analysed as model rhizobia in the present study. The relative neutrality plot showed that selection pressure played a role in codon usage in the genus Rhizobium. Spearman's rank correlation analysis combined with correspondence analysis (COA) showed that the codon adaptation index and the effective number of codons (ENC) had strong correlation with the first axis of the COA, which indicated the important role of gene expression level and the ENC in the codon usage patterns in this genus. The relative synonymous codon usage of Cys codons had the strongest correlation with the second axis of the COA. Accordingly, the usage of Cys codons was another important factor that shaped the codon usage patterns in Rhizobium genomes and was a conserved feature of the genus. Moreover, the comparison of codon usage between highly and lowly expressed genes showed that 20 unique preferred codons were shared among Rhizobium genomes, revealing another conserved feature of the genus. This is the first report of the codon usage patterns in the genus Rhizobium.

Genome-wide comparative analysis of codon usage bias and codon context patterns among cyanobacterial genomes.

PubMed

Prabha, Ratna; Singh, Dhananjaya P; Sinha, Swati; Ahmad, Khurshid; Rai, Anil

2017-04-01

With the increasing accumulation of genomic sequence information of prokaryotes, the study of codon usage bias has gained renewed attention. The purpose of this study was to examine codon selection pattern within and across cyanobacterial species belonging to diverse taxonomic orders and habitats. We performed detailed comparative analysis of cyanobacterial genomes with respect to codon bias. Our analysis reflects that in cyanobacterial genomes, A- and/or T-ending codons were used predominantly in the genes whereas G- and/or C-ending codons were largely avoided. Variation in the codon context usage of cyanobacterial genes corresponded to the clustering of cyanobacteria as per their GC content. Analysis of codon adaptation index (CAI) and synonymous codon usage order (SCUO) revealed that majority of genes are associated with low codon bias. Codon selection pattern in cyanobacterial genomes reflected compositional constraints as major influencing factor. It is also identified that although, mutational constraint may play some role in affecting codon usage bias in cyanobacteria, compositional constraint in terms of genomic GC composition coupled with environmental factors affected codon selection pattern in cyanobacterial genomes. Copyright © 2016 Elsevier B.V. All rights reserved.

Analysis of four families with the Stickler syndrome by linkage studies. Identification of a new premature stop codon in the COL2A1 gene in a family

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bonaventure, J.; Lasselin, C.; Toutain, A.

1994-09-01

The Stickler syndrome is an arthro-ophthalmopathy which associates progressive myopia with vitreal degeneration and retinal detachment. Cleft palate, cranio-facial abnormalities, deafness and osteoarthritis are often associated symptoms. Genetic heterogeneity of this autosomal dominant disease was consistent with its large clinical variability. Linkage studies have provided evidence for cosegregation of the disease with COL2A1, the gene coding for type II collagen, in about 50% of the families. Four additional families are reported here. Linkage analyses by using a VNTR located in the 3{prime} region of the gene were achieved. In three families, positive lod scores were obtained with a cumulative maximalmore » value of 3.5 at a recombination fraction of 0. In one of these families, single strand conformation analysis of 25 exons disclosed a new mutation in exon 42. Codon for glutamic acid at position a1-803 was converted into a stop codon. The mutation was detected in DNA samples from all the affected members of the family but not in the unaffected. This result confirms that most of the Stickler syndromes linked to COL2A1 are due to premature stop codons. In a second family, an abnormal SSCP pattern of exon 34 was detected in all the affected individuals. The mutation is likely to correspond to a splicing defect in the acceptor site of intron 33. In one family the disease did not segregate with the COL2A1 locus. Further linkage studies with intragenic dimorphic sites in the COL10A1 gene and highly polymorphic markers close to the COL9A1 locus indicated that this disorder did not result from defects in these two genes.« less

Codon usage bias in prokaryotic pyrimidine-ending codons is associated with the degeneracy of the encoded amino acids

PubMed Central

Wald, Naama; Alroy, Maya; Botzman, Maya; Margalit, Hanah

2012-01-01

Synonymous codons are unevenly distributed among genes, a phenomenon termed codon usage bias. Understanding the patterns of codon bias and the forces shaping them is a major step towards elucidating the adaptive advantage codon choice can confer at the level of individual genes and organisms. Here, we perform a large-scale analysis to assess codon usage bias pattern of pyrimidine-ending codons in highly expressed genes in prokaryotes. We find a bias pattern linked to the degeneracy of the encoded amino acid. Specifically, we show that codon-pairs that encode two- and three-fold degenerate amino acids are biased towards the C-ending codon while codons encoding four-fold degenerate amino acids are biased towards the U-ending codon. This codon usage pattern is widespread in prokaryotes, and its strength is correlated with translational selection both within and between organisms. We show that this bias is associated with an improved correspondence with the tRNA pool, avoidance of mis-incorporation errors during translation and moderate stability of codon–anticodon interaction, all consistent with more efficient translation. PMID:22581775

The helicase Ded1p controls use of near-cognate translation initiation codons in 5' UTRs.

PubMed

Guenther, Ulf-Peter; Weinberg, David E; Zubradt, Meghan M; Tedeschi, Frank A; Stawicki, Brittany N; Zagore, Leah L; Brar, Gloria A; Licatalosi, Donny D; Bartel, David P; Weissman, Jonathan S; Jankowsky, Eckhard

2018-06-27

The conserved and essential DEAD-box RNA helicase Ded1p from yeast and its mammalian orthologue DDX3 are critical for the initiation of translation 1 . Mutations in DDX3 are linked to tumorigenesis 2-4 and intellectual disability 5 , and the enzyme is targeted by a range of viruses 6 . How Ded1p and its orthologues engage RNAs during the initiation of translation is unknown. Here we show, by integrating transcriptome-wide analyses of translation, RNA structure and Ded1p-RNA binding, that the effects of Ded1p on the initiation of translation are connected to near-cognate initiation codons in 5' untranslated regions. Ded1p associates with the translation pre-initiation complex at the mRNA entry channel and repressing the activity of Ded1p leads to the accumulation of RNA structure in 5' untranslated regions, the initiation of translation from near-cognate start codons immediately upstream of these structures and decreased protein synthesis from the corresponding main open reading frames. The data reveal a program for the regulation of translation that links Ded1p, the activation of near-cognate start codons and mRNA structure. This program has a role in meiosis, in which a marked decrease in the levels of Ded1p is accompanied by the activation of the alternative translation initiation sites that are seen when the activity of Ded1p is repressed. Our observations indicate that Ded1p affects translation initiation by controlling the use of near-cognate initiation codons that are proximal to mRNA structure in 5' untranslated regions.

Expression of chicken parvovirus VP2 in chicken embryo fibroblasts requires codon optimization for production of naked DNA and vectored meleagrid herpesvirus type 1 vaccines.

PubMed

Spatz, Stephen J; Volkening, Jeremy D; Mullis, Robert; Li, Fenglan; Mercado, John; Zsak, Laszlo

2013-10-01

Meleagrid herpesvirus type 1 (MeHV-1) is an ideal vector for the expression of antigens from pathogenic avian organisms in order to generate vaccines. Chicken parvovirus (ChPV) is a widespread infectious virus that causes serious disease in chickens. It is one of the etiological agents largely suspected in causing Runting Stunting Syndrome (RSS) in chickens. Initial attempts to express the wild-type gene encoding the capsid protein VP2 of ChPV by insertion into the thymidine kinase gene of MeHV-1 were unsuccessful. However, transient expression of a codon-optimized synthetic VP2 gene cloned into the bicistronic vector pIRES2-Ds-Red2, could be demonstrated by immunocytochemical staining of transfected chicken embryo fibroblasts (CEFs). Red fluorescence could also be detected in these transfected cells since the red fluorescent protein gene is downstream from the internal ribosome entry site (IRES). Strikingly, fluorescence could not be demonstrated in cells transiently transfected with the bicistronic vector containing the wild-type or non-codon-optimized VP2 gene. Immunocytochemical staining of these cells also failed to demonstrate expression of wild-type VP2, indicating that the lack of expression was at the RNA level and the VP2 protein was not toxic to CEFs. Chickens vaccinated with a DNA vaccine consisting of the bicistronic vector containing the codon-optimized VP2 elicited a humoral immune response as measured by a VP2-specific ELISA. This VP2 codon-optimized bicistronic cassette was rescued into the MeHV-1 genome generating a vectored vaccine against ChPV disease.

The Selenocysteine-Specific Elongation Factor Contains Unique Sequences That Are Required for Both Nuclear Export and Selenocysteine Incorporation.

PubMed

Dubey, Aditi; Copeland, Paul R

2016-01-01

Selenocysteine (Sec) is a critical residue in at least 25 human proteins that are essential for antioxidant defense and redox signaling in cells. Sec is inserted into proteins cotranslationally by the recoding of an in-frame UGA termination codon to a Sec codon. In eukaryotes, this recoding event requires several specialized factors, including a dedicated, Sec-specific elongation factor called eEFSec, which binds Sec-tRNASec with high specificity and delivers it to the ribosome for selenoprotein production. Unlike most translation factors, including the canonical elongation factor eEF1A, eEFSec readily localizes to the nucleus of mammalian cells and shuttles between the cytoplasmic and nuclear compartments. The functional significance of eEFSec's nuclear localization has remained unclear. In this study, we have examined the subcellular localization of eEFSec in the context of altered Sec incorporation to demonstrate that reduced selenoprotein production does not correlate with changes in the nuclear localization of eEFSec. In addition, we identify several novel sequences of the protein that are essential for localization as well as Sec insertion activity, and show that eEFSec utilizes CRM1-mediated nuclear export pathway. Our findings argue for two distinct pools of eEFSec in the cell, where the cytoplasmic pool participates in Sec incorporation and the nuclear pool may be involved in an as yet unknown function.

Optimizing doped libraries by using genetic algorithms

NASA Astrophysics Data System (ADS)

Tomandl, Dirk; Schober, Andreas; Schwienhorst, Andreas

1997-01-01

The insertion of random sequences into protein-encoding genes in combination with biologicalselection techniques has become a valuable tool in the design of molecules that have usefuland possibly novel properties. By employing highly effective screening protocols, a functionaland unique structure that had not been anticipated can be distinguished among a hugecollection of inactive molecules that together represent all possible amino acid combinations.This technique is severely limited by its restriction to a library of manageable size. Oneapproach for limiting the size of a mutant library relies on `doping schemes', where subsetsof amino acids are generated that reveal only certain combinations of amino acids in a proteinsequence. Three mononucleotide mixtures for each codon concerned must be designed, suchthat the resulting codons that are assembled during chemical gene synthesis represent thedesired amino acid mixture on the level of the translated protein. In this paper we present adoping algorithm that `reverse translates' a desired mixture of certain amino acids into threemixtures of mononucleotides. The algorithm is designed to optimally bias these mixturestowards the codons of choice. This approach combines a genetic algorithm with localoptimization strategies based on the downhill simplex method. Disparate relativerepresentations of all amino acids (and stop codons) within a target set can be generated.Optional weighing factors are employed to emphasize the frequencies of certain amino acidsand their codon usage, and to compensate for reaction rates of different mononucleotidebuilding blocks (synthons) during chemical DNA synthesis. The effect of statistical errors thataccompany an experimental realization of calculated nucleotide mixtures on the generatedmixtures of amino acids is simulated. These simulations show that the robustness of differentoptima with respect to small deviations from calculated values depends on their concomitantfitness. Furthermore, the calculations probe the fitness landscape locally and allow apreliminary assessment of its structure.

Integrated analysis of individual codon contribution to protein biosynthesis reveals a new approach to improving the basis of rational gene design

PubMed Central

Villada, Juan C.; Brustolini, Otávio José Bernardes

2017-01-01

Abstract Gene codon optimization may be impaired by the misinterpretation of frequency and optimality of codons. Although recent studies have revealed the effects of codon usage bias (CUB) on protein biosynthesis, an integrated perspective of the biological role of individual codons remains unknown. Unlike other previous studies, we show, through an integrated framework that attributes of codons such as frequency, optimality and positional dependency should be combined to unveil individual codon contribution for protein biosynthesis. We designed a codon quantification method for assessing CUB as a function of position within genes with a novel constraint: the relativity of position-dependent codon usage shaped by coding sequence length. Thus, we propose a new way of identifying the enrichment, depletion and non-uniform positional distribution of codons in different regions of yeast genes. We clustered codons that shared attributes of frequency and optimality. The cluster of non-optimal codons with rare occurrence displayed two remarkable characteristics: higher codon decoding time than frequent–non-optimal cluster and enrichment at the 5′-end region, where optimal codons with the highest frequency are depleted. Interestingly, frequent codons with non-optimal adaptation to tRNAs are uniformly distributed in the Saccharomyces cerevisiae genes, suggesting their determinant role as a speed regulator in protein elongation. PMID:28449100

Integrated analysis of individual codon contribution to protein biosynthesis reveals a new approach to improving the basis of rational gene design.

PubMed

Villada, Juan C; Brustolini, Otávio José Bernardes; Batista da Silveira, Wendel

2017-08-01

Gene codon optimization may be impaired by the misinterpretation of frequency and optimality of codons. Although recent studies have revealed the effects of codon usage bias (CUB) on protein biosynthesis, an integrated perspective of the biological role of individual codons remains unknown. Unlike other previous studies, we show, through an integrated framework that attributes of codons such as frequency, optimality and positional dependency should be combined to unveil individual codon contribution for protein biosynthesis. We designed a codon quantification method for assessing CUB as a function of position within genes with a novel constraint: the relativity of position-dependent codon usage shaped by coding sequence length. Thus, we propose a new way of identifying the enrichment, depletion and non-uniform positional distribution of codons in different regions of yeast genes. We clustered codons that shared attributes of frequency and optimality. The cluster of non-optimal codons with rare occurrence displayed two remarkable characteristics: higher codon decoding time than frequent-non-optimal cluster and enrichment at the 5'-end region, where optimal codons with the highest frequency are depleted. Interestingly, frequent codons with non-optimal adaptation to tRNAs are uniformly distributed in the Saccharomyces cerevisiae genes, suggesting their determinant role as a speed regulator in protein elongation. © The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

First complete mitochondrial genome of the South American annual fish Austrolebias charrua (Cyprinodontiformes: Rivulidae): peculiar features among cyprinodontiforms mitogenomes.

PubMed

Gutiérrez, Verónica; Rego, Natalia; Naya, Hugo; García, Graciela

2015-10-28

Among teleosts, the South American genus Austrolebias (Cyprinodontiformes: Rivulidae) includes 42 taxa of annual fishes divided into five different species groups. It is a monophyletic genus, but morphological and molecular data do not resolve the relationship among intrageneric clades and high rates of substitution have been previously described in some mitochondrial genes. In this work, the complete mitogenome of a species of the genus was determined for the first time. We determined its structure, gene order and evolutionary peculiar features, which will allow us to evaluate the performance of mitochondrial genes in the phylogenetic resolution at different taxonomic levels. Regarding gene content and order, the circular mitogenome of A. charrua (17,271 pb) presents the typical pattern of vertebrate mitogenomes. It contains the full complement of 13 proteins-coding genes, 22 tRNA, 2 rRNA and one non-coding control region. Notably, the tRNA-Cys was only 57 bp in length and lacks the D-loop arm. In three full sibling individuals, heteroplasmatic condition was detected due to a total of 12 variable sites in seven protein-coding genes. Among cyprinodontiforms, the mitogenome of A. charrua exhibits the lowest G+C content (37 %) and GCskew, as well as the highest strand asymmetry with a net difference of T over A at 1st and 3rd codon positions. Considering the 12 coding-genes of the H strand, correspondence analyses of nucleotide composition and codon usage show that A and T at 1st and 3rd codon positions have the highest weight in the first axis, and segregate annual species from the other cyprinodontiforms analyzed. Given the annual life-style, their mitogenomes could be under different selective pressures. All 13 protein-coding genes are under strong purifying selection and we did not find any significant evidence of nucleotide sites showing episodic selection (dN >dS) at annual lineages. When fast evolving third codon positions were removed from alignments, the "supergene" tree recovers our reference species phylogeny as well as the Cytb, ND4L and ND6 genes. Therefore, third codon positions seem to be saturated in the aforementioned coding regions at intergeneric Cyprinodontiformes comparisons. The complete mitogenome obtained in present work, offers relevant data for further comparative studies on molecular phylogeny and systematics of this taxonomic controversial endemic genus of annual fishes.

Partial attenuation of Marek's disease virus by manipulation of Di-codon bias

USDA-ARS?s Scientific Manuscript database

All species studied to date demonstrate a preference for certain codons over other synonymous codons (codon bias), a preference which is also observed for pairs of codons (di-codon bias). Previous studies using poliovirus and influenza virus as models have demonstrated the ability to cause attenuat...

Novel splice site mutation in the growth hormone receptor gene in Turkish patients with Laron-type dwarfism.

PubMed

Arman, Ahmet; Ozon, Alev; Isguven, Pinar S; Coker, Ajda; Peker, Ismail; Yordam, Nursen

2008-01-01

Growth hormone (GH) is involved in growth, and fat and carbohydrate metabolism. Interaction of GH with the GH receptor (GHR) is necessary for systemic and local production of insulin-like growth factor-I (IGF-I) which mediates GH actions. Mutations in the GHR cause severe postnatal growth failure; the disorder is an autosomal recessive genetic disease resulting in GH insensitivity, called Laron syndrome. It is characterized by dwarfism with elevated serum GH and low levels of IGF-I. We analyzed the GHR gene for mutations and polymorphisms in eight patients with Laron-type dwarfism from six families. We found three missense mutations (S40L, V125A, I526L), one nonsense mutation (W157X), and one splice site mutation in the extracellular domain of GHR. Furthermore, G168G and exon 3 deletion polymorphisms were detected in patients with Laron syndrome. The splice site mutation, which is a novel mutation, was located at the donor splice site of exon 2/ intron 2 within GHR. Although this mutation changed the highly conserved donor splice site consensus sequence GT to GGT by insertion of a G residue, the intron splicing between exon 2 and exon 3 was detected in the patient. These results imply that the splicing occurs arthe GT site in intron 2, leaving the extra inserted G residue at the end of exon 2, thus changing the open reading frame of GHR resulting in a premature termination codon in exon 3.

Transcriptome Analysis of Core Dinoflagellates Reveals a Universal Bias towards "GC" Rich Codons.

PubMed

Williams, Ernest; Place, Allen; Bachvaroff, Tsvetan

2017-04-27

Although dinoflagellates are a potential source of pharmaceuticals and natural products, the mechanisms for regulating and producing these compounds are largely unknown because of extensive post-transcriptional control of gene expression. One well-documented mechanism for controlling gene expression during translation is codon bias, whereby specific codons slow or even terminate protein synthesis. Approximately 10,000 annotatable genes from fifteen "core" dinoflagellate transcriptomes along a range of overall guanine and cytosine (GC) content were used for codonW analysis to determine the relative synonymous codon usage (RSCU) and the GC content at each codon position. GC bias in the analyzed dataset and at the third codon position varied from 51% and 54% to 66% and 88%, respectively. Codons poor in GC were observed to be universally absent, but bias was most pronounced for codons ending in uracil followed by adenine (UA). GC bias at the third codon position was able to explain low abundance codons as well as the low effective number of codons. Thus, we propose that a bias towards codons rich in GC bases is a universal feature of core dinoflagellates, possibly relating to their unique chromosome structure, and not likely a major mechanism for controlling gene expression.

Multiple Evolutionary Selections Involved in Synonymous Codon Usages in the Streptococcus agalactiae Genome.

PubMed

Ma, Yan-Ping; Ke, Hao; Liang, Zhi-Ling; Liu, Zhen-Xing; Hao, Le; Ma, Jiang-Yao; Li, Yu-Gu

2016-02-24

Streptococcus agalactiae is an important human and animal pathogen. To better understand the genetic features and evolution of S. agalactiae, multiple factors influencing synonymous codon usage patterns in S. agalactiae were analyzed in this study. A- and U-ending rich codons were used in S. agalactiae function genes through the overall codon usage analysis, indicating that Adenine (A)/Thymine (T) compositional constraints might contribute an important role to the synonymous codon usage pattern. The GC3% against the effective number of codon (ENC) value suggested that translational selection was the important factor for codon bias in the microorganism. Principal component analysis (PCA) showed that (i) mutational pressure was the most important factor in shaping codon usage of all open reading frames (ORFs) in the S. agalactiae genome; (ii) strand specific mutational bias was not capable of influencing the codon usage bias in the leading and lagging strands; and (iii) gene length was not the important factor in synonymous codon usage pattern in this organism. Additionally, the high correlation between tRNA adaptation index (tAI) value and codon adaptation index (CAI), frequency of optimal codons (Fop) value, reinforced the role of natural selection for efficient translation in S. agalactiae. Comparison of synonymous codon usage pattern between S. agalactiae and susceptible hosts (human and tilapia) showed that synonymous codon usage of S. agalactiae was independent of the synonymous codon usage of susceptible hosts. The study of codon usage in S. agalactiae may provide evidence about the molecular evolution of the bacterium and a greater understanding of evolutionary relationships between S. agalactiae and its hosts.

Multiple Evolutionary Selections Involved in Synonymous Codon Usages in the Streptococcus agalactiae Genome

PubMed Central

Ma, Yan-Ping; Ke, Hao; Liang, Zhi-Ling; Liu, Zhen-Xing; Hao, Le; Ma, Jiang-Yao; Li, Yu-Gu

2016-01-01

Streptococcus agalactiae is an important human and animal pathogen. To better understand the genetic features and evolution of S. agalactiae, multiple factors influencing synonymous codon usage patterns in S. agalactiae were analyzed in this study. A- and U-ending rich codons were used in S. agalactiae function genes through the overall codon usage analysis, indicating that Adenine (A)/Thymine (T) compositional constraints might contribute an important role to the synonymous codon usage pattern. The GC3% against the effective number of codon (ENC) value suggested that translational selection was the important factor for codon bias in the microorganism. Principal component analysis (PCA) showed that (i) mutational pressure was the most important factor in shaping codon usage of all open reading frames (ORFs) in the S. agalactiae genome; (ii) strand specific mutational bias was not capable of influencing the codon usage bias in the leading and lagging strands; and (iii) gene length was not the important factor in synonymous codon usage pattern in this organism. Additionally, the high correlation between tRNA adaptation index (tAI) value and codon adaptation index (CAI), frequency of optimal codons (Fop) value, reinforced the role of natural selection for efficient translation in S. agalactiae. Comparison of synonymous codon usage pattern between S. agalactiae and susceptible hosts (human and tilapia) showed that synonymous codon usage of S. agalactiae was independent of the synonymous codon usage of susceptible hosts. The study of codon usage in S. agalactiae may provide evidence about the molecular evolution of the bacterium and a greater understanding of evolutionary relationships between S. agalactiae and its hosts. PMID:26927064

Translation efficiencies of synonymous codons are not always correlated with codon usage in tobacco chloroplasts.

PubMed

Nakamura, Masayuki; Sugiura, Masahiro

2007-01-01

Codon usage in chloroplasts is different from that in prokaryotic and eukaryotic nuclear genomes. However, no experimental approach has been made to analyse the translation efficiency of individual codons in chloroplasts. We devised an in vitro assay for translation efficiencies using synthetic mRNAs, and measured the translation efficiencies of five synonymous codon groups in tobacco chloroplasts. Among four alanine codons (GCN, where N is U, C, A or G), GCU was the most efficient for translation, whereas the chloroplast genome lacks tRNA genes corresponding to GCU. Phenylalanine and tyrosine are each encoded by two codons (UUU/C and UAU/C, respectively). Phenylalanine UUC and tyrosine UAC were translated more than twice as efficiently than UUU and UAU, respectively, contrary to their codon usage, whereas translation efficiencies of synonymous codons for alanine, aspartic acid and asparagine were parallel to their codon usage. These observations indicate that translation efficiencies of individual codons are not always correlated with codon usage in vitro in chloroplasts. This raises an important issue for foreign gene expression in chloroplasts.

Two novel mutations in the alpha-galactosidase gene in Japanese classical hemizygotes with Fabry disease.

PubMed

Okumiya, T; Takenaka, T; Ishii, S; Kase, R; Kamei, S; Sakuraba, H

1996-09-01

Four alpha-galactosidase gene mutations were identified in Japanese male patients with Fabry disease who had no detectable alpha-galactosidase activity. Two of them were novel mutations, an 11-bp deletion in exon 2 and a g-1 to t substitution at the 3' end of the splice acceptor site in intron 1. The former caused a frameshift and led to the creation of a new stop codon at codon 118. The latter was predicted to provoke aberrant mRNA splicing followed by accelerated degradation of the mRNA. A nonsense mutation, R301X, and a 2-bp deletion starting at nucleotide position 718, which were reported previously, were also identified in unrelated patients.

Towards a comprehensive picture of C-to-U RNA editing sites in angiosperm mitochondria.

PubMed

Edera, Alejandro A; Gandini, Carolina L; Sanchez-Puerta, M Virginia

2018-05-14

Our understanding of the dynamic and evolution of RNA editing in angiosperms is in part limited by the few editing sites identified to date. This study identified 10,217 editing sites from 17 diverse angiosperms. Our analyses confirmed the universality of certain features of RNA editing, and offer new evidence behind the loss of editing sites in angiosperms. RNA editing is a post-transcriptional process that substitutes cytidines (C) for uridines (U) in organellar transcripts of angiosperms. These substitutions mostly take place in mitochondrial messenger RNAs at specific positions called editing sites. By means of publicly available RNA-seq data, this study identified 10,217 editing sites in mitochondrial protein-coding genes of 17 diverse angiosperms. Even though other types of mismatches were also identified, we did not find evidence of non-canonical editing processes. The results showed an uneven distribution of editing sites among species, genes, and codon positions. The analyses revealed that editing sites were conserved across angiosperms but there were some species-specific sites. Non-synonymous editing sites were particularly highly conserved (~ 80%) across the plant species and were efficiently edited (80% editing extent). In contrast, editing sites at third codon positions were poorly conserved (~ 30%) and only partially edited (~ 40% editing extent). We found that the loss of editing sites along angiosperm evolution is mainly occurring by replacing editing sites with thymidines, instead of a degradation of the editing recognition motif around editing sites. Consecutive and highly conserved editing sites had been replaced by thymidines as result of retroprocessing, by which edited transcripts are reverse transcribed to cDNA and then integrated into the genome by homologous recombination. This phenomenon was more pronounced in eudicots, and in the gene cox1. These results suggest that retroprocessing is a widespread driving force underlying the loss of editing sites in angiosperm mitochondria.

Functional Versatility of AGY Serine Codons in Immunoglobulin Variable Region Genes

PubMed Central

Detanico, Thiago; Phillips, Matthew; Wysocki, Lawrence J.

2016-01-01

In systemic autoimmunity, autoantibodies directed against nuclear antigens (Ags) often arise by somatic hypermutation (SHM) that converts AGT and AGC (AGY) Ser codons into Arg codons. This can occur by three different single-base changes. Curiously, AGY Ser codons are far more abundant in complementarity-determining regions (CDRs) of IgV-region genes than expected for random codon use or from species-specific codon frequency data. CDR AGY codons are also more abundant than TCN Ser codons. We show that these trends hold even in cartilaginous fishes. Because AGC is a preferred target for SHM by activation-induced cytidine deaminase, we asked whether the AGY abundance was solely due to a selection pressure to conserve high mutability in CDRs regardless of codon context but found that this was not the case. Instead, AGY triplets were selectively enriched in the Ser codon reading frame. Motivated by reports implicating a functional role for poly/autoreactive specificities in antiviral antibodies, we also analyzed mutations at AGY in antibodies directed against a number of different viruses and found that mutations producing Arg codons in antiviral antibodies were indeed frequent. Unexpectedly, however, we also found that AGY codons mutated often to encode nearly all of the amino acids that are reported to provide the most frequent contacts with Ag. In many cases, mutations producing codons for these alternative amino acids in antiviral antibodies were more frequent than those producing Arg codons. Mutations producing each of these key amino acids required only single-base changes in AGY. AGY is the only codon group in which two-thirds of random mutations generate codons for these key residues. Finally, by directly analyzing X-ray structures of immune complexes from the RCSB protein database, we found that Ag-contact residues generated via SHM occurred more often at AGY than at any other codon group. Thus, preservation of AGY codons in antibody genes appears to have been driven by their exceptional functional versatility, despite potential autoreactive consequences. PMID:27920779

Chloroplast DNA codon use: evidence for selection at the psb A locus based on tRNA availability.

PubMed

Morton, B R

1993-09-01

Codon use in the three sequenced chloroplast genomes (Marchantia, Oryza, and Nicotiana) is examined. The chloroplast has a bias in that codons NNA and NNT are favored over synonymous NNC and NNG codons. This appears to be a consequence of an overall high A + T content of the genome. This pattern of codon use is not followed by the psb A gene of all three genomes and other psb A sequences examined. In this gene, the codon use favors NNC over NNT for twofold degenerate amino acids. In each case the only tRNA coded by the genome is complementary to the NNC codon. This codon use is similar to the codon use by chloroplast genes examined from Chlamydomonas reinhardtii. Since psb A is the major translation product of the chloroplast, this suggests that selection is acting on the codon use of this gene to adapt codons to tRNA availability, as previously suggested for unicellular organisms.

Genomic analysis of codon usage shows influence of mutation pressure, natural selection, and host features on Marburg virus evolution.

PubMed

Nasrullah, Izza; Butt, Azeem M; Tahir, Shifa; Idrees, Muhammad; Tong, Yigang

2015-08-26

The Marburg virus (MARV) has a negative-sense single-stranded RNA genome, belongs to the family Filoviridae, and is responsible for several outbreaks of highly fatal hemorrhagic fever. Codon usage patterns of viruses reflect a series of evolutionary changes that enable viruses to shape their survival rates and fitness toward the external environment and, most importantly, their hosts. To understand the evolution of MARV at the codon level, we report a comprehensive analysis of synonymous codon usage patterns in MARV genomes. Multiple codon analysis approaches and statistical methods were performed to determine overall codon usage patterns, biases in codon usage, and influence of various factors, including mutation pressure, natural selection, and its two hosts, Homo sapiens and Rousettus aegyptiacus. Nucleotide composition and relative synonymous codon usage (RSCU) analysis revealed that MARV shows mutation bias and prefers U- and A-ended codons to code amino acids. Effective number of codons analysis indicated that overall codon usage among MARV genomes is slightly biased. The Parity Rule 2 plot analysis showed that GC and AU nucleotides were not used proportionally which accounts for the presence of natural selection. Codon usage patterns of MARV were also found to be influenced by its hosts. This indicates that MARV have evolved codon usage patterns that are specific to both of its hosts. Moreover, selection pressure from R. aegyptiacus on the MARV RSCU patterns was found to be dominant compared with that from H. sapiens. Overall, mutation pressure was found to be the most important and dominant force that shapes codon usage patterns in MARV. To our knowledge, this is the first detailed codon usage analysis of MARV and extends our understanding of the mechanisms that contribute to codon usage and evolution of MARV.

Transcriptome Analysis of Core Dinoflagellates Reveals a Universal Bias towards “GC” Rich Codons

PubMed Central

Williams, Ernest; Place, Allen; Bachvaroff, Tsvetan

2017-01-01

Although dinoflagellates are a potential source of pharmaceuticals and natural products, the mechanisms for regulating and producing these compounds are largely unknown because of extensive post-transcriptional control of gene expression. One well-documented mechanism for controlling gene expression during translation is codon bias, whereby specific codons slow or even terminate protein synthesis. Approximately 10,000 annotatable genes from fifteen “core” dinoflagellate transcriptomes along a range of overall guanine and cytosine (GC) content were used for codonW analysis to determine the relative synonymous codon usage (RSCU) and the GC content at each codon position. GC bias in the analyzed dataset and at the third codon position varied from 51% and 54% to 66% and 88%, respectively. Codons poor in GC were observed to be universally absent, but bias was most pronounced for codons ending in uracil followed by adenine (UA). GC bias at the third codon position was able to explain low abundance codons as well as the low effective number of codons. Thus, we propose that a bias towards codons rich in GC bases is a universal feature of core dinoflagellates, possibly relating to their unique chromosome structure, and not likely a major mechanism for controlling gene expression. PMID:28448468

Neutropenia-associated ELANE mutations disrupting translation initiation produce novel neutrophil elastase isoforms

PubMed Central

Tidwell, Timothy; Wechsler, Jeremy; Nayak, Ramesh C.; Trump, Lisa; Salipante, Stephen J.; Cheng, Jerry C.; Donadieu, Jean; Glaubach, Taly; Corey, Seth J.; Grimes, H. Leighton; Lutzko, Carolyn; Cancelas, Jose A.

2014-01-01

Hereditary neutropenia is usually caused by heterozygous germline mutations in the ELANE gene encoding neutrophil elastase (NE). How mutations cause disease remains uncertain, but two hypotheses have been proposed. In one, ELANE mutations lead to mislocalization of NE. In the other, ELANE mutations disturb protein folding, inducing an unfolded protein response in the endoplasmic reticulum (ER). In this study, we describe new types of mutations that disrupt the translational start site. At first glance, they should block translation and are incompatible with either the mislocalization or misfolding hypotheses, which require mutant protein for pathogenicity. We find that start-site mutations, instead, force translation from downstream in-frame initiation codons, yielding amino-terminally truncated isoforms lacking ER-localizing (pre) and zymogen-maintaining (pro) sequences, yet retain essential catalytic residues. Patient-derived induced pluripotent stem cells recapitulate hematopoietic and molecular phenotypes. Expression of the amino-terminally deleted isoforms in vitro reduces myeloid cell clonogenic capacity. We define an internal ribosome entry site (IRES) within ELANE and demonstrate that adjacent mutations modulate IRES activity, independently of protein-coding sequence alterations. Some ELANE mutations, therefore, appear to cause neutropenia via the production of amino-terminally deleted NE isoforms rather than by altering the coding sequence of the full-length protein. PMID:24184683

Characterization of the porcine epidemic diarrhea virus codon usage bias.

PubMed

Chen, Ye; Shi, Yuzhen; Deng, Hongjuan; Gu, Ting; Xu, Jian; Ou, Jinxin; Jiang, Zhiguo; Jiao, Yiren; Zou, Tan; Wang, Chong

2014-12-01

Porcine epidemic diarrhea virus (PEDV) has been responsible for several recent outbreaks of porcine epidemic diarrhea (PED) and has caused great economic loss in the swine-raising industry. Considering the significance of PEDV, a systemic analysis was performed to study its codon usage patterns. The relative synonymous codon usage value of each codon revealed that codon usage bias exists and that PEDV tends to use codons that end in T. The mean ENC value of 47.91 indicates that the codon usage bias is low. However, we still wanted to identify the cause of this codon usage bias. A correlation analysis between the codon compositions (A3s, T3s, G3s, C3s, and GC3s), the ENC values, and the nucleotide contents (A%, T%, G%, C%, and GC%) indicated that mutational bias plays role in shaping the PEDV codon usage bias. This was further confirmed by a principal component analysis between the codon compositions and the axis values. Using the Gravy, Aroma, and CAI values, a role of natural selection in the PEDV codon usage pattern was also identified. Neutral analysis indicated that natural selection pressure plays a more important role than mutational bias in codon usage bias. Natural selection also plays an increasingly significant role during PEDV evolution. Additionally, gene function and geographic distribution also influence the codon usage bias to a degree. Copyright © 2014 Elsevier B.V. All rights reserved.

Mutations in the Treacher Collins syndrome gene lead to mislocalization of the nucleolar protein treacle.

PubMed

Marsh, K L; Dixon, J; Dixon, M J

1998-10-01

Treacher Collins syndrome (TCS) is an autosomal dominant disorder of craniofacial development, the features of which include conductive hearing loss and cleft palate. The TCS gene ( TCOF1 ), which is localized to chromosome 5q32-q33.1, recently has been identified by positional cloning. Analysis of TCOF1 revealed that the majority of TCS mutations result in the creation of a premature termination codon. The function of the predicted protein, treacle, is unknown, although indirect evidence from database analyses suggests that it may function as a shuttling nucleolar phosphoprotein. In the current study, we provide the first direct evidence that treacle is a nucleolar protein. An antibody generated against treacle shows that it localizes to the nucleolus. Fusion proteins tagged to a green fluorescent protein reporter were shown to localize to different compartments of the cell when putative nuclear localization signals were deleted. Parallel experiments using conserved regions of the murine homologue of TCOF1 confirmed these results. Site-directed mutagenesis has been used to recreate mutations observed in individuals with TCS. The resulting truncated proteins are mislocalized within the cell, which further supports the hypothesis that an integral part of treacle's function involves shuttling between the nucleolus and the cytoplasm. TCS is, therefore, the first Mendelian disorder resulting from mutations which lead to aberrant expression of a nucleolar protein.

Genome-wide analysis of codon usage bias in Ebolavirus.

PubMed

Cristina, Juan; Moreno, Pilar; Moratorio, Gonzalo; Musto, Héctor

2015-01-22

Ebola virus (EBOV) is a member of the family Filoviridae and its genome consists of a 19-kb, single-stranded, negative sense RNA. EBOV is subdivided into five distinct species with different pathogenicities, being Zaire ebolavirus (ZEBOV) the most lethal species. The interplay of codon usage among viruses and their hosts is expected to affect overall viral survival, fitness, evasion from host's immune system and evolution. In the present study, we performed comprehensive analyses of codon usage and composition of ZEBOV. Effective number of codons (ENC) indicates that the overall codon usage among ZEBOV strains is slightly biased. Different codon preferences in ZEBOV genes in relation to codon usage of human genes were found. Highly preferred codons are all A-ending triplets, which strongly suggests that mutational bias is a main force shaping codon usage in ZEBOV. Dinucleotide composition also plays a role in the overall pattern of ZEBOV codon usage. ZEBOV does not seem to use the most abundant tRNAs present in the human cells for most of their preferred codons. Copyright © 2014 Elsevier B.V. All rights reserved.

Minigene-like inhibition of protein synthesis mediated by hungry codons near the start codon

PubMed Central

Jacinto-Loeza, Eva; Vivanco-Domínguez, Serafín; Guarneros, Gabriel; Hernández-Sánchez, Javier

2008-01-01

Rare AGA or AGG codons close to the initiation codon inhibit protein synthesis by a tRNA-sequestering mechanism as toxic minigenes do. To further understand this mechanism, a parallel analysis of protein synthesis and peptidyl-tRNA accumulation was performed using both a set of lacZ constructs where AGAAGA codons were moved codon by codon from +2, +3 up to +7, +8 positions and a series of 3–8 codon minigenes containing AGAAGA codons before the stop codon. β-Galactosidase synthesis from the AGAAGA lacZ constructs (in a Pth defective in vitro system without exogenous tRNA) diminished as the AGAAGA codons were closer to AUG codon. Likewise, β-galactosidase expression from the reporter +7 AGA lacZ gene (plus tRNA, 0.25 μg/μl) waned as the AGAAGAUAA minigene shortened. Pth counteracted both the length-dependent minigene effect on the expression of β-galactosidase from the +7 AGA lacZ reporter gene and the positional effect from the AGAAGA lacZ constructs. The +2, +3 AGAAGA lacZ construct and the shortest +2, +3 AGAAGAUAA minigene accumulated the highest percentage of peptidyl-tRNAArg4. These observations lead us to propose that hungry codons at early positions, albeit with less strength, inhibit protein synthesis by a minigene-like mechanism involving accumulation of peptidyl-tRNA. PMID:18583364

Synonymous codon usage of genes in polymerase complex of Newcastle disease virus.

PubMed

Kumar, Chandra Shekhar; Kumar, Sachin

2017-06-01

Newcastle disease virus (NDV) is pathogenic to both avian and non-avian species but extensively finds poultry as its primary host and causes heavy economic losses in the poultry industry. In this study, a total of 186 polymerase complex comprising of nucleoprotein (N), phosphoprotein (P), and large polymerase (L) genes of NDV was analyzed for synonymous codon usage. The relative synonymous codon usage and effective number of codons (ENC) values were used to estimate codon usage variation in each gene. Correspondence analysis (COA) was used to study the major trend in codon usage variation. Analyzing the ENC plot values against GC3s (at synonymous third codon position) we concluded that mutational pressure was the main factor determining codon usage bias than translational selection in NDV N, P, and L genes. Moreover, correlation analysis indicated, that aromaticity of N, P, and L genes also influenced the codon usage variation. The varied distribution of pathotypes for N, P, and L gene clearly suggests that change in codon usage for NDV is pathotype specific. The codon usage preference similarity in N, P, and L gene might be detrimental for polymerase complex functioning. The study represents a comprehensive analysis to date of N, P, and L genes codon usage pattern of NDV and provides a basic understanding of the mechanisms for codon usage bias. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evolution of Synonymous Codon Usage in Neurospora tetrasperma and Neurospora discreta

PubMed Central

Whittle, C. A.; Sun, Y.; Johannesson, H.

2011-01-01

Neurospora comprises a primary model system for the study of fungal genetics and biology. In spite of this, little is known about genome evolution in Neurospora. For example, the evolution of synonymous codon usage is largely unknown in this genus. In the present investigation, we conducted a comprehensive analysis of synonymous codon usage and its relationship to gene expression and gene length (GL) in Neurospora tetrasperma and Neurospora discreta. For our analysis, we examined codon usage among 2,079 genes per organism and assessed gene expression using large-scale expressed sequenced tag (EST) data sets (279,323 and 453,559 ESTs for N. tetrasperma and N. discreta, respectively). Data on relative synonymous codon usage revealed 24 codons (and two putative codons) that are more frequently used in genes with high than with low expression and thus were defined as optimal codons. Although codon-usage bias was highly correlated with gene expression, it was independent of selectively neutral base composition (introns); thus demonstrating that translational selection drives synonymous codon usage in these genomes. We also report that GL (coding sequences [CDS]) was inversely associated with optimal codon usage at each gene expression level, with highly expressed short genes having the greatest frequency of optimal codons. Optimal codon frequency was moderately higher in N. tetrasperma than in N. discreta, which might be due to variation in selective pressures and/or mating systems. PMID:21402862

Codon usage bias in phylum Actinobacteria: relevance to environmental adaptation and host pathogenicity.

PubMed

Lal, Devi; Verma, Mansi; Behura, Susanta K; Lal, Rup

2016-10-01

Actinobacteria are Gram-positive bacteria commonly found in soil, freshwater and marine ecosystems. In this investigation, bias in codon usages of ninety actinobacterial genomes was analyzed by estimating different indices of codon bias such as Nc (effective number of codons), SCUO (synonymous codon usage order), RSCU (relative synonymous codon usage), as well as sequence patterns of codon contexts. The results revealed several characteristic features of codon usage in Actinobacteria, as follows: 1) C- or G-ending codons are used frequently in comparison with A- and U ending codons; 2) there is a direct relationship of GC content with use of specific amino acids such as alanine, proline and glycine; 3) there is an inverse relationship between GC content and Nc estimates, 4) there is low SCUO value (<0.5) for most genes; and 5) GCC-GCC, GCC-GGC, GCC-GAG and CUC-GAC are the frequent context sequences among codons. This study highlights the fact that: 1) in Actinobacteria, extreme GC content and codon bias are driven by mutation rather than natural selection; (2) traits like aerobicity are associated with effective natural selection and therefore low GC content and low codon bias, demonstrating the role of both mutational bias and translational selection in shaping the habitat and phenotype of actinobacterial species. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Electrochemical studies of a truncated laccase produced in Pichia pastoris

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gelo-Pujic, M.; Kim, H.H.; Butlin, N.G.

1999-12-01

The cDNA that encodes an isoform is laccase from Trametes versicolor (LCCI), as well as a truncated version (LCCIa), was subcloned and expressed by using the yeast Pichia pastoris as the heterologous host. The amino acid sequence of LCCIa is identical to that of LCCI except that the final 11 amino acids at the C terminus of LCCI are replaced with a single cysteine residue. This modification was introduced for the purpose of improving the kinetics of electron transfer between an electrode and the copper-containing active site of laccase. The two laccases (LCCI and LCCIa) are compared in terms ofmore » their relative activity with two substrates that have different redox potentials. Results from electrochemical studies on solutions containing LCCI and LCCIa indicate that the redox potential of the active site of LCCIa is shifted to more negative values (411 mV versus normal hydrogen electrode voltage) than that found in other fungal laccases. In addition, replacing the 11 codons at the C terminus of the laccase gene with a single cysteine codon influences the rate of heterogeneous electron transfer between and electrode and the copper-containing active site. These results demonstrate for the first time that the rate of electron transfer between an oxidoreductase and an electrode can be enhanced by changes to the primary structure of a protein via site-directed mutagenesis.« less

Molecular mimicry of human tRNALys anti-codon domain by HIV-1 RNA genome facilitates tRNA primer annealing.

PubMed

Jones, Christopher P; Saadatmand, Jenan; Kleiman, Lawrence; Musier-Forsyth, Karin

2013-02-01

The primer for initiating reverse transcription in human immunodeficiency virus type 1 (HIV-1) is tRNA(Lys3). Host cell tRNA(Lys) is selectively packaged into HIV-1 through a specific interaction between the major tRNA(Lys)-binding protein, human lysyl-tRNA synthetase (hLysRS), and the viral proteins Gag and GagPol. Annealing of the tRNA primer onto the complementary primer-binding site (PBS) in viral RNA is mediated by the nucleocapsid domain of Gag. The mechanism by which tRNA(Lys3) is targeted to the PBS and released from hLysRS prior to annealing is unknown. Here, we show that hLysRS specifically binds to a tRNA anti-codon-like element (TLE) in the HIV-1 genome, which mimics the anti-codon loop of tRNA(Lys) and is located proximal to the PBS. Mutation of the U-rich sequence within the TLE attenuates binding of hLysRS in vitro and reduces the amount of annealed tRNA(Lys3) in virions. Thus, LysRS binds specifically to the TLE, which is part of a larger LysRS binding domain in the viral RNA that includes elements of the Psi packaging signal. Our results suggest that HIV-1 uses molecular mimicry of the anti-codon of tRNA(Lys) to increase the efficiency of tRNA(Lys3) annealing to viral RNA.

Molecular evolution of the enzymes involved in the sphingolipid metabolism of Leishmania: selection pressure in relation to functional divergence and conservation.

PubMed

Mandlik, Vineetha; Shinde, Sonali; Singh, Shailza

2014-06-21

Selection pressure governs the relative mutability and the conservedness of a protein across the protein family. Biomolecules (DNA, RNA and proteins) continuously evolve under the effect of evolutionary pressure that arises as a consequence of the host parasite interaction. IPCS (Inositol phosphorylceramide synthase), SPL (Sphingosine-1-P lyase) and SPT (Serine palmitoyl transferase) represent three important enzymes involved in the sphingolipid metabolism of Leishmania. These enzymes are responsible for maintaining the viability and infectivity of the parasite and have been classified as druggable targets in the parasite metabolome. The present work relates to the role of selection pressure deciding functional conservedness and divergence of the drug targets. IPCS and SPL protein families appear to diverge from the SPT family. The three protein families were largely under the influence of purifying selection and were moderately conserved baring two residues in the IPCS protein which were under the influence of positive selection. To further explore the selection pressure at the codon level, codon usage bias indices were calculated to analyze genes for their synonymous codon usage pattern. IPCS gene exhibited slightly lower codon bias as compared to SPL and SPT protein families. Evolutionary tracing of the proposed drug targets has been done with a viewpoint that the amino-acids lining the drug binding pocket should have a lower evolvability. Sites under positive selection (HIS20 and CYS30 of IPCS) should be avoided during devising strategies for inhibitor design.

Molecular mimicry of human tRNALys anti-codon domain by HIV-1 RNA genome facilitates tRNA primer annealing

PubMed Central

Jones, Christopher P.; Saadatmand, Jenan; Kleiman, Lawrence; Musier-Forsyth, Karin

2013-01-01

The primer for initiating reverse transcription in human immunodeficiency virus type 1 (HIV-1) is tRNALys3. Host cell tRNALys is selectively packaged into HIV-1 through a specific interaction between the major tRNALys-binding protein, human lysyl-tRNA synthetase (hLysRS), and the viral proteins Gag and GagPol. Annealing of the tRNA primer onto the complementary primer-binding site (PBS) in viral RNA is mediated by the nucleocapsid domain of Gag. The mechanism by which tRNALys3 is targeted to the PBS and released from hLysRS prior to annealing is unknown. Here, we show that hLysRS specifically binds to a tRNA anti-codon-like element (TLE) in the HIV-1 genome, which mimics the anti-codon loop of tRNALys and is located proximal to the PBS. Mutation of the U-rich sequence within the TLE attenuates binding of hLysRS in vitro and reduces the amount of annealed tRNALys3 in virions. Thus, LysRS binds specifically to the TLE, which is part of a larger LysRS binding domain in the viral RNA that includes elements of the Psi packaging signal. Our results suggest that HIV-1 uses molecular mimicry of the anti-codon of tRNALys to increase the efficiency of tRNALys3 annealing to viral RNA. PMID:23264568

Analysis of transcriptome data reveals multifactor constraint on codon usage in Taenia multiceps.

PubMed

Huang, Xing; Xu, Jing; Chen, Lin; Wang, Yu; Gu, Xiaobin; Peng, Xuerong; Yang, Guangyou

2017-04-20

Codon usage bias (CUB) is an important evolutionary feature in genomes that has been widely observed in many organisms. However, the synonymous codon usage pattern in the genome of T. multiceps remains to be clarified. In this study, we analyzed the codon usage of T. multiceps based on the transcriptome data to reveal the constraint factors and to gain an improved understanding of the mechanisms that shape synonymous CUB. Analysis of a total of 8,620 annotated mRNA sequences from T. multiceps indicated only a weak codon bias, with mean GC and GC3 content values of 49.29% and 51.43%, respectively. Our analysis indicated that nucleotide composition, mutational pressure, natural selection, gene expression level, amino acids with grand average of hydropathicity (GRAVY) and aromaticity (Aromo) and the effective selection of amino-acids all contributed to the codon usage in T. multiceps. Among these factors, natural selection was implicated as the major factor affecting the codon usage variation in T. multiceps. The codon usage of ribosome genes was affected mainly by mutations, while the essential genes were affected mainly by selection. In addition, 21codons were identified as "optimal codons". Overall, the optimal codons were GC-rich (GC:AU, 41:22), and ended with G or C (except CGU). Furthermore, different degrees of variation in codon usage were found between T. multiceps and Escherichia coli, yeast, Homo sapiens. However, little difference was found between T. multiceps and Taenia pisiformis. In this study, the codon usage pattern of T. multiceps was analyzed systematically and factors affected CUB were also identified. This is the first study of codon biology in T. multiceps. Understanding the codon usage pattern in T. multiceps can be helpful for the discovery of new genes, molecular genetic engineering and evolutionary studies.

Codon usage patterns in Nematoda: analysis based on over 25 million codons in thirty-two species

PubMed Central

2006-01-01

Background Codon usage has direct utility in molecular characterization of species and is also a marker for molecular evolution. To understand codon usage within the diverse phylum Nematoda, we analyzed a total of 265,494 expressed sequence tags (ESTs) from 30 nematode species. The full genomes of Caenorhabditis elegans and C. briggsae were also examined. A total of 25,871,325 codons were analyzed and a comprehensive codon usage table for all species was generated. This is the first codon usage table available for 24 of these organisms. Results Codon usage similarity in Nematoda usually persists over the breadth of a genus but then rapidly diminishes even within each clade. Globodera, Meloidogyne, Pristionchus, and Strongyloides have the most highly derived patterns of codon usage. The major factor affecting differences in codon usage between species is the coding sequence GC content, which varies in nematodes from 32% to 51%. Coding GC content (measured as GC3) also explains much of the observed variation in the effective number of codons (R = 0.70), which is a measure of codon bias, and it even accounts for differences in amino acid frequency. Codon usage is also affected by neighboring nucleotides (N1 context). Coding GC content correlates strongly with estimated noncoding genomic GC content (R = 0.92). On examining abundant clusters in five species, candidate optimal codons were identified that may be preferred in highly expressed transcripts. Conclusion Evolutionary models indicate that total genomic GC content, probably the product of directional mutation pressure, drives codon usage rather than the converse, a conclusion that is supported by examination of nematode genomes. PMID:26271136

Molecular evolution of ependymin and the phylogenetic resolution of early divergences among euteleost fishes.

PubMed

Ortí, G; Meyer, A

1996-04-01

The rate and pattern of DNA evolution of ependymin, a single-copy gene coding for a highly expressed glycoprotein in the brain matrix of teleost fishes, is characterized and its phylogenetic utility for fish systematics is assessed. DNA sequences were determined from catfish, electric fish, and characiforms and compared with published ependymin sequences from cyprinids, salmon, pike, and herring. Among these groups, ependymin amino acid sequences were highly divergent (up to 60% sequence difference), but had surprisingly similar hydropathy profiles and invariant glycosylation sites, suggesting that functional properties of the proteins are conserved. Comparison of base composition at third codon positions and introns revealed AT-rich introns and GC-rich third codon positions, suggesting that the biased codon usage observed might not be due to mutational bias. Phylogenetic information content of third codon positions was surprisingly high and sufficient to recover the most basal nodes of the tree, in spite of the observation that pairwise distances (at third codon positions) were well above the presumed saturation level. This finding can be explained by the high proportion of phylogenetically informative nonsynonymous changes at third codon positions among these highly divergent proteins. Ependymin DNA sequences have established the first molecular evidence for the monophyly of a group containing salmonids and esociforms. In addition, ependymin suggests a sister group relationship of electric fish (Gymnotiformes) and Characiformes, constituting a significant departure from currently accepted classifications. However, relationships among characiform lineages were not completely resolved by ependymin sequences in spite of seemingly appropriate levels of variation among taxa and considerably low levels of homoplasy in the data (consistency index = 0.7). If the diversification of Characiformes took place in an "explosive" manner, over a relatively short period of time this pattern should also be observed using other phylogenetic markers. Poor conservation of ependymin's primary structure hinders the design of efficient primers for PCR that could be used in wide-ranging fish systematic studies. However, alternative methods like PCR amplification from cDNA used here should provide promising comparative sequence data for the resolution of phylogenetic relationships among other basal lineages of teleost fishes.

Thermostable proteins bioprocesses: The activity of restriction endonuclease-methyltransferase from Thermus thermophilus (RM.TthHB27I) cloned in Escherichia coli is critically affected by the codon composition of the synthetic gene.

PubMed

Krefft, Daria; Papkov, Aliaksei; Zylicz-Stachula, Agnieszka; Skowron, Piotr M

2017-01-01

Obtaining thermostable enzymes (thermozymes) is an important aspect of biotechnology. As thermophiles have adapted their genomes to high temperatures, their cloned genes' expression in mesophiles is problematic. This is mainly due to their high GC content, which leads to the formation of unfavorable secondary mRNA structures and codon usage in Escherichia coli (E. coli). RM.TthHB27I is a member of a family of bifunctional thermozymes, containing a restriction endonuclease (REase) and a methyltransferase (MTase) in a single polypeptide. Thermus thermophilus HB27 (T. thermophilus) produces low amounts of RM.TthHB27I with a unique DNA cleavage specificity. We have previously cloned the wild type (wt) gene into E. coli, which increased the production of RM.TthHB27I over 100-fold. However, its enzymatic activities were extremely low for an ORF expressed under a T7 promoter. We have designed and cloned a fully synthetic tthHB27IRM gene, using a modified 'codon randomization' strategy. Codons with a high GC content and of low occurrence in E. coli were eliminated. We incorporated a stem-loop circuit, devised to negatively control the expression of this highly toxic gene by partially hiding the ribosome-binding site (RBS) and START codon in mRNA secondary structures. Despite having optimized 59% of codons, the amount of produced RM.TthHB27I protein was similar for both recombinant tthHB27IRM gene variants. Moreover, the recombinant wt RM.TthHB27I is very unstable, while the RM.TthHB27I resulting from the expression of the synthetic gene exhibited enzymatic activities and stability equal to the native thermozyme isolated from T. thermophilus. Thus, we have developed an efficient purification protocol using the synthetic tthHB27IRM gene variant only. This suggests the effect of co-translational folding kinetics, possibly affected by the frequency of translational errors. The availability of active RM.TthHB27I is of practical importance in molecular biotechnology, extending the palette of available REase specificities.

A detailed analysis of codon usage patterns and influencing factors in Zika virus.

PubMed

Singh, Niraj K; Tyagi, Anuj

2017-07-01

Recent outbreaks of Zika virus (ZIKV) in Africa, Latin America, Europe, and Southeast Asia have resulted in serious health concerns. To understand more about evolution and transmission of ZIKV, detailed codon usage analysis was performed for all available strains. A high effective number of codons (ENC) value indicated the presence of low codon usage bias in ZIKV. The effect of mutational pressure on codon usage bias was confirmed by significant correlations between nucleotide compositions at third codon positions and ENCs. Correlation analysis between Gravy values, Aroma values and nucleotide compositions at third codon positions also indicated some influence of natural selection. However, the low codon adaptation index (CAI) value of ZIKV with reference to human and mosquito indicated poor adaptation of ZIKV codon usage towards its hosts, signifying that natural selection has a weaker influence than mutational pressure. Additionally, relative dinucleotide frequencies, geographical distribution, and evolutionary processes also influenced the codon usage pattern to some extent.

Structure of the human gene encoding the protein repair L-isoaspartyl (D-aspartyl) O-methyltransferase.

PubMed

DeVry, C G; Tsai, W; Clarke, S

1996-11-15

The protein L-isoaspartyl/D-aspartyl O-methyltransferase (EC 2.1.1.77) catalyzes the first step in the repair of proteins damaged in the aging process by isomerization or racemization reactions at aspartyl and asparaginyl residues. A single gene has been localized to human chromosome 6 and multiple transcripts arising through alternative splicing have been identified. Restriction enzyme mapping, subcloning, and DNA sequence analysis of three overlapping clones from a human genomic library in bacteriophage P1 indicate that the gene spans approximately 60 kb and is composed of 8 exons interrupted by 7 introns. Analysis of intron/exon splice junctions reveals that all of the donor and acceptor splice sites are in agreement with the mammalian consensus splicing sequence. Determination of transcription initiation sites by primer extension analysis of poly(A)+ mRNA from human brain identifies multiple start sites, with a major site 159 nucleotides upstream from the ATG start codon. Sequence analysis of the 5'-untranslated region demonstrates several potential cis-acting DNA elements including SP1, ETF, AP1, AP2, ARE, XRE, CREB, MED-1, and half-palindromic ERE motifs. The promoter of this methyltransferase gene lacks an identifiable TATA box but is characterized by a CpG island which begins approximately 723 nucleotides upstream of the major transcriptional start site and extends through exon 1 and into the first intron. These features are characteristic of housekeeping genes and are consistent with the wide tissue distribution observed for this methyltransferase activity.

Biased Gene Conversion and GC-Content Evolution in the Coding Sequences of Reptiles and Vertebrates

PubMed Central

Figuet, Emeric; Ballenghien, Marion; Romiguier, Jonathan; Galtier, Nicolas

2015-01-01

Mammalian and avian genomes are characterized by a substantial spatial heterogeneity of GC-content, which is often interpreted as reflecting the effect of local GC-biased gene conversion (gBGC), a meiotic repair bias that favors G and C over A and T alleles in high-recombining genomic regions. Surprisingly, the first fully sequenced nonavian sauropsid (i.e., reptile), the green anole Anolis carolinensis, revealed a highly homogeneous genomic GC-content landscape, suggesting the possibility that gBGC might not be at work in this lineage. Here, we analyze GC-content evolution at third-codon positions (GC3) in 44 vertebrates species, including eight newly sequenced transcriptomes, with a specific focus on nonavian sauropsids. We report that reptiles, including the green anole, have a genome-wide distribution of GC3 similar to that of mammals and birds, and we infer a strong GC3-heterogeneity to be already present in the tetrapod ancestor. We further show that the dynamic of coding sequence GC-content is largely governed by karyotypic features in vertebrates, notably in the green anole, in agreement with the gBGC hypothesis. The discrepancy between third-codon positions and noncoding DNA regarding GC-content dynamics in the green anole could not be explained by the activity of transposable elements or selection on codon usage. This analysis highlights the unique value of third-codon positions as an insertion/deletion-free marker of nucleotide substitution biases that ultimately affect the evolution of proteins. PMID:25527834

Novel BRCA1 splice-site mutation in ovarian cancer patients of Slavic origin.

PubMed

Krivokuca, Ana; Dragos, Vita Setrajcic; Stamatovic, Ljiljana; Blatnik, Ana; Boljevic, Ivana; Stegel, Vida; Rakobradovic, Jelena; Skerl, Petra; Jovandic, Stevo; Krajc, Mateja; Magic, Mirjana Brankovic; Novakovic, Srdjan

2018-04-01

Mutations in breast cancer susceptibility gene 1 (BRCA1) lead to defects in a number of cellular pathways including DNA damage repair and transcriptional regulation, resulting in the elevated genome instability and predisposing to breast and ovarian cancers. We report a novel mutation LRG_292t1:c.4356delA,p.(Ala1453Glnfs*3) in the 12th exon of BRCA1, in the splice site region near the donor site of intron 12. It is a frameshift mutation with the termination codon generated on the third amino acid position from the site of deletion. Human Splice Finder 3.0 and MutationTaster have assessed this variation as disease causing, based on the alteration of splicing, creation of premature stop codon and other potential alterations initiated by nucleotide deletion. Among the most important alterations are frameshift and splice site changes (score of the newly created donor splice site: 0.82). c.4356delA was associated with two ovarian cancer cases in two families of Slavic origin. It was detected by next generation sequencing, and confirmed with Sanger sequencing in both cases. Because of the fact that it changes the reading frame of the protein, novel mutation c.4356delA p.(Ala1453Glnfs*3) in BRCA1 gene might be of clinical significance for hereditary ovarian cancer. Further functional as well as segregation analyses within the families are necessary for appropriate clinical classification of this variant. Since it has been detected in two ovarian cancer patients of Slavic origin, it is worth investigating founder effect of this mutation in Slavic populations.

Dengue virus type 1 clade replacement in recurring homotypic outbreaks

PubMed Central

2013-01-01

Background Recurring dengue outbreaks occur in cyclical pattern in most endemic countries. The recurrences of dengue virus (DENV) infection predispose the population to increased risk of contracting the severe forms of dengue. Understanding the DENV evolutionary mechanism underlying the recurring dengue outbreaks has important implications for epidemic prediction and disease control. Results We used a set of viral envelope (E) gene to reconstruct the phylogeny of DENV-1 isolated between the periods of 1987–2011 in Malaysia. Phylogenetic analysis of DENV-1 E gene revealed that genotype I virus clade replacements were associated with the cyclical pattern of major DENV-1 outbreaks in Malaysia. A total of 9 non-conservative amino acid substitutions in the DENV-1 E gene consensus were identified; 4 in domain I, 3 in domain II and 2 in domain III. Selection pressure analyses did not reveal any positively selected codon site within the full length E gene sequences (1485 nt, 495 codons). A total of 183 (mean dN/dS = 0.0413) negatively selected sites were found within the Malaysian isolates; neither positive nor negative selection was noted for the remaining 312 codons. All the viruses were cross-neutralized by the respective patient sera suggesting no strong support for immunological advantage of any of the amino acid substitutions. Conclusion DENV-1 clade replacement is associated with recurrences of major DENV-1 outbreaks in Malaysia. Our findings are consistent with those of other studies that the DENV-1 clade replacement is a stochastic event independent of positive selection. PMID:24073945

Translational autocontrol of the Escherichia coli ribosomal protein S15.

PubMed

Portier, C; Dondon, L; Grunberg-Manago, M

1990-01-20

When rpsO, the gene encoding the ribosomal protein S15 in Escherichia coli, is carried by a multicopy plasmid, the mRNA synthesis rate of S15 increases with the gene dosage but the rate of synthesis of S15 does not rise. A translational fusion between S15 and beta-galactosidase was introduced on the chromosome in a delta lac strain and the expression of beta-galactosidase studied under different conditions. The presence of S15 in trans represses the beta-galactosidase level five- to sixfold, while the synthesis rate of the S15-beta-galactosidase mRNA decreases by only 30 to 50%. These data indicate that S15 is subject to autogenous translational control. Derepressed mutants were isolated and sequenced. All the point mutations map in the second codon of S15, suggesting a location for the operator site that is very near to the translation initiation codon. However, the creation of deletion mutations shows that the operator extends into the 5' non-coding part of the message, thus overlapping the ribosome loading site.

Optimizing Restriction Site Placement for Synthetic Genomes

NASA Astrophysics Data System (ADS)

Montes, Pablo; Memelli, Heraldo; Ward, Charles; Kim, Joondong; Mitchell, Joseph S. B.; Skiena, Steven

Restriction enzymes are the workhorses of molecular biology. We introduce a new problem that arises in the course of our project to design virus variants to serve as potential vaccines: we wish to modify virus-length genomes to introduce large numbers of unique restriction enzyme recognition sites while preserving wild-type function by substitution of synonymous codons. We show that the resulting problem is NP-Complete, give an exponential-time algorithm, and propose effective heuristics, which we show give excellent results for five sample viral genomes. Our resulting modified genomes have several times more unique restriction sites and reduce the maximum gap between adjacent sites by three to nine-fold.

A nucleolus-predominant piggyBac transposase, NP-mPB, mediates elevated transposition efficiency in mammalian cells.

PubMed

Hong, Jin-Bon; Chou, Fu-Ju; Ku, Amy T; Fan, Hsiang-Hsuan; Lee, Tung-Lung; Huang, Yung-Hsin; Yang, Tsung-Lin; Su, I-Chang; Yu, I-Shing; Lin, Shu-Wha; Chien, Chung-Liang; Ho, Hong-Nerng; Chen, You-Tzung

2014-01-01

PiggyBac is a prevalent transposon system used to deliver transgenes and functionally explore the mammalian untouched genomic territory. The important features of piggyBac transposon are the relatively low insertion site preference and the ability of seamless removal from genome, which allow its potential uses in functional genomics and regenerative medicine. Efforts to increase its transposition efficiency in mammals were made through engineering the corresponding transposase (PBase) codon usage to enhance its expression level and through screening for mutant PBase variants with increased enzyme activity. To improve the safety for its potential use in regenerative medicine applications, site-specific transposition was achieved by using engineered zinc finger- and Gal4-fused PBases. An excision-prone PBase variant has also been successfully developed. Here we describe the construction of a nucleolus-predominant PBase, NP-mPB, by adding a nucleolus-predominant (NP) signal peptide from HIV-1 TAT protein to a mammalian codon-optimized PBase (mPB). Although there is a predominant fraction of the NP-mPB-tGFP fusion proteins concentrated in the nucleoli, an insertion site preference toward nucleolar organizer regions is not detected. Instead a 3-4 fold increase in piggyBac transposition efficiency is reproducibly observed in mouse and human cells.

Genome-wide analysis of codon usage bias in four sequenced cotton species.

PubMed

Wang, Liyuan; Xing, Huixian; Yuan, Yanchao; Wang, Xianlin; Saeed, Muhammad; Tao, Jincai; Feng, Wei; Zhang, Guihua; Song, Xianliang; Sun, Xuezhen

2018-01-01

Codon usage bias (CUB) is an important evolutionary feature in a genome which provides important information for studying organism evolution, gene function and exogenous gene expression. The CUB and its shaping factors in the nuclear genomes of four sequenced cotton species, G. arboreum (A2), G. raimondii (D5), G. hirsutum (AD1) and G. barbadense (AD2) were analyzed in the present study. The effective number of codons (ENC) analysis showed the CUB was weak in these four species and the four subgenomes of the two tetraploids. Codon composition analysis revealed these four species preferred to use pyrimidine-rich codons more frequently than purine-rich codons. Correlation analysis indicated that the base content at the third position of codons affect the degree of codon preference. PR2-bias plot and ENC-plot analyses revealed that the CUB patterns in these genomes and subgenomes were influenced by combined effects of translational selection, directional mutation and other factors. The translational selection (P2) analysis results, together with the non-significant correlation between GC12 and GC3, further revealed that translational selection played the dominant role over mutation pressure in the codon usage bias. Through relative synonymous codon usage (RSCU) analysis, we detected 25 high frequency codons preferred to end with T or A, and 31 low frequency codons inclined to end with C or G in these four species and four subgenomes. Finally, 19 to 26 optimal codons with 19 common ones were determined for each species and subgenomes, which preferred to end with A or T. We concluded that the codon usage bias was weak and the translation selection was the main shaping factor in nuclear genes of these four cotton genomes and four subgenomes.

Polypyrimidine tract binding protein 1 protects mRNAs from recognition by the nonsense-mediated mRNA decay pathway

PubMed Central

Ge, Zhiyun; Quek, Bao Lin; Beemon, Karen L; Hogg, J Robert

2016-01-01

The nonsense-mediated mRNA decay (NMD) pathway degrades mRNAs containing long 3'UTRs to perform dual roles in mRNA quality control and gene expression regulation. However, expansion of vertebrate 3'UTR functions has required a physical expansion of 3'UTR lengths, complicating the process of detecting nonsense mutations. We show that the polypyrimidine tract binding protein 1 (PTBP1) shields specific retroviral and cellular transcripts from NMD. When bound near a stop codon, PTBP1 blocks the NMD protein UPF1 from binding 3'UTRs. PTBP1 can thus mark specific stop codons as genuine, preserving both the ability of NMD to accurately detect aberrant mRNAs and the capacity of long 3'UTRs to regulate gene expression. Illustrating the wide scope of this mechanism, we use RNA-seq and transcriptome-wide analysis of PTBP1 binding sites to show that many human mRNAs are protected by PTBP1 and that PTBP1 enrichment near stop codons correlates with 3'UTR length and resistance to NMD. DOI: http://dx.doi.org/10.7554/eLife.11155.001 PMID:26744779

Translation of the first upstream ORF in the hepatitis B virus pregenomic RNA modulates translation at the core and polymerase initiation codons

PubMed Central

Chen, Augustine; Kao, Y. F.; Brown, Chris M.

2005-01-01

The human hepatitis B virus (HBV) has a compact genome encoding four major overlapping coding regions: the core, polymerase, surface and X. The polymerase initiation codon is preceded by the partially overlapping core and four or more upstream initiation codons. There is evidence that several mechanisms are used to enable the synthesis of the polymerase protein, including leaky scanning and ribosome reinitiation. We have examined the first AUG in the pregenomic RNA, it precedes that of the core. It initiates an uncharacterized short upstream open reading frame (uORF), highly conserved in all HBV subtypes, we designated the C0 ORF. This arrangement suggested that expression of the core and polymerase may be affected by this uORF. Initiation at the C0 ORF was confirmed in reporter constructs in transfected cells. The C0 ORF had an inhibitory role in downstream expression from the core initiation site in HepG2 cells and in vitro, but also stimulated reinitiation at the polymerase start when in an optimal context. Our results indicate that the C0 ORF is a determinant in balancing the synthesis of the core and polymerase proteins. PMID:15731337

Sequence Polishing Library (SPL) v10.0

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oberortner, Ernst

The Sequence Polishing Library (SPL) is a suite of software tools in order to automate "Design for Synthesis and Assembly" workflows. Specifically: The SPL "Converter" tool converts files among the following sequence data exchange formats: CSV, FASTA, GenBank, and Synthetic Biology Open Language (SBOL); The SPL "Juggler" tool optimizes the codon usages of DNA coding sequences according to an optimization strategy, a user-specific codon usage table and genetic code. In addition, the SPL "Juggler" can translate amino acid sequences into DNA sequences.:The SPL "Polisher" verifies NA sequences against DNA synthesis constraints, such as GC content, repeating k-mers, and restriction sites.more » In case of violations, the "Polisher" reports the violations in a comprehensive manner. The "Polisher" tool can also modify the violating regions according to an optimization strategy, a user-specific codon usage table and genetic code;The SPL "Partitioner" decomposes large DNA sequences into smaller building blocks with partial overlaps that enable an efficient assembly. The "Partitioner" enables the user to configure the characteristics of the overlaps, which are mostly determined by the utilized assembly protocol, such as length, GC content, or melting temperature.« less

Structural insights into eRF3 and stop codon recognition by eRF1

PubMed Central

Cheng, Zhihong; Saito, Kazuki; Pisarev, Andrey V.; Wada, Miki; Pisareva, Vera P.; Pestova, Tatyana V.; Gajda, Michal; Round, Adam; Kong, Chunguang; Lim, Mengkiat; Nakamura, Yoshikazu; Svergun, Dmitri I.; Ito, Koichi; Song, Haiwei

2009-01-01

Eukaryotic translation termination is mediated by two interacting release factors, eRF1 and eRF3, which act cooperatively to ensure efficient stop codon recognition and fast polypeptide release. The crystal structures of human and Schizosaccharomyces pombe full-length eRF1 in complex with eRF3 lacking the GTPase domain revealed details of the interaction between these two factors and marked conformational changes in eRF1 that occur upon binding to eRF3, leading eRF1 to resemble a tRNA molecule. Small-angle X-ray scattering analysis of the eRF1/eRF3/GTP complex suggested that eRF1's M domain contacts eRF3's GTPase domain. Consistently, mutation of Arg192, which is predicted to come in close contact with the switch regions of eRF3, revealed its important role for eRF1's stimulatory effect on eRF3's GTPase activity. An ATP molecule used as a crystallization additive was bound in eRF1's putative decoding area. Mutational analysis of the ATP-binding site shed light on the mechanism of stop codon recognition by eRF1. PMID:19417105

Codon 219 polymorphism of PRNP in healthy caucasians and Creutzfeldt-Jakob disease patients

DOE Office of Scientific and Technical Information (OSTI.GOV)

Petraroli, R.; Pocchiari, M.

1996-04-01

A number of point and insert mutations of the PrP gene (PRNP) have been linked to familial Creutzfeldt-Jakob disease (CJD) and Gerstmann-Straussler-Scheinker disease (GSS). Moreover, the methionine/valine homozygosity at the polymorphic codon 129 of PRNP may cause a predisposition to sporadic and iatrogenic CJD or may control the age at onset of familial cases carrying either the 144-bp insertion or codon 178, codon 198, and codon 210 pathogenic mutations in PRNP. In addition, the association of methionine or valine at codon 129 and the point mutation at codon 178 on the same allele seem to play an important role inmore » determining either fatal familial insomnia or CJD. However, it is noteworthy that a relationship between codon 129 polymorphism and accelerated pathogenesis (early age at onset or shorter duration of the disease) has not been seen in familial CJD patients with codon 200 mutation or in GSS patients with codon 102 mutation, arguing that other, as yet unidentified, gene products or environmental factors, or both, may influence the clinical expression of these diseases. 17 refs.« less

Exploring codon context bias for synthetic gene design of a thermostable invertase in Escherichia coli.

PubMed

Pek, Han Bin; Klement, Maximilian; Ang, Kok Siong; Chung, Bevan Kai-Sheng; Ow, Dave Siak-Wei; Lee, Dong-Yup

2015-01-01

Various isoforms of invertases from prokaryotes, fungi, and higher plants has been expressed in Escherichia coli, and codon optimisation is a widely-adopted strategy for improvement of heterologous enzyme expression. Successful synthetic gene design for recombinant protein expression can be done by matching its translational elongation rate against heterologous host organisms via codon optimization. Amongst the various design parameters considered for the gene synthesis, codon context bias has been relatively overlooked compared to individual codon usage which is commonly adopted in most of codon optimization tools. In addition, matching the rates of transcription and translation based on secondary structure may lead to enhanced protein folding. In this study, we evaluated codon context fitness as design criterion for improving the expression of thermostable invertase from Thermotoga maritima in Escherichia coli and explored the relevance of secondary structure regions for folding and expression. We designed three coding sequences by using (1) a commercial vendor optimized gene algorithm, (2) codon context for the whole gene, and (3) codon context based on the secondary structure regions. Then, the codon optimized sequences were transformed and expressed in E. coli. From the resultant enzyme activities and protein yield data, codon context fitness proved to have the highest activity as compared to the wild-type control and other criteria while secondary structure-based strategy is comparable to the control. Codon context bias was shown to be a relevant parameter for enhancing enzyme production in Escherichia coli by codon optimization. Thus, we can effectively design synthetic genes within heterologous host organisms using this criterion. Copyright © 2015 Elsevier Inc. All rights reserved.

The Effect of an Alternate Start Codon on Heterologous Expression of a PhoA Fusion Protein in Mycoplasma gallisepticum

PubMed Central

Panicker, Indu S.; Browning, Glenn F.; Markham, Philip F.

2015-01-01

While the genomes of many Mycoplasma species have been sequenced, there are no collated data on translational start codon usage, and the effects of alternate start codons on gene expression have not been studied. Analysis of the annotated genomes found that ATG was the most prevalent translational start codon among Mycoplasma spp. However in Mycoplasma gallisepticum a GTG start codon is commonly used in the vlhA multigene family, which encodes a highly abundant, phase variable lipoprotein adhesin. Therefore, the effect of this alternate start codon on expression of a reporter PhoA lipoprotein was examined in M. gallisepticum. Mutation of the start codon from ATG to GTG resulted in a 2.5 fold reduction in the level of transcription of the phoA reporter, but the level of PhoA activity in the transformants containing phoA with a GTG start codon was only 63% of that of the transformants with a phoA with an ATG start codon, suggesting that GTG was a more efficient translational initiation codon. The effect of swapping the translational start codon in phoA reporter gene expression was less in M. gallisepticum than has been seen previously in Escherichia coli or Bacillus subtilis, suggesting the process of translational initiation in mycoplasmas may have some significant differences from those used in other bacteria. This is the first study of translational start codon usage in mycoplasmas and the impact of the use of an alternate start codon on expression in these bacteria. PMID:26010086

A codon-optimized green fluorescent protein for live cell imaging in Zymoseptoria tritici☆

PubMed Central

Kilaru, S.; Schuster, M.; Studholme, D.; Soanes, D.; Lin, C.; Talbot, N.J.; Steinberg, G.

2015-01-01

Fluorescent proteins (FPs) are powerful tools to investigate intracellular dynamics and protein localization. Cytoplasmic expression of FPs in fungal pathogens allows greater insight into invasion strategies and the host-pathogen interaction. Detection of their fluorescent signal depends on the right combination of microscopic setup and signal brightness. Slow rates of photo-bleaching are pivotal for in vivo observation of FPs over longer periods of time. Here, we test green-fluorescent proteins, including Aequorea coerulescens GFP (AcGFP), enhanced GFP (eGFP) from Aequorea victoria and a novel Zymoseptoria tritici codon-optimized eGFP (ZtGFP), for their usage in conventional and laser-enhanced epi-fluorescence, and confocal laser-scanning microscopy. We show that eGFP, expressed cytoplasmically in Z. tritici, is significantly brighter and more photo-stable than AcGFP. The codon-optimized ZtGFP performed even better than eGFP, showing significantly slower bleaching and a 20–30% further increase in signal intensity. Heterologous expression of all GFP variants did not affect pathogenicity of Z. tritici. Our data establish ZtGFP as the GFP of choice to investigate intracellular protein dynamics in Z. tritici, but also infection stages of this wheat pathogen inside host tissue. PMID:26092799

The functional readthrough extension of malate dehydrogenase reveals a modification of the genetic code

PubMed Central

Hofhuis, Julia; Schueren, Fabian; Nötzel, Christopher; Lingner, Thomas; Gärtner, Jutta; Jahn, Olaf

2016-01-01

Translational readthrough gives rise to C-terminally extended proteins, thereby providing the cell with new protein isoforms. These may have different properties from the parental proteins if the extensions contain functional domains. While for most genes amino acid incorporation at the stop codon is far lower than 0.1%, about 4% of malate dehydrogenase (MDH1) is physiologically extended by translational readthrough and the actual ratio of MDH1x (extended protein) to ‘normal' MDH1 is dependent on the cell type. In human cells, arginine and tryptophan are co-encoded by the MDH1x UGA stop codon. Readthrough is controlled by the 7-nucleotide high-readthrough stop codon context without contribution of the subsequent 50 nucleotides encoding the extension. All vertebrate MDH1x is directed to peroxisomes via a hidden peroxisomal targeting signal (PTS) in the readthrough extension, which is more highly conserved than the extension of lactate dehydrogenase B. The hidden PTS of non-mammalian MDH1x evolved to be more efficient than the PTS of mammalian MDH1x. These results provide insight into the genetic and functional co-evolution of these dually localized dehydrogenases. PMID:27881739

Molecular cloning, structure, and chromosomal localization of the mouse LIM/homeobox gene Lhx5

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bertuzzi, S.; Sheng, Hui Z.; Westphal, H.

1996-09-01

Lhx5, the mouse ortholog of the Xenopus Xlim-5, is a LIM/homeobox gene expressed in the central nervous system during both embryonic development and adulthood. During development its domain of expression is mainly localized at the most anterior portion of the neural tube, and it precedes the morphological differentiation of the forebrain; for this reason we believe that Lhx5 could play an important role in forebrain patterning. Here we present the structural organization and the chromosomal localization of the Lhx5 gene. The gene is composed of five exons spanning more than 10 kb of genomic sequence. The first and second LIMmore » domains are encoded by the first and second exon, while the codons of the homeobox are split between the third and the fourth exons. The structure of Lhx5 is similar to that of other LIM/homeodomain proteins, Lxh1/lim1 and Lhx3/lim3, but differs from that of other LIM genes, such as mec3 and LMO1/Rbtn1, in which the codons for the LIM domains are interrupted by introns. We have mapped Lhx5 to the central region of mouse chromosome 5. 38 refs., 4 figs.« less

Phylogeny and evolution of Newcastle disease virus genotypes isolated in Asia during 2008-2011.

PubMed

Ebrahimi, Mohammad Majid; Shahsavandi, Shahla; Moazenijula, Gholamreza; Shamsara, Mahdi

2012-08-01

The full-length fusion (F) genes of 51 Newcastle disease (ND) strains isolated from chickens in Asia during the period 2008-2011 were genetically analyzed. Phylogenetic analysis showed that genotype VII of NDV still predominant in the domestic poultry of Asia. The sub-genotype VIIb circulated in the Iran and Indian sub-continent countries, whereas VIId sub-genotype existed in Far East countries. The non-synonymous to synonymous substitutions ratio was calculated 0.27 for VIId sub-genotype and 0.51 for VIIb sub-genotype indicates purifying/stabilizing selection which resulted in a low evolution rate in F gene of VIIb sub-genotype. There is evidence of localized positive selection when comparing these sub-genotypes protein sequences. Five codons in F gene of ND viruses had a posterior probability >90% using the Bayesian method, indicating these sites were under positive selection. To identify sites under positive selection; amino acid substitution classified depends on their radicalism and neutrality. The results indicate that although most positions were under purifying selection and can be eliminated, a few positions located in sub-genotype specific regions were subject to positive selection.

Generate Optimized Genetic Rhythm for Enzyme Expression in Non-native systems

DOE Office of Scientific and Technical Information (OSTI.GOV)

2016-11-03

Most amino acids are represented by more than one codon, resulting in redundancy in the genetic code. Silent codon substitutions that do not alter the amino acid sequence still have an effect on protein expression. We have developed an algorithm, GoGREEN, to enhance the expression of foreign proteins in a host organism. GoGREEN selects codons according to frequency patterns seen in the gene of interest using the codon usage table from the host organism. GoGREEN is also designed to accommodate gaps in the sequence.This software takes for input (1) the aligned protein sequences for genes the user wishes to express,more » (2) the codon usage table for the host organism, (3) and the DNA sequence for the target protein found in the host organism. The program will select codons based on codon usage patterns for the target DNA sequence. The program will also select codons for “gaps” found in the aligned protein sequences using the codon usage table from the host organism.« less

Genetic Variation of Goat Interferon Regulatory Factor 3 Gene and Its Implication in Goat Evolution

PubMed Central

Shu, Liping; Zhang, Yesheng; Wang, Yangzi; Sanni, Timothy M.; Imumorin, Ikhide G.; Peters, Sunday O.; Zhang, Jiajin; Dong, Yang; Wang, Wen

2016-01-01

The immune systems are fundamentally vital for evolution and survival of species; as such, selection patterns in innate immune loci are of special interest in molecular evolutionary research. The interferon regulatory factor (IRF) gene family control many different aspects of the innate and adaptive immune responses in vertebrates. Among these, IRF3 is known to take active part in very many biological processes. We assembled and evaluated 1356 base pairs of the IRF3 gene coding region in domesticated goats from Africa (Nigeria, Ethiopia and South Africa) and Asia (Iran and China) and the wild goat (Capra aegagrus). Five segregating sites with θ value of 0.0009 for this gene demonstrated a low diversity across the goats’ populations. Fu and Li tests were significantly positive but Tajima’s D test was significantly negative, suggesting its deviation from neutrality. Neighbor joining tree of IRF3 gene in domesticated goats, wild goat and sheep showed that all domesticated goats have a closer relationship than with the wild goat and sheep. Maximum likelihood tree of the gene showed that different domesticated goats share a common ancestor and suggest single origin. Four unique haplotypes were observed across all the sequences, of which, one was particularly common to African goats (MOCH-K14-0425, Poitou and WAD). In assessing the evolution mode of the gene, we found that the codon model dN/dS ratio for all goats was greater than one. Phylogenetic Analysis by Maximum Likelihood (PAML) gave a ω0 (dN/dS) value of 0.067 with LnL value of -6900.3 for the first Model (M1) while ω2 = 1.667 in model M2 with LnL value of -6900.3 with positive selection inferred in 3 codon sites. Mechanistic empirical combination (MEC) model for evaluating adaptive selection pressure on particular codons also confirmed adaptive selection pressure in three codons (207, 358 and 408) in IRF3 gene. Positive diversifying selection inferred with recent evolutionary changes in domesticated goat IRF3 led us to conclude that the gene evolution may have been influenced by domestication processes in goats. PMID:27598391

Genetic Variation of Goat Interferon Regulatory Factor 3 Gene and Its Implication in Goat Evolution.

PubMed

Okpeku, Moses; Esmailizadeh, Ali; Adeola, Adeniyi C; Shu, Liping; Zhang, Yesheng; Wang, Yangzi; Sanni, Timothy M; Imumorin, Ikhide G; Peters, Sunday O; Zhang, Jiajin; Dong, Yang; Wang, Wen

2016-01-01

The immune systems are fundamentally vital for evolution and survival of species; as such, selection patterns in innate immune loci are of special interest in molecular evolutionary research. The interferon regulatory factor (IRF) gene family control many different aspects of the innate and adaptive immune responses in vertebrates. Among these, IRF3 is known to take active part in very many biological processes. We assembled and evaluated 1356 base pairs of the IRF3 gene coding region in domesticated goats from Africa (Nigeria, Ethiopia and South Africa) and Asia (Iran and China) and the wild goat (Capra aegagrus). Five segregating sites with θ value of 0.0009 for this gene demonstrated a low diversity across the goats' populations. Fu and Li tests were significantly positive but Tajima's D test was significantly negative, suggesting its deviation from neutrality. Neighbor joining tree of IRF3 gene in domesticated goats, wild goat and sheep showed that all domesticated goats have a closer relationship than with the wild goat and sheep. Maximum likelihood tree of the gene showed that different domesticated goats share a common ancestor and suggest single origin. Four unique haplotypes were observed across all the sequences, of which, one was particularly common to African goats (MOCH-K14-0425, Poitou and WAD). In assessing the evolution mode of the gene, we found that the codon model dN/dS ratio for all goats was greater than one. Phylogenetic Analysis by Maximum Likelihood (PAML) gave a ω0 (dN/dS) value of 0.067 with LnL value of -6900.3 for the first Model (M1) while ω2 = 1.667 in model M2 with LnL value of -6900.3 with positive selection inferred in 3 codon sites. Mechanistic empirical combination (MEC) model for evaluating adaptive selection pressure on particular codons also confirmed adaptive selection pressure in three codons (207, 358 and 408) in IRF3 gene. Positive diversifying selection inferred with recent evolutionary changes in domesticated goat IRF3 led us to conclude that the gene evolution may have been influenced by domestication processes in goats.

Contribution of DNA Repair Xeroderma Pigmentosum Group D Genotype to Gastric Cancer Risk in Taiwan.

PubMed

Ji, Hong-Xue; Chang, Wen-Shin; Tsai, Chia-Wen; Wang, Ju-Yu; Huang, Nai-Kuei; Lee, An-Sheng; Shen, Ming-Yi; Chen, Wei-Yu; Chiang, Yao-Chang; Shih, Tzu-Ching; Hsu, Chin-Mu; Bau, Da-Tian

2015-09-01

It has been proposed that genetic variations of DNA repair genes confer susceptibility to cancer, and the DNA repair gene xeroderma pigmentosum group D (XPD), the caretaker of genome stability, is thought to play a major role in the nucleotide excision repair system. We investigated three genotypes of XPD, at promoter -114 (rs3810366), and codon 312 (rs1799793), 751 (rs13181), and their associated with gastric cancer susceptibility in a Taiwanese population. In the present study, 121 patients with gastric cancer and 363 gender- and age-matched healthy controls were recruited and genotyped for XPD by polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) methodology, and the association of XPD genotype with gastric cancer risk was investigated. We found a significant difference in the distribution of A allele-bearing XPD codon 312 genotypes [odds ratio (OR)=1.64, 95% confidence interval (CI)=1.20-2.25, p=0.0019], but not in XPD codon 751 or promoter -114 sites, between the gastric cancer and control groups. Those who had G/A or A/A at XPD codon 312 had a 1.83-fold (95% CI=1.14-2.95, p=0.0159) and 1.87-fold (95% CI=1.04-3.34, p=0.0378) increased risk of gastric cancer compared to those with G/G. The risk for G/A and A/A genotypes had synergistic effects with alcohol drinking (OR=11.27, 95% CI=3.72-34.17, p=0.0001), cigarette smoking (OR=23.20, 95% CI=6.24-86.23, p=0.0001) and Helicobacter pylori infection (OR=5.38, 95% CI=2.76-10.52, p=0.0001) on gastric cancer susceptibility. Our findings suggest that the A allele of XPD codon 312 may contribute to gastric carcinogenesis and may be useful for early detection and prevention of gastric cancer. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Human tRNA(Lys3)(UUU) is pre-structured by natural modifications for cognate and wobble codon binding through keto-enol tautomerism.

PubMed

Vendeix, Franck A P; Murphy, Frank V; Cantara, William A; Leszczyńska, Grażyna; Gustilo, Estella M; Sproat, Brian; Malkiewicz, Andrzej; Agris, Paul F

2012-03-02

Human tRNA(Lys3)(UUU) (htRNA(Lys3)(UUU)) decodes the lysine codons AAA and AAG during translation and also plays a crucial role as the primer for HIV-1 (human immunodeficiency virus type 1) reverse transcription. The posttranscriptional modifications 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U(34)), 2-methylthio-N(6)-threonylcarbamoyladenosine (ms(2)t(6)A(37)), and pseudouridine (Ψ(39)) in the tRNA's anticodon domain are critical for ribosomal binding and HIV-1 reverse transcription. To understand the importance of modified nucleoside contributions, we determined the structure and function of this tRNA's anticodon stem and loop (ASL) domain with these modifications at positions 34, 37, and 39, respectively (hASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37);Ψ(39)). Ribosome binding assays in vitro revealed that the hASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37);Ψ(39) bound AAA and AAG codons, whereas binding of the unmodified ASL(Lys3)(UUU) was barely detectable. The UV hyperchromicity, the circular dichroism, and the structural analyses indicated that Ψ(39) enhanced the thermodynamic stability of the ASL through base stacking while ms(2)t(6)A(37) restrained the anticodon to adopt an open loop conformation that is required for ribosomal binding. The NMR-restrained molecular-dynamics-derived solution structure revealed that the modifications provided an open, ordered loop for codon binding. The crystal structures of the hASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37);Ψ(39) bound to the 30S ribosomal subunit with each codon in the A site showed that the modified nucleotides mcm(5)s(2)U(34) and ms(2)t(6)A(37) participate in the stability of the anticodon-codon interaction. Importantly, the mcm(5)s(2)U(34)·G(3) wobble base pair is in the Watson-Crick geometry, requiring unusual hydrogen bonding to G in which mcm(5)s(2)U(34) must shift from the keto to the enol form. The results unambiguously demonstrate that modifications pre-structure the anticodon as a key prerequisite for efficient and accurate recognition of cognate and wobble codons. Copyright © 2011 Elsevier Ltd. All rights reserved.

Whole genome sequencing based characterization of extensively drug-resistant Mycobacterium tuberculosis isolates from Pakistan.

PubMed

Ali, Asho; Hasan, Zahra; McNerney, Ruth; Mallard, Kim; Hill-Cawthorne, Grant; Coll, Francesc; Nair, Mridul; Pain, Arnab; Clark, Taane G; Hasan, Rumina

2015-01-01

Improved molecular diagnostic methods for detection drug resistance in Mycobacterium tuberculosis (MTB) strains are required. Resistance to first- and second- line anti-tuberculous drugs has been associated with single nucleotide polymorphisms (SNPs) in particular genes. However, these SNPs can vary between MTB lineages therefore local data is required to describe different strain populations. We used whole genome sequencing (WGS) to characterize 37 extensively drug-resistant (XDR) MTB isolates from Pakistan and investigated 40 genes associated with drug resistance. Rifampicin resistance was attributable to SNPs in the rpoB hot-spot region. Isoniazid resistance was most commonly associated with the katG codon 315 (92%) mutation followed by inhA S94A (8%) however, one strain did not have SNPs in katG, inhA or oxyR-ahpC. All strains were pyrazimamide resistant but only 43% had pncA SNPs. Ethambutol resistant strains predominantly had embB codon 306 (62%) mutations, but additional SNPs at embB codons 406, 378 and 328 were also present. Fluoroquinolone resistance was associated with gyrA 91-94 codons in 81% of strains; four strains had only gyrB mutations, while others did not have SNPs in either gyrA or gyrB. Streptomycin resistant strains had mutations in ribosomal RNA genes; rpsL codon 43 (42%); rrs 500 region (16%), and gidB (34%) while six strains did not have mutations in any of these genes. Amikacin/kanamycin/capreomycin resistance was associated with SNPs in rrs at nt1401 (78%) and nt1484 (3%), except in seven (19%) strains. We estimate that if only the common hot-spot region targets of current commercial assays were used, the concordance between phenotypic and genotypic testing for these XDR strains would vary between rifampicin (100%), isoniazid (92%), flouroquinolones (81%), aminoglycoside (78%) and ethambutol (62%); while pncA sequencing would provide genotypic resistance in less than half the isolates. This work highlights the importance of expanded targets for drug resistance detection in MTB isolates.

Amino acid repeats avert mRNA folding through conservative substitutions and synonymous codons, regardless of codon bias.

PubMed

Barik, Sailen

2017-12-01

A significant number of proteins in all living species contains amino acid repeats (AARs) of various lengths and compositions, many of which play important roles in protein structure and function. Here, I have surveyed select homopolymeric single [(A)n] and double [(AB)n] AARs in the human proteome. A close examination of their codon pattern and analysis of RNA structure propensity led to the following set of empirical rules: (1) One class of amino acid repeats (Class I) uses a mixture of synonymous codons, some of which approximate the codon bias ratio in the overall human proteome; (2) The second class (Class II) disregards the codon bias ratio, and appears to have originated by simple repetition of the same codon (or just a few codons); and finally, (3) In all AARs (including Class I, Class II, and the in-betweens), the codons are chosen in a manner that precludes the formation of RNA secondary structure. It appears that the AAR genes have evolved by orchestrating a balance between codon usage and mRNA secondary structure. The insights gained here should provide a better understanding of AAR evolution and may assist in designing synthetic genes.

Complex codon usage pattern and compositional features of retroviruses.

PubMed

RoyChoudhury, Sourav; Mukherjee, Debaprasad

2013-01-01

Retroviruses infect a wide range of organisms including humans. Among them, HIV-1, which causes AIDS, has now become a major threat for world health. Some of these viruses are also potential gene transfer vectors. In this study, the patterns of synonymous codon usage in retroviruses have been studied through multivariate statistical methods on ORFs sequences from the available 56 retroviruses. The principal determinant for evolution of the codon usage pattern in retroviruses seemed to be the compositional constraints, while selection for translation of the viral genes plays a secondary role. This was further supported by multivariate analysis on relative synonymous codon usage. Thus, it seems that mutational bias might have dominated role over translational selection in shaping the codon usage of retroviruses. Codon adaptation index was used to identify translationally optimal codons among genes from retroviruses. The comparative analysis of the preferred and optimal codons among different retroviral groups revealed that four codons GAA, AAA, AGA, and GGA were significantly more frequent in most of the retroviral genes inspite of some differences. Cluster analysis also revealed that phylogenetically related groups of retroviruses have probably evolved their codon usage in a concerted manner under the influence of their nucleotide composition.

Prion protein gene analysis in three kindreds with fatal familial insomnia (FFI): Codon 178 mutation and codon 129 polymorphism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Medori, R.; Tritschler, H.J.

1993-10-01

Fatal familial insomnia (FFI) is a disease linked to a GAC(Asp) [yields] AAC(Asn) mutation in codon 178 of the prion protein (PrP) gene. FFI is characterized clinically by untreatable progressive insomnia, dysautonomia, and motor dysfunctions and is characterized pathologically by selective thalamic atrophy. The authors confirmed the 178[sup Asn] mutation in the PrP gene of a third FFI family of French ancestry. Three family members who are under 40 years of age and who inherited the mutation showed only reduced perfusion in the basal ganglia on single photon emission computerized tomography. Some FFI features differ from the clinical and neuropathologicmore » findings associated with 178[sup Asn] reported elsewhere. However, additional intragenic mutations accounting for the phenotypic differences were not observed in two affected individuals. In other sporadic and familial forms of Creutzfeldt-Jakob disease and Gerstmann-Straeussler syndrome, Met or Val homozygosity at polymorphic codon 129 is associated with a more severe phenotype, younger age at onset, and faster progression. In FFI, young and old individuals at disease onset had 129[sup Met/Val]. Moreover, of five 178[sup Asn] individuals who are above age-at-onset range and who are well, two have 129[sup Met] and three have 129[sup Met/Val], suggesting that polymorphic site 129 does not modulate FFI phenotypic expression. Genetic heterogeneity and environment may play an important role in inter- and intrafamilial variability of the 178[sup Asn] mutation. 32 refs., 5 figs., 1 tab.« less

Viral morphogenesis is the dominant source of sequence censorship in M13 combinatorial peptide phage display.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rodi, D. J.; Soares, A. S.; Makowski, L.

Novel statistical methods have been developed and used to quantitate and annotate the sequence diversity within combinatorial peptide libraries on the basis of small numbers (1-200) of sequences selected at random from commercially available M13 p3-based phage display libraries. These libraries behave statistically as though they correspond to populations containing roughly 4.0{+-}1.6% of the random dodecapeptides and 7.9{+-}2.6% of the random constrained heptapeptides that are theoretically possible within the phage populations. Analysis of amino acid residue occurrence patterns shows no demonstrable influence on sequence censorship by Escherichia coli tRNA isoacceptor profiles or either overall codon or Class II codon usagemore » patterns, suggesting no metabolic constraints on recombinant p3 synthesis. There is an overall depression in the occurrence of cysteine, arginine and glycine residues and an overabundance of proline, threonine and histidine residues. The majority of position-dependent amino acid sequence bias is clustered at three positions within the inserted peptides of the dodecapeptide library, +1, +3 and +12 downstream from the signal peptidase cleavage site. Conformational tendency measures of the peptides indicate a significant preference for inserts favoring a {beta}-turn conformation. The observed protein sequence limitations can primarily be attributed to genetic codon degeneracy and signal peptidase cleavage preferences. These data suggest that for applications in which maximal sequence diversity is essential, such as epitope mapping or novel receptor identification, combinatorial peptide libraries should be constructed using codon-corrected trinucleotide cassettes within vector-host systems designed to minimize morphogenesis-related censorship.« less

Codon usage bias: causative factors, quantification methods and genome-wide patterns: with emphasis on insect genomes.

PubMed

Behura, Susanta K; Severson, David W

2013-02-01

Codon usage bias refers to the phenomenon where specific codons are used more often than other synonymous codons during translation of genes, the extent of which varies within and among species. Molecular evolutionary investigations suggest that codon bias is manifested as a result of balance between mutational and translational selection of such genes and that this phenomenon is widespread across species and may contribute to genome evolution in a significant manner. With the advent of whole-genome sequencing of numerous species, both prokaryotes and eukaryotes, genome-wide patterns of codon bias are emerging in different organisms. Various factors such as expression level, GC content, recombination rates, RNA stability, codon position, gene length and others (including environmental stress and population size) can influence codon usage bias within and among species. Moreover, there has been a continuous quest towards developing new concepts and tools to measure the extent of codon usage bias of genes. In this review, we outline the fundamental concepts of evolution of the genetic code, discuss various factors that may influence biased usage of synonymous codons and then outline different principles and methods of measurement of codon usage bias. Finally, we discuss selected studies performed using whole-genome sequences of different insect species to show how codon bias patterns vary within and among genomes. We conclude with generalized remarks on specific emerging aspects of codon bias studies and highlight the recent explosion of genome-sequencing efforts on arthropods (such as twelve Drosophila species, species of ants, honeybee, Nasonia and Anopheles mosquitoes as well as the recent launch of a genome-sequencing project involving 5000 insects and other arthropods) that may help us to understand better the evolution of codon bias and its biological significance. © 2012 The Authors. Biological Reviews © 2012 Cambridge Philosophical Society.

Pandemic influenza A virus codon usage revisited: biases, adaptation and implications for vaccine strain development

PubMed Central

2012-01-01

Background Influenza A virus (IAV) is a member of the family Orthomyxoviridae and contains eight segments of a single-stranded RNA genome with negative polarity. The first influenza pandemic of this century was declared in April of 2009, with the emergence of a novel H1N1 IAV strain (H1N1pdm) in Mexico and USA. Understanding the extent and causes of biases in codon usage is essential to the understanding of viral evolution. A comprehensive study to investigate the effect of selection pressure imposed by the human host on the codon usage of an emerging, pandemic IAV strain and the trends in viral codon usage involved over the pandemic time period is much needed. Results We performed a comprehensive codon usage analysis of 310 IAV strains from the pandemic of 2009. Highly biased codon usage for Ala, Arg, Pro, Thr and Ser were found. Codon usage is strongly influenced by underlying biases in base composition. When correspondence analysis (COA) on relative synonymous codon usage (RSCU) is applied, the distribution of IAV ORFs in the plane defined by the first two major dimensional factors showed that different strains are located at different places, suggesting that IAV codon usage also reflects an evolutionary process. Conclusions A general association between codon usage bias, base composition and poor adaptation of the virus to the respective host tRNA pool, suggests that mutational pressure is the main force shaping H1N1 pdm IAV codon usage. A dynamic process is observed in the variation of codon usage of the strains enrolled in these studies. These results suggest a balance of mutational bias and natural selection, which allow the virus to explore and re-adapt its codon usage to different environments. Recoding of IAV taking into account codon bias, base composition and adaptation to host tRNA may provide important clues to develop new and appropriate vaccines. PMID:23134595

Frequent Nuclear/Cytoplasmic Localization of β-Catenin without Exon 3 Mutations in Malignant Melanoma

PubMed Central

Rimm, David L.; Caca, Karel; Hu, Gang; Harrison, Frank B.; Fearon, Eric R.

1999-01-01

β-Catenin has a critical role in E-cadherin-mediated cell-cell adhesion, and it also functions as a downstream signaling molecule in the wnt pathway. Mutations in the putative glycogen synthase kinase 3β phosphorylation sites near the β-catenin amino terminus have been found in some cancers and cancer cell lines. The mutations render β-catenin resistant to regulation by a complex containing the glycogen synthase kinase 3β, adenomatous polyposis coli, and axin proteins. As a result, β-catenin accumulates in the cytosol and nucleus and activates T-cell factor/lymphoid enhancing factor transcription factors. Previously, 6 of 27 melanoma cell lines were found to have β-catenin exon 3 mutations affecting the N-terminal phosphorylation sites (Rubinfeld B, Robbins P, Elgamil M, Albert I, Porfiri E, Polakis P: Stabilization of beta-catenin by genetic defects in melanoma cell lines. Science 1997, 275:1790–1792). To assess the role of β-catenin defects in primary melanomas, we undertook immunohistochemical and DNA sequencing studies in 65 melanoma specimens. Nuclear and/or cytoplasmic localization of β-catenin, a potential indicator of wnt pathway activation, was seen focally within roughly one third of the tumors, though a clonal somatic mutation in β-catenin was found in only one case (codon 45 Ser→Pro). Our findings demonstrate that β-catenin mutations are rare in primary melanoma, in contrast to the situation in melanoma cell lines. Nonetheless, activation of β-catenin, as indicated by its nuclear and/or cytoplasmic localization, appears to be frequent in melanoma, and in some cases, it may reflect focal and transient activation of the wnt pathway within the tumor. PMID:10027390

Drosophila muller f elements maintain a distinct set of genomic properties over 40 million years of evolution.

PubMed

Leung, Wilson; Shaffer, Christopher D; Reed, Laura K; Smith, Sheryl T; Barshop, William; Dirkes, William; Dothager, Matthew; Lee, Paul; Wong, Jeannette; Xiong, David; Yuan, Han; Bedard, James E J; Machone, Joshua F; Patterson, Seantay D; Price, Amber L; Turner, Bryce A; Robic, Srebrenka; Luippold, Erin K; McCartha, Shannon R; Walji, Tezin A; Walker, Chelsea A; Saville, Kenneth; Abrams, Marita K; Armstrong, Andrew R; Armstrong, William; Bailey, Robert J; Barberi, Chelsea R; Beck, Lauren R; Blaker, Amanda L; Blunden, Christopher E; Brand, Jordan P; Brock, Ethan J; Brooks, Dana W; Brown, Marie; Butzler, Sarah C; Clark, Eric M; Clark, Nicole B; Collins, Ashley A; Cotteleer, Rebecca J; Cullimore, Peterson R; Dawson, Seth G; Docking, Carter T; Dorsett, Sasha L; Dougherty, Grace A; Downey, Kaitlyn A; Drake, Andrew P; Earl, Erica K; Floyd, Trevor G; Forsyth, Joshua D; Foust, Jonathan D; Franchi, Spencer L; Geary, James F; Hanson, Cynthia K; Harding, Taylor S; Harris, Cameron B; Heckman, Jonathan M; Holderness, Heather L; Howey, Nicole A; Jacobs, Dontae A; Jewell, Elizabeth S; Kaisler, Maria; Karaska, Elizabeth A; Kehoe, James L; Koaches, Hannah C; Koehler, Jessica; Koenig, Dana; Kujawski, Alexander J; Kus, Jordan E; Lammers, Jennifer A; Leads, Rachel R; Leatherman, Emily C; Lippert, Rachel N; Messenger, Gregory S; Morrow, Adam T; Newcomb, Victoria; Plasman, Haley J; Potocny, Stephanie J; Powers, Michelle K; Reem, Rachel M; Rennhack, Jonathan P; Reynolds, Katherine R; Reynolds, Lyndsey A; Rhee, Dong K; Rivard, Allyson B; Ronk, Adam J; Rooney, Meghan B; Rubin, Lainey S; Salbert, Luke R; Saluja, Rasleen K; Schauder, Taylor; Schneiter, Allison R; Schulz, Robert W; Smith, Karl E; Spencer, Sarah; Swanson, Bryant R; Tache, Melissa A; Tewilliager, Ashley A; Tilot, Amanda K; VanEck, Eve; Villerot, Matthew M; Vylonis, Megan B; Watson, David T; Wurzler, Juliana A; Wysocki, Lauren M; Yalamanchili, Monica; Zaborowicz, Matthew A; Emerson, Julia A; Ortiz, Carlos; Deuschle, Frederic J; DiLorenzo, Lauren A; Goeller, Katie L; Macchi, Christopher R; Muller, Sarah E; Pasierb, Brittany D; Sable, Joseph E; Tucci, Jessica M; Tynon, Marykathryn; Dunbar, David A; Beken, Levent H; Conturso, Alaina C; Danner, Benjamin L; DeMichele, Gabriella A; Gonzales, Justin A; Hammond, Maureen S; Kelley, Colleen V; Kelly, Elisabeth A; Kulich, Danielle; Mageeney, Catherine M; McCabe, Nikie L; Newman, Alyssa M; Spaeder, Lindsay A; Tumminello, Richard A; Revie, Dennis; Benson, Jonathon M; Cristostomo, Michael C; DaSilva, Paolo A; Harker, Katherine S; Jarrell, Jenifer N; Jimenez, Luis A; Katz, Brandon M; Kennedy, William R; Kolibas, Kimberly S; LeBlanc, Mark T; Nguyen, Trung T; Nicolas, Daniel S; Patao, Melissa D; Patao, Shane M; Rupley, Bryan J; Sessions, Bridget J; Weaver, Jennifer A; Goodman, Anya L; Alvendia, Erica L; Baldassari, Shana M; Brown, Ashley S; Chase, Ian O; Chen, Maida; Chiang, Scott; Cromwell, Avery B; Custer, Ashley F; DiTommaso, Tia M; El-Adaimi, Jad; Goscinski, Nora C; Grove, Ryan A; Gutierrez, Nestor; Harnoto, Raechel S; Hedeen, Heather; Hong, Emily L; Hopkins, Barbara L; Huerta, Vilma F; Khoshabian, Colin; LaForge, Kristin M; Lee, Cassidy T; Lewis, Benjamin M; Lydon, Anniken M; Maniaci, Brian J; Mitchell, Ryan D; Morlock, Elaine V; Morris, William M; Naik, Priyanka; Olson, Nicole C; Osterloh, Jeannette M; Perez, Marcos A; Presley, Jonathan D; Randazzo, Matt J; Regan, Melanie K; Rossi, Franca G; Smith, Melanie A; Soliterman, Eugenia A; Sparks, Ciani J; Tran, Danny L; Wan, Tiffany; Welker, Anne A; Wong, Jeremy N; Sreenivasan, Aparna; Youngblom, Jim; Adams, Andrew; Alldredge, Justin; Bryant, Ashley; Carranza, David; Cifelli, Alyssa; Coulson, Kevin; Debow, Calise; Delacruz, Noelle; Emerson, Charlene; Farrar, Cassandra; Foret, Don; Garibay, Edgar; Gooch, John; Heslop, Michelle; Kaur, Sukhjit; Khan, Ambreen; Kim, Van; Lamb, Travis; Lindbeck, Peter; Lucas, Gabi; Macias, Elizabeth; Martiniuc, Daniela; Mayorga, Lissett; Medina, Joseph; Membreno, Nelson; Messiah, Shady; Neufeld, Lacey; Nguyen, San Francisco; Nichols, Zachary; Odisho, George; Peterson, Daymon; Rodela, Laura; Rodriguez, Priscilla; Rodriguez, Vanessa; Ruiz, Jorge; Sherrill, Will; Silva, Valeria; Sparks, Jeri; Statton, Geeta; Townsend, Ashley; Valdez, Isabel; Waters, Mary; Westphal, Kyle; Winkler, Stacey; Zumkehr, Joannee; DeJong, Randall J; Hoogewerf, Arlene J; Ackerman, Cheri M; Armistead, Isaac O; Baatenburg, Lara; Borr, Matthew J; Brouwer, Lindsay K; Burkhart, Brandon J; Bushhouse, Kelsey T; Cesko, Lejla; Choi, Tiffany Y Y; Cohen, Heather; Damsteegt, Amanda M; Darusz, Jess M; Dauphin, Cory M; Davis, Yelena P; Diekema, Emily J; Drewry, Melissa; Eisen, Michelle E M; Faber, Hayley M; Faber, Katherine J; Feenstra, Elizabeth; Felzer-Kim, Isabella T; Hammond, Brandy L; Hendriksma, Jesse; Herrold, Milton R; Hilbrands, Julia A; Howell, Emily J; Jelgerhuis, Sarah A; Jelsema, Timothy R; Johnson, Benjamin K; Jones, Kelly K; Kim, Anna; Kooienga, Ross D; Menyes, Erika E; Nollet, Eric A; Plescher, Brittany E; Rios, Lindsay; Rose, Jenny L; Schepers, Allison J; Scott, Geoff; Smith, Joshua R; Sterling, Allison M; Tenney, Jenna C; Uitvlugt, Chris; VanDyken, Rachel E; VanderVennen, Marielle; Vue, Samantha; Kokan, Nighat P; Agbley, Kwabea; Boham, Sampson K; Broomfield, Daniel; Chapman, Kayla; Dobbe, Ali; Dobbe, Ian; Harrington, William; Ibrahem, Marwan; Kennedy, Andre; Koplinsky, Chad A; Kubricky, Cassandra; Ladzekpo, Danielle; Pattison, Claire; Ramirez, Roman E; Wande, Lucia; Woehlke, Sarah; Wawersik, Matthew; Kiernan, Elizabeth; Thompson, Jeffrey S; Banker, Roxanne; Bartling, Justina R; Bhatiya, Chinmoy I; Boudoures, Anna L; Christiansen, Lena; Fosselman, Daniel S; French, Kristin M; Gill, Ishwar S; Havill, Jessen T; Johnson, Jaelyn L; Keny, Lauren J; Kerber, John M; Klett, Bethany M; Kufel, Christina N; May, Francis J; Mecoli, Jonathan P; Merry, Callie R; Meyer, Lauren R; Miller, Emily G; Mullen, Gregory J; Palozola, Katherine C; Pfeil, Jacob J; Thomas, Jessica G; Verbofsky, Evan M; Spana, Eric P; Agarwalla, Anant; Chapman, Julia; Chlebina, Ben; Chong, Insun; Falk, I N; Fitzgibbons, John D; Friedman, Harrison; Ighile, Osagie; Kim, Andrew J; Knouse, Kristin A; Kung, Faith; Mammo, Danny; Ng, Chun Leung; Nikam, Vinayak S; Norton, Diana; Pham, Philip; Polk, Jessica W; Prasad, Shreya; Rankin, Helen; Ratliff, Camille D; Scala, Victoria; Schwartz, Nicholas U; Shuen, Jessica A; Xu, Amy; Xu, Thomas Q; Zhang, Yi; Rosenwald, Anne G; Burg, Martin G; Adams, Stephanie J; Baker, Morgan; Botsford, Bobbi; Brinkley, Briana; Brown, Carter; Emiah, Shadie; Enoch, Erica; Gier, Chad; Greenwell, Alyson; Hoogenboom, Lindsay; Matthews, Jordan E; McDonald, Mitchell; Mercer, Amanda; Monsma, Nicholaus; Ostby, Kristine; Ramic, Alen; Shallman, Devon; Simon, Matthew; Spencer, Eric; Tomkins, Trisha; Wendland, Pete; Wylie, Anna; Wolyniak, Michael J; Robertson, Gregory M; Smith, Samuel I; DiAngelo, Justin R; Sassu, Eric D; Bhalla, Satish C; Sharif, Karim A; Choeying, Tenzin; Macias, Jason S; Sanusi, Fareed; Torchon, Karvyn; Bednarski, April E; Alvarez, Consuelo J; Davis, Kristen C; Dunham, Carrie A; Grantham, Alaina J; Hare, Amber N; Schottler, Jennifer; Scott, Zackary W; Kuleck, Gary A; Yu, Nicole S; Kaehler, Marian M; Jipp, Jacob; Overvoorde, Paul J; Shoop, Elizabeth; Cyrankowski, Olivia; Hoover, Betsy; Kusner, Matt; Lin, Devry; Martinov, Tijana; Misch, Jonathan; Salzman, Garrett; Schiedermayer, Holly; Snavely, Michael; Zarrasola, Stephanie; Parrish, Susan; Baker, Atlee; Beckett, Alissa; Belella, Carissa; Bryant, Julie; Conrad, Turner; Fearnow, Adam; Gomez, Carolina; Herbstsomer, Robert A; Hirsch, Sarah; Johnson, Christen; Jones, Melissa; Kabaso, Rita; Lemmon, Eric; Vieira, Carolina Marques Dos Santos; McFarland, Darryl; McLaughlin, Christopher; Morgan, Abbie; Musokotwane, Sepo; Neutzling, William; Nietmann, Jana; Paluskievicz, Christina; Penn, Jessica; Peoples, Emily; Pozmanter, Caitlin; Reed, Emily; Rigby, Nichole; Schmidt, Lasse; Shelton, Micah; Shuford, Rebecca; Tirasawasdichai, Tiara; Undem, Blair; Urick, Damian; Vondy, Kayla; Yarrington, Bryan; Eckdahl, Todd T; Poet, Jeffrey L; Allen, Alica B; Anderson, John E; Barnett, Jason M; Baumgardner, Jordan S; Brown, Adam D; Carney, Jordan E; Chavez, Ramiro A; Christgen, Shelbi L; Christie, Jordan S; Clary, Andrea N; Conn, Michel A; Cooper, Kristen M; Crowley, Matt J; Crowley, Samuel T; Doty, Jennifer S; Dow, Brian A; Edwards, Curtis R; Elder, Darcie D; Fanning, John P; Janssen, Bridget M; Lambright, Anthony K; Lane, Curtiss E; Limle, Austin B; Mazur, Tammy; McCracken, Marly R; McDonough, Alexa M; Melton, Amy D; Minnick, Phillip J; Musick, Adam E; Newhart, William H; Noynaert, Joseph W; Ogden, Bradley J; Sandusky, Michael W; Schmuecker, Samantha M; Shipman, Anna L; Smith, Anna L; Thomsen, Kristen M; Unzicker, Matthew R; Vernon, William B; Winn, Wesley W; Woyski, Dustin S; Zhu, Xiao; Du, Chunguang; Ament, Caitlin; Aso, Soham; Bisogno, Laura Simone; Caronna, Jason; Fefelova, Nadezhda; Lopez, Lenin; Malkowitz, Lorraine; Marra, Jonathan; Menillo, Daniella; Obiorah, Ifeanyi; Onsarigo, Eric Nyabeta; Primus, Shekerah; Soos, Mahdi; Tare, Archana; Zidan, Ameer; Jones, Christopher J; Aronhalt, Todd; Bellush, James M; Burke, Christa; DeFazio, Steve; Does, Benjamin R; Johnson, Todd D; Keysock, Nicholas; Knudsen, Nelson H; Messler, James; Myirski, Kevin; Rekai, Jade Lea; Rempe, Ryan Michael; Salgado, Michael S; Stagaard, Erica; Starcher, Justin R; Waggoner, Andrew W; Yemelyanova, Anastasia K; Hark, Amy T; Bertolet, Anne; Kuschner, Cyrus E; Parry, Kesley; Quach, Michael; Shantzer, Lindsey; Shaw, Mary E; Smith, Mary A; Glenn, Omolara; Mason, Portia; Williams, Charlotte; Key, S Catherine Silver; Henry, Tyneshia C P; Johnson, Ashlee G; White, Jackie X; Haberman, Adam; Asinof, Sam; Drumm, Kelly; Freeburg, Trip; Safa, Nadia; Schultz, Darrin; Shevin, Yakov; Svoronos, Petros; Vuong, Tam; Wellinghoff, Jules; Hoopes, Laura L M; Chau, Kim M; Ward, Alyssa; Regisford, E Gloria C; Augustine, LaJerald; Davis-Reyes, Brionna; Echendu, Vivienne; Hales, Jasmine; Ibarra, Sharon; Johnson, Lauriaun; Ovu, Steven; Braverman, John M; Bahr, Thomas J; Caesar, Nicole M; Campana, Christopher; Cassidy, Daniel W; Cognetti, Peter A; English, Johnathan D; Fadus, Matthew C; Fick, Cameron N; Freda, Philip J; Hennessy, Bryan M; Hockenberger, Kelsey; Jones, Jennifer K; King, Jessica E; Knob, Christopher R; Kraftmann, Karen J; Li, Linghui; Lupey, Lena N; Minniti, Carl J; Minton, Thomas F; Moran, Joseph V; Mudumbi, Krishna; Nordman, Elizabeth C; Puetz, William J; Robinson, Lauren M; Rose, Thomas J; Sweeney, Edward P; Timko, Ashley S; Paetkau, Don W; Eisler, Heather L; Aldrup, Megan E; Bodenberg, Jessica M; Cole, Mara G; Deranek, Kelly M; DeShetler, Megan; Dowd, Rose M; Eckardt, Alexandra K; Ehret, Sharon C; Fese, Jessica; Garrett, Amanda D; Kammrath, Anna; Kappes, Michelle L; Light, Morgan R; Meier, Anne C; O'Rouke, Allison; Perella, Mallory; Ramsey, Kimberley; Ramthun, Jennifer R; Reilly, Mary T; Robinett, Deirdre; Rossi, Nadine L; Schueler, Mary Grace; Shoemaker, Emma; Starkey, Kristin M; Vetor, Ashley; Vrable, Abby; Chandrasekaran, Vidya; Beck, Christopher; Hatfield, Kristen R; Herrick, Douglas A; Khoury, Christopher B; Lea, Charlotte; Louie, Christopher A; Lowell, Shannon M; Reynolds, Thomas J; Schibler, Jeanine; Scoma, Alexandra H; Smith-Gee, Maxwell T; Tuberty, Sarah; Smith, Christopher D; Lopilato, Jane E; Hauke, Jeanette; Roecklein-Canfield, Jennifer A; Corrielus, Maureen; Gilman, Hannah; Intriago, Stephanie; Maffa, Amanda; Rauf, Sabya A; Thistle, Katrina; Trieu, Melissa; Winters, Jenifer; Yang, Bib; Hauser, Charles R; Abusheikh, Tariq; Ashrawi, Yara; Benitez, Pedro; Boudreaux, Lauren R; Bourland, Megan; Chavez, Miranda; Cruz, Samantha; Elliott, GiNell; Farek, Jesse R; Flohr, Sarah; Flores, Amanda H; Friedrichs, Chelsey; Fusco, Zach; Goodwin, Zane; Helmreich, Eric; Kiley, John; Knepper, John Mark; Langner, Christine; Martinez, Megan; Mendoza, Carlos; Naik, Monal; Ochoa, Andrea; Ragland, Nicolas; Raimey, England; Rathore, Sunil; Reza, Evangelina; Sadovsky, Griffin; Seydoux, Marie-Isabelle B; Smith, Jonathan E; Unruh, Anna K; Velasquez, Vicente; Wolski, Matthew W; Gosser, Yuying; Govind, Shubha; Clarke-Medley, Nicole; Guadron, Leslie; Lau, Dawn; Lu, Alvin; Mazzeo, Cheryl; Meghdari, Mariam; Ng, Simon; Pamnani, Brad; Plante, Olivia; Shum, Yuki Kwan Wa; Song, Roy; Johnson, Diana E; Abdelnabi, Mai; Archambault, Alexi; Chamma, Norma; Gaur, Shailly; Hammett, Deborah; Kandahari, Adrese; Khayrullina, Guzal; Kumar, Sonali; Lawrence, Samantha; Madden, Nigel; Mandelbaum, Max; Milnthorp, Heather; Mohini, Shiv; Patel, Roshni; Peacock, Sarah J; Perling, Emily; Quintana, Amber; Rahimi, Michael; Ramirez, Kristen; Singhal, Rishi; Weeks, Corinne; Wong, Tiffany; Gillis, Aubree T; Moore, Zachary D; Savell, Christopher D; Watson, Reece; Mel, Stephanie F; Anilkumar, Arjun A; Bilinski, Paul; Castillo, Rostislav; Closser, Michael; Cruz, Nathalia M; Dai, Tiffany; Garbagnati, Giancarlo F; Horton, Lanor S; Kim, Dongyeon; Lau, Joyce H; Liu, James Z; Mach, Sandy D; Phan, Thu A; Ren, Yi; Stapleton, Kenneth E; Strelitz, Jean M; Sunjed, Ray; Stamm, Joyce; Anderson, Morgan C; Bonifield, Bethany Grace; Coomes, Daniel; Dillman, Adam; Durchholz, Elaine J; Fafara-Thompson, Antoinette E; Gross, Meleah J; Gygi, Amber M; Jackson, Lesley E; Johnson, Amy; Kocsisova, Zuzana; Manghelli, Joshua L; McNeil, Kylie; Murillo, Michael; Naylor, Kierstin L; Neely, Jessica; Ogawa, Emmy E; Rich, Ashley; Rogers, Anna; Spencer, J Devin; Stemler, Kristina M; Throm, Allison A; Van Camp, Matt; Weihbrecht, Katie; Wiles, T Aaron; Williams, Mallory A; Williams, Matthew; Zoll, Kyle; Bailey, Cheryl; Zhou, Leming; Balthaser, Darla M; Bashiri, Azita; Bower, Mindy E; Florian, Kayla A; Ghavam, Nazanin; Greiner-Sosanko, Elizabeth S; Karim, Helmet; Mullen, Victor W; Pelchen, Carly E; Yenerall, Paul M; Zhang, Jiayu; Rubin, Michael R; Arias-Mejias, Suzette M; Bermudez-Capo, Armando G; Bernal-Vega, Gabriela V; Colon-Vazquez, Mariela; Flores-Vazquez, Arelys; Gines-Rosario, Mariela; Llavona-Cartagena, Ivan G; Martinez-Rodriguez, Javier O; Ortiz-Fuentes, Lionel; Perez-Colomba, Eliezer O; Perez-Otero, Joseph; Rivera, Elisandra; Rodriguez-Giron, Luke J; Santiago-Sanabria, Arnaldo J; Senquiz-Gonzalez, Andrea M; delValle, Frank R Soto; Vargas-Franco, Dorianmarie; Velázquez-Soto, Karla I; Zambrana-Burgos, Joan D; Martinez-Cruzado, Juan Carlos; Asencio-Zayas, Lillyann; Babilonia-Figueroa, Kevin; Beauchamp-Pérez, Francis D; Belén-Rodríguez, Juliana; Bracero-Quiñones, Luciann; Burgos-Bula, Andrea P; Collado-Méndez, Xavier A; Colón-Cruz, Luis R; Correa-Muller, Ana I; Crooke-Rosado, Jonathan L; Cruz-García, José M; Defendini-Ávila, Marianna; Delgado-Peraza, Francheska M; Feliciano-Cancela, Alex J; Gónzalez-Pérez, Valerie M; Guiblet, Wilfried; Heredia-Negrón, Aldo; Hernández-Muñiz, Jennifer; Irizarry-González, Lourdes N; Laboy-Corales, Ángel L; Llaurador-Caraballo, Gabriela A; Marín-Maldonado, Frances; Marrero-Llerena, Ulises; Martell-Martínez, Héctor A; Martínez-Traverso, Idaliz M; Medina-Ortega, Kiara N; Méndez-Castellanos, Sonya G; Menéndez-Serrano, Krizia C; Morales-Caraballo, Carol I; Ortiz-DeChoudens, Saryleine; Ortiz-Ortiz, Patricia; Pagán-Torres, Hendrick; Pérez-Afanador, Diana; Quintana-Torres, Enid M; Ramírez-Aponte, Edwin G; Riascos-Cuero, Carolina; Rivera-Llovet, Michelle S; Rivera-Pagán, Ingrid T; Rivera-Vicéns, Ramón E; Robles-Juarbe, Fabiola; Rodríguez-Bonilla, Lorraine; Rodríguez-Echevarría, Brian O; Rodríguez-García, Priscila M; Rodríguez-Laboy, Abneris E; Rodríguez-Santiago, Susana; Rojas-Vargas, Michael L; Rubio-Marrero, Eva N; Santiago-Colón, Albeliz; Santiago-Ortiz, Jorge L; Santos-Ramos, Carlos E; Serrano-González, Joseline; Tamayo-Figueroa, Alina M; Tascón-Peñaranda, Edna P; Torres-Castillo, José L; Valentín-Feliciano, Nelson A; Valentín-Feliciano, Yashira M; Vargas-Barreto, Nadyan M; Vélez-Vázquez, Miguel; Vilanova-Vélez, Luis R; Zambrana-Echevarría, Cristina; MacKinnon, Christy; Chung, Hui-Min; Kay, Chris; Pinto, Anthony; Kopp, Olga R; Burkhardt, Joshua; Harward, Chris; Allen, Robert; Bhat, Pavan; Chang, Jimmy Hsiang-Chun; Chen, York; Chesley, Christopher; Cohn, Dara; DuPuis, David; Fasano, Michael; Fazzio, Nicholas; Gavinski, Katherine; Gebreyesus, Heran; Giarla, Thomas; Gostelow, Marcus; Greenstein, Rachel; Gunasinghe, Hashini; Hanson, Casey; Hay, Amanda; He, Tao Jian; Homa, Katie; Howe, Ruth; Howenstein, Jeff; Huang, Henry; Khatri, Aaditya; Kim, Young Lu; Knowles, Olivia; Kong, Sarah; Krock, Rebecca; Kroll, Matt; Kuhn, Julia; Kwong, Matthew; Lee, Brandon; Lee, Ryan; Levine, Kevin; Li, Yedda; Liu, Bo; Liu, Lucy; Liu, Max; Lousararian, Adam; Ma, Jimmy; Mallya, Allyson; Manchee, Charlie; Marcus, Joseph; McDaniel, Stephen; Miller, Michelle L; Molleston, Jerome M; Diez, Cristina Montero; Ng, Patrick; Ngai, Natalie; Nguyen, Hien; Nylander, Andrew; Pollack, Jason; Rastogi, Suchita; Reddy, Himabindu; Regenold, Nathaniel; Sarezky, Jon; Schultz, Michael; Shim, Jien; Skorupa, Tara; Smith, Kenneth; Spencer, Sarah J; Srikanth, Priya; Stancu, Gabriel; Stein, Andrew P; Strother, Marshall; Sudmeier, Lisa; Sun, Mengyang; Sundaram, Varun; Tazudeen, Noor; Tseng, Alan; Tzeng, Albert; Venkat, Rohit; Venkataram, Sandeep; Waldman, Leah; Wang, Tracy; Yang, Hao; Yu, Jack Y; Zheng, Yin; Preuss, Mary L; Garcia, Angelica; Juergens, Matt; Morris, Robert W; Nagengast, Alexis A; Azarewicz, Julie; Carr, Thomas J; Chichearo, Nicole; Colgan, Mike; Donegan, Megan; Gardner, Bob; Kolba, Nik; Krumm, Janice L; Lytle, Stacey; MacMillian, Laurell; Miller, Mary; Montgomery, Andrew; Moretti, Alysha; Offenbacker, Brittney; Polen, Mike; Toth, John; Woytanowski, John; Kadlec, Lisa; Crawford, Justin; Spratt, Mary L; Adams, Ashley L; Barnard, Brianna K; Cheramie, Martin N; Eime, Anne M; Golden, Kathryn L; Hawkins, Allyson P; Hill, Jessica E; Kampmeier, Jessica A; Kern, Cody D; Magnuson, Emily E; Miller, Ashley R; Morrow, Cody M; Peairs, Julia C; Pickett, Gentry L; Popelka, Sarah A; Scott, Alexis J; Teepe, Emily J; TerMeer, Katie A; Watchinski, Carmen A; Watson, Lucas A; Weber, Rachel E; Woodard, Kate A; Barnard, Daron C; Appiah, Isaac; Giddens, Michelle M; McNeil, Gerard P; Adebayo, Adeola; Bagaeva, Kate; Chinwong, Justina; Dol, Chrystel; George, Eunice; Haltaufderhyde, Kirk; Haye, Joanna; Kaur, Manpreet; Semon, Max; Serjanov, Dmitri; Toorie, Anika; Wilson, Christopher; Riddle, Nicole C; Buhler, Jeremy; Mardis, Elaine R; Elgin, Sarah C R

2015-03-04

The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25-50%) than euchromatic reference regions (3-11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11-27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4-3.6 vs. 8.4-8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu. Copyright © 2015 Leung et al.

Revelation of Influencing Factors in Overall Codon Usage Bias of Equine Influenza Viruses

PubMed Central

Bhatia, Sandeep; Sood, Richa; Selvaraj, Pavulraj

2016-01-01

Equine influenza viruses (EIVs) of H3N8 subtype are culprits of severe acute respiratory infections in horses, and are still responsible for significant outbreaks worldwide. Adaptability of influenza viruses to a particular host is significantly influenced by their codon usage preference, due to an absolute dependence on the host cellular machinery for their replication. In the present study, we analyzed genome-wide codon usage patterns in 92 EIV strains, including both H3N8 and H7N7 subtypes by computing several codon usage indices and applying multivariate statistical methods. Relative synonymous codon usage (RSCU) analysis disclosed bias of preferred synonymous codons towards A/U-ended codons. The overall codon usage bias in EIVs was slightly lower, and mainly affected by the nucleotide compositional constraints as inferred from the RSCU and effective number of codon (ENc) analysis. Our data suggested that codon usage pattern in EIVs is governed by the interplay of mutation pressure, natural selection from its hosts and undefined factors. The H7N7 subtype was found less fit to its host (horse) in comparison to H3N8, by possessing higher codon bias, lower mutation pressure and much less adaptation to tRNA pool of equine cells. To the best of our knowledge, this is the first report describing the codon usage analysis of the complete genomes of EIVs. The outcome of our study is likely to enhance our understanding of factors involved in viral adaptation, evolution, and fitness towards their hosts. PMID:27119730

The evolutionary radiation of Arvicolinae rodents (voles and lemmings): relative contribution of nuclear and mitochondrial DNA phylogenies

PubMed Central

Galewski, Thomas; Tilak, Marie-ka; Sanchez, Sophie; Chevret, Pascale; Paradis, Emmanuel; Douzery, Emmanuel JP

2006-01-01

Background Mitochondrial and nuclear genes have generally been employed for different purposes in molecular systematics, the former to resolve relationships within recently evolved groups and the latter to investigate phylogenies at a deeper level. In the case of rapid and recent evolutionary radiations, mitochondrial genes like cytochrome b (CYB) are often inefficient for resolving phylogenetic relationships. One of the best examples is illustrated by Arvicolinae rodents (Rodentia; Muridae), the most impressive mammalian radiation of the Northern Hemisphere which produced voles, lemmings and muskrats. Here, we compare the relative contribution of a nuclear marker – the exon 10 of the growth hormone receptor (GHR) gene – to the one of the mitochondrial CYB for inferring phylogenetic relationships among the major lineages of arvicoline rodents. Results The analysis of GHR sequences improves the overall resolution of the Arvicolinae phylogeny. Our results show that the Caucasian long-clawed vole (Prometheomys schaposnikowi) is one of the basalmost arvicolines, and confirm that true lemmings (Lemmus) and collared lemmings (Dicrostonyx) are not closely related as suggested by morphology. Red-backed voles (Myodini) are found as the sister-group of a clade encompassing water vole (Arvicola), snow vole (Chionomys), and meadow voles (Microtus and allies). Within the latter, no support is recovered for the generic recognition of Blanfordimys, Lasiopodomys, Neodon, and Phaiomys as suggested by morphology. Comparisons of parameter estimates for branch lengths, base composition, among sites rate heterogeneity, and GTR relative substitution rates indicate that CYB sequences consistently exhibit more heterogeneity among codon positions than GHR. By analyzing the contribution of each codon position to node resolution, we show that the apparent higher efficiency of GHR is due to their third positions. Although we focus on speciation events spanning the last 10 million years (Myr), CYB sequences display highly saturated codon positions contrary to the nuclear exon. Lastly, variable length bootstrap predicts a significant increase in resolution of arvicoline phylogeny through the sequencing of nuclear data in an order of magnitude three to five times greater than the size of GHR exon 10. Conclusion Our survey provides a first resolved gene tree for Arvicolinae. The comparison of CYB and GHR phylogenetic efficiency supports recent assertions that nuclear genes are useful for resolving relationships of recently evolved animals. The superiority of nuclear exons may reside both in (i) less heterogeneity among sites, and (ii) the presence of highly informative sites in third codon positions, that evolve rapidly enough to accumulate synapomorphies, but slow enough to avoid substitutional saturation. PMID:17029633

Electrochemical Studies of a Truncated Laccase Produced in Pichia pastoris

PubMed Central

Gelo-Pujic, Mirjana; Kim, Hyug-Han; Butlin, Nathan G.; Palmore, G. Tayhas R.

1999-01-01

The cDNA that encodes an isoform of laccase from Trametes versicolor (LCCI), as well as a truncated version (LCCIa), was subcloned and expressed by using the yeast Pichia pastoris as the heterologous host. The amino acid sequence of LCCIa is identical to that of LCCI except that the final 11 amino acids at the C terminus of LCCI are replaced with a single cysteine residue. This modification was introduced for the purpose of improving the kinetics of electron transfer between an electrode and the copper-containing active site of laccase. The two laccases (LCCI and LCCIa) are compared in terms of their relative activity with two substrates that have different redox potentials. Results from electrochemical studies on solutions containing LCCI and LCCIa indicate that the redox potential of the active site of LCCIa is shifted to more negative values (411 mV versus normal hydrogen electrode voltage) than that found in other fungal laccases. In addition, replacing the 11 codons at the C terminus of the laccase gene with a single cysteine codon (i.e., LCCI→LCCIa) influences the rate of heterogeneous electron transfer between an electrode and the copper-containing active site (khet for LCCIa = 1.3 × 10−4 cm s−1). These results demonstrate for the first time that the rate of electron transfer between an oxidoreductase and an electrode can be enhanced by changes to the primary structure of a protein via site-directed mutagenesis. PMID:10584012

A GTPase reaction accompanying the rejection of Leu-tRNA2 by UUU-programmed ribosomes. Proofreading of the codon-anticodon interaction by ribosomes.

PubMed

Thompson, R C; Dix, D B; Gerson, R B; Karim, A M

1981-01-10

The characteristics of a GTPase reaction between poly(U)-programmed ribosomes, EFTu . GTP, and the near-cognate aminoacyl (aa)-tRNA, Leu-tRNA Leu 2, have been studied to assess the role of this reaction in proofreading of the codon-anticodon interaction. The reaction resembles the GTPase reaction with cognate aa-tRNAs and EFTu . GTP in its substrate requirements, in its involving EFTu . GTP . aa-tRNA ternary complexes, and in its requiring a free ribosomal A-site. The noncognate reaction differs from the cognate one in that aa-tRNA becomes stably bound to the ribosomes only 5% of the time; it therefore seems best characterized as an abortive enzymatic binding reaction. The rate of reaction is a significant fraction (4%) of that of the cognate aa-tRNA, indicating that recognition of ternary complexes by ribosomes involves a level of error greater than that of translation as a whole. The rejection of the noncognate aa-tRNA following GTP hydrolysis is therefore a vital step in the translation process and fulfills the criteria set for a proofreading reaction. Leu-tRNA Leu 2 which escapes rejection through proofreading, forms a stable complex with the ribosomal A-site, so it appears that the Leu-tRNA2 which was rejected never reached the A-site and that proofreading precedes full A-site binding.

The Human Cytomegalovirus IE2 and UL112-113 Proteins Accumulate in Viral DNA Replication Compartments That Initiate from the Periphery of Promyelocytic Leukemia Protein-Associated Nuclear Bodies (PODs or ND10)

PubMed Central

Ahn, Jin-Hyun; Jang, Won-Jong; Hayward, Gary S.

1999-01-01

During human cytomegalovirus (HCMV) infection, the periphery of promyelocytic leukemia protein (PML)-associated nuclear bodies (also known as PML oncogenic domains [PODs] or ND10) are sites for both input viral genome deposition and immediate-early (IE) gene transcription. At very early times after infection, the IE1 protein localizes to and subsequently disrupts PODs, whereas the IE2 protein localizes within or adjacent to PODs. This process appears to be required for efficient viral gene expression and DNA replication. We have investigated the initiation of viral DNA replication compartment formation by studying the localization of viral IE proteins, DNA replication proteins, and the PML protein during productive infection. Localization of IE2 adjacent to PODs between 2 and 6 h after infection was confirmed by confocal microscopy of human fibroblasts (HF cells) infected with both wild-type HCMV(Towne) and with an IE1-deletion mutant HCMV(CR208) that fails to disrupt PODs. In HCMV(Towne)-infected HF cells at 24 to 48 h, IE2 also accumulated in newly formed viral DNA replication compartments containing the polymerase processivity factor (UL44), the single-stranded DNA binding protein (SSB; UL57), the UL112-113 accessory protein, and newly incorporated bromodeoxyuridine (BrdU). Double labeling of the HCMV(CR208)-infected HF cells demonstrated that formation of viral DNA replication compartments initiates within granular structures that bud from the periphery of some of the PODs and subsequently coalesce into larger structures that are flanked by PODs. In transient DNA transfection assays, both the N terminus (codons 136 to 290) and the C terminus (codons 379 to 579) of IE2 exon 5, but not the central region between them, were found to be necessary for both the punctate distribution of IE2 and its association with PODs. Like IE2, the UL112-113 accessory replication protein was also distributed in a POD-associated pattern in both DNA-transfected and virus-infected cells beginning at 6 h. Furthermore, when all six replication core machinery proteins (polymerase complex, SSB, and helicase-primase complex) were expressed together in the presence of UL112-113, they also accumulated at POD-associated sites, suggesting that the UL112-113 protein (but not IE2) may play a role in recruitment of viral replication fork proteins into the periphery of PODs. These results show that (i) subsequent to accumulating at the periphery of PODs, IE2 is incorporated together with the core proteins into viral DNA replication compartments that initiate from the periphery of PODs and then grow to fill the space between groups of PODs, and (ii) the UL112-113 protein appears to have a key role in assembling and recruiting the core replication machinery proteins in the initial stages of viral replication compartment formation. PMID:10559364

Comparison of Insertional RNA Editing in Myxomycetes

PubMed Central

Chen, Cai; Frankhouser, David; Bundschuh, Ralf

2012-01-01

RNA editing describes the process in which individual or short stretches of nucleotides in a messenger or structural RNA are inserted, deleted, or substituted. A high level of RNA editing has been observed in the mitochondrial genome of Physarum polycephalum. The most frequent editing type in Physarum is the insertion of individual Cs. RNA editing is extremely accurate in Physarum; however, little is known about its mechanism. Here, we demonstrate how analyzing two organisms from the Myxomycetes, namely Physarum polycephalum and Didymium iridis, allows us to test hypotheses about the editing mechanism that can not be tested from a single organism alone. First, we show that using the recently determined full transcriptome information of Physarum dramatically improves the accuracy of computational editing site prediction in Didymium. We use this approach to predict genes in the mitochondrial genome of Didymium and identify six new edited genes as well as one new gene that appears unedited. Next we investigate sequence conservation in the vicinity of editing sites between the two organisms in order to identify sites that harbor the information for the location of editing sites based on increased conservation. Our results imply that the information contained within only nine or ten nucleotides on either side of the editing site (a distance previously suggested through experiments) is not enough to locate the editing sites. Finally, we show that the codon position bias in C insertional RNA editing of these two organisms is correlated with the selection pressure on the respective genes thereby directly testing an evolutionary theory on the origin of this codon bias. Beyond revealing interesting properties of insertional RNA editing in Myxomycetes, our work suggests possible approaches to be used when finding sequence motifs for any biological process fails. PMID:22383871

Codon usage affects the structure and function of the Drosophila circadian clock protein PERIOD.

PubMed

Fu, Jingjing; Murphy, Katherine A; Zhou, Mian; Li, Ying H; Lam, Vu H; Tabuloc, Christine A; Chiu, Joanna C; Liu, Yi

2016-08-01

Codon usage bias is a universal feature of all genomes, but its in vivo biological functions in animal systems are not clear. To investigate the in vivo role of codon usage in animals, we took advantage of the sensitivity and robustness of the Drosophila circadian system. By codon-optimizing parts of Drosophila period (dper), a core clock gene that encodes a critical component of the circadian oscillator, we showed that dper codon usage is important for circadian clock function. Codon optimization of dper resulted in conformational changes of the dPER protein, altered dPER phosphorylation profile and stability, and impaired dPER function in the circadian negative feedback loop, which manifests into changes in molecular rhythmicity and abnormal circadian behavioral output. This study provides an in vivo example that demonstrates the role of codon usage in determining protein structure and function in an animal system. These results suggest a universal mechanism in eukaryotes that uses a codon usage "code" within genetic codons to regulate cotranslational protein folding. © 2016 Fu et al.; Published by Cold Spring Harbor Laboratory Press.

Absence of opioid stress-induced analgesia in mice lacking beta-endorphin by site-directed mutagenesis.

PubMed

Rubinstein, M; Mogil, J S; Japón, M; Chan, E C; Allen, R G; Low, M J

1996-04-30

A physiological role for beta-endorphin in endogenous pain inhibition was investigated by targeted mutagenesis of the proopiomelanocortin gene in mouse embryonic stem cells. The tyrosine codon at position 179 of the proopiomelanocortin gene was converted to a premature translational stop codon. The resulting transgenic mice display no overt developmental or behavioral alterations and have a normally functioning hypothalamic-pituitary-adrenal axis. Homozygous transgenic mice with a selective deficiency of beta-endorphin exhibit normal analgesia in response to morphine, indicating the presence of functional mu-opiate receptors. However, these mice lack the opioid (naloxone reversible) analgesia induced by mild swim stress. Mutant mice also display significantly greater nonopioid analgesia in response to cold water swim stress compared with controls and display paradoxical naloxone-induced analgesia. These changes may reflect compensatory upregulation of alternative pain inhibitory mechanisms.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ogle, James M.; Brodersen, Ditlev E.; Clemons, William M.

Crystal structures of the 30S ribosomal subunit in complex with messenger RNA and cognate transfer RNA in the A site, both in the presence and absence of the antibiotic paromomycin, have been solved at between 3.1 and 3.3 angstroms resolution. Cognate transfer RNA (tRNA) binding induces global domain movements of the 30S subunit and changes in the conformation of the universally conserved and essential bases A1492, A1493, and G530 of 16S RNA. These bases interact intimately with the minor groove of the first two base pairs between the codon and anticodon, thus sensing Watson-Crick base-pairing geometry and discriminating against near-cognatemore » tRNA. The third, or 'wobble,' position of the codon is free to accommodate certain noncanonical base pairs. By partially inducing these structural changes, paromomycin facilitates binding of near-cognate tRNAs.« less

The fuzzy polynucleotide space: basic properties.

PubMed

Torres, Angela; Nieto, Juan J

2003-03-22

Any triplet codon may be regarded as a 12-dimensional fuzzy code. Sufficient information about a particular sequence may not be available in certain situations. The investigator will be confronted with imprecise sequences, yet want to make comparisons of sequences. Fuzzy polynucleotides can be compared by using geometrical interpretation of fuzzy sets as points in a hypercube. We introduce the space of fuzzy polynucleotides and a means of measuring dissimilitudes between them. We establish mathematical principles to measure dissimilarities between fuzzy polynucleotides and present several examples in this metric space. We calculate the frequencies of the nucleotides at the three base sites of a codon in the coding sequences of Escherichia coli K-12 and Mycobacterium tuberculosis H37Rv, and consider them as points in that fuzzy space. We compute the distance between the genomes of E.coli and M.tuberculosis.

Analyses of clinicopathological, molecular, and prognostic associations of KRAS codon 61 and codon 146 mutations in colorectal cancer: cohort study and literature review

PubMed Central

2014-01-01

Background KRAS mutations in codons 12 and 13 are established predictive biomarkers for anti-EGFR therapy in colorectal cancer. Previous studies suggest that KRAS codon 61 and 146 mutations may also predict resistance to anti-EGFR therapy in colorectal cancer. However, clinicopathological, molecular, and prognostic features of colorectal carcinoma with KRAS codon 61 or 146 mutation remain unclear. Methods We utilized a molecular pathological epidemiology database of 1267 colon and rectal cancers in the Nurse’s Health Study and the Health Professionals Follow-up Study. We examined KRAS mutations in codons 12, 13, 61 and 146 (assessed by pyrosequencing), in relation to clinicopathological features, and tumor molecular markers, including BRAF and PIK3CA mutations, CpG island methylator phenotype (CIMP), LINE-1 methylation, and microsatellite instability (MSI). Survival analyses were performed in 1067 BRAF-wild-type cancers to avoid confounding by BRAF mutation. Cox proportional hazards models were used to compute mortality hazard ratio, adjusting for potential confounders, including disease stage, PIK3CA mutation, CIMP, LINE-1 hypomethylation, and MSI. Results KRAS codon 61 mutations were detected in 19 cases (1.5%), and codon 146 mutations in 40 cases (3.2%). Overall KRAS mutation prevalence in colorectal cancers was 40% (=505/1267). Of interest, compared to KRAS-wild-type, overall, KRAS-mutated cancers more frequently exhibited cecal location (24% vs. 12% in KRAS-wild-type; P < 0.0001), CIMP-low (49% vs. 32% in KRAS-wild-type; P < 0.0001), and PIK3CA mutations (24% vs. 11% in KRAS-wild-type; P < 0.0001). These trends were evident irrespective of mutated codon, though statistical power was limited for codon 61 mutants. Neither KRAS codon 61 nor codon 146 mutation was significantly associated with clinical outcome or prognosis in univariate or multivariate analysis [colorectal cancer-specific mortality hazard ratio (HR) = 0.81, 95% confidence interval (CI) = 0.29-2.26 for codon 61 mutation; colorectal cancer-specific mortality HR = 0.86, 95% CI = 0.42-1.78 for codon 146 mutation]. Conclusions Tumors with KRAS mutations in codons 61 and 146 account for an appreciable proportion (approximately 5%) of colorectal cancers, and their clinicopathological and molecular features appear generally similar to KRAS codon 12 or 13 mutated cancers. To further assess clinical utility of KRAS codon 61 and 146 testing, large-scale trials are warranted. PMID:24885062

Codon adaptation and synonymous substitution rate in diatom plastid genes.

PubMed

Morton, Brian R; Sorhannus, Ulf; Fox, Martin

2002-07-01

Diatom plastid genes are examined with respect to codon adaptation and rates of silent substitution (Ks). It is shown that diatom genes follow the same pattern of codon usage as other plastid genes studied previously. Highly expressed diatom genes display codon adaptation, or a bias toward specific major codons, and these major codons are the same as those in red algae, green algae, and land plants. It is also found that there is a strong correlation between Ks and variation in codon adaptation across diatom genes, providing the first evidence for such a relationship in the algae. It is argued that this finding supports the notion that the correlation arises from selective constraints, not from variation in mutation rate among genes. Finally, the diatom genes are examined with respect to variation in Ks among different synonymous groups. Diatom genes with strong codon adaptation do not show the same variation in synonymous substitution rate among codon groups as the flowering plant psbA gene which, previous studies have shown, has strong codon adaptation but unusually high rates of silent change in certain synonymous groups. The lack of a similar finding in diatoms supports the suggestion that the feature is unique to the flowering plant psbA due to recent relaxations in selective pressure in that lineage.

Genetic Code Optimization for Cotranslational Protein Folding: Codon Directional Asymmetry Correlates with Antiparallel Betasheets, tRNA Synthetase Classes.

PubMed

Seligmann, Hervé; Warthi, Ganesh

2017-01-01

A new codon property, codon directional asymmetry in nucleotide content (CDA), reveals a biologically meaningful genetic code dimension: palindromic codons (first and last nucleotides identical, codon structure XZX) are symmetric (CDA = 0), codons with structures ZXX/XXZ are 5'/3' asymmetric (CDA = - 1/1; CDA = - 0.5/0.5 if Z and X are both purines or both pyrimidines, assigning negative/positive (-/+) signs is an arbitrary convention). Negative/positive CDAs associate with (a) Fujimoto's tetrahedral codon stereo-table; (b) tRNA synthetase class I/II (aminoacylate the 2'/3' hydroxyl group of the tRNA's last ribose, respectively); and (c) high/low antiparallel (not parallel) betasheet conformation parameters. Preliminary results suggest CDA-whole organism associations (body temperature, developmental stability, lifespan). Presumably, CDA impacts spatial kinetics of codon-anticodon interactions, affecting cotranslational protein folding. Some synonymous codons have opposite CDA sign (alanine, leucine, serine, and valine), putatively explaining how synonymous mutations sometimes affect protein function. Correlations between CDA and tRNA synthetase classes are weaker than between CDA and antiparallel betasheet conformation parameters. This effect is stronger for mitochondrial genetic codes, and potentially drives mitochondrial codon-amino acid reassignments. CDA reveals information ruling nucleotide-protein relations embedded in reversed (not reverse-complement) sequences (5'-ZXX-3'/5'-XXZ-3').

Monoclonal origin of peritoneal implants and lymph node deposits in serous borderline ovarian tumors (s-BOT) with high intratumoral homogeneity.

PubMed

Horn, Lars-Christian; Höhn, Anne K; Einenkel, Jens; Siebolts, Udo

2014-11-01

Molecular studies have shown that the most prevalent mutations in serous ovarian borderline tumors (s-BOT) are BRAF and/or KRAS alterations. About one third of s-BOT represent peritoneal implants and/or lymph node involvement. These extraovarian deposits may be monoclonal or polyclonal in origin. To test both the hypotheses, mutational analyses using pyrosequencing for BRAF codon 600 and KRAS codon 12/13 and 61 of microdissected tissue was performed in 15 s-BOT and their invasive and noninvasive peritoneal implants. Two to 6 implants from different peritoneal sites were examined in 13 cases. Lymph node deposits were available for the analysis in 3 cases. Six s-BOT showed mutation in exon 2 codon 12 of the KRAS proto-oncogen. Five additional cases showed BRAF p.V600E mutation representing an overall mutation rate of 73.3%. Multiple (2-6) peritoneal implants were analyzed after microdissection in 13 of 15 cases. All showed identical mutational results when compared with the ovarian site of the disease. All lymph node deposits, including those with multiple deposits in different nodes, showed identical results, suggesting high intratumoral mutational homogeneity. The evidence presented in this study and the majority of data reported in the literature support the hypothesis that s-BOT with their peritoneal implants and lymph node deposits show identical mutational status of BRAF and KRAS suggesting a monoclonal rather than a polyclonal disease regarding these both tested genetic loci. In addition, a high intratumoral genetic homogeneity can be suggested. In conclusion, the results of the present study support the monoclonal origin of s-BOT and their peritoneal implants and lymph node deposits.

Development of a codon optimization strategy using the efor RED reporter gene as a test case

NASA Astrophysics Data System (ADS)

Yip, Chee-Hoo; Yarkoni, Orr; Ajioka, James; Wan, Kiew-Lian; Nathan, Sheila

2018-04-01

Synthetic biology is a platform that enables high-level synthesis of useful products such as pharmaceutically related drugs, bioplastics and green fuels from synthetic DNA constructs. Large-scale expression of these products can be achieved in an industrial compliant host such as Escherichia coli. To maximise the production of recombinant proteins in a heterologous host, the genes of interest are usually codon optimized based on the codon usage of the host. However, the bioinformatics freeware available for standard codon optimization might not be ideal in determining the best sequence for the synthesis of synthetic DNA. Synthesis of incorrect sequences can prove to be a costly error and to avoid this, a codon optimization strategy was developed based on the E. coli codon usage using the efor RED reporter gene as a test case. This strategy replaces codons encoding for serine, leucine, proline and threonine with the most frequently used codons in E. coli. Furthermore, codons encoding for valine and glycine are substituted with the second highly used codons in E. coli. Both the optimized and original efor RED genes were ligated to the pJS209 plasmid backbone using Gibson Assembly and the recombinant DNAs were transformed into E. coli E. cloni 10G strain. The fluorescence intensity per cell density of the optimized sequence was improved by 20% compared to the original sequence. Hence, the developed codon optimization strategy is proposed when designing an optimal sequence for heterologous protein production in E. coli.

Analysis of Synonymous Codon Usage Bias of Zika Virus and Its Adaption to the Hosts

PubMed Central

Wang, Hongju; Liu, Siqing; Zhang, Bo

2016-01-01

Zika virus (ZIKV) is a mosquito-borne virus (arbovirus) in the family Flaviviridae, and the symptoms caused by ZIKV infection in humans include rash, fever, arthralgia, myalgia, asthenia and conjunctivitis. Codon usage bias analysis can reveal much about the molecular evolution and host adaption of ZIKV. To gain insight into the evolutionary characteristics of ZIKV, we performed a comprehensive analysis on the codon usage pattern in 46 ZIKV strains by calculating the effective number of codons (ENc), codon adaptation index (CAI), relative synonymous codon usage (RSCU), and other indicators. The results indicate that the codon usage bias of ZIKV is relatively low. Several lines of evidence support the hypothesis that translational selection plays a role in shaping the codon usage pattern of ZIKV. The results from a correspondence analysis (CA) indicate that other factors, such as base composition, aromaticity, and hydrophobicity may also be involved in shaping the codon usage pattern of ZIKV. Additionally, the results from a comparative analysis of RSCU between ZIKV and its hosts suggest that ZIKV tends to evolve codon usage patterns that are comparable to those of its hosts. Moreover, selection pressure from Homo sapiens on the ZIKV RSCU patterns was found to be dominant compared with that from Aedes aegypti and Aedes albopictus. Taken together, both natural translational selection and mutation pressure are important for shaping the codon usage pattern of ZIKV. Our findings contribute to understanding the evolution of ZIKV and its adaption to its hosts. PMID:27893824

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vendeix, Franck A.P.; Murphy, IV, Frank V.; Cantara, William A.

Human tRNA Lys3 UUU (htRNA Lys3 UUU) decodes the lysine codons AAA and AAG during translation and also plays a crucial role as the primer for HIV-1 (human immunodeficiency virus type 1) reverse transcription. The posttranscriptional modifications 5-methoxycarbonylmethyl-2-thiouridine (mcm 5s 2U 34), 2-methylthio-N 6-threonylcarbamoyladenosine (ms 2t 6A 37), and pseudouridine (Ψ 39) in the tRNA's anticodon domain are critical for ribosomal binding and HIV-1 reverse transcription. To understand the importance of modified nucleoside contributions, we determined the structure and function of this tRNA's anticodon stem and loop (ASL) domain with these modifications at positions 34, 37, and 39, respectively (hASLmore » Lys3 UUU-mcm 5s 2U 34;ms 2t 6A 37;Ψ 39). Ribosome binding assays in vitro revealed that the hASL Lys3 UUU-mcm 5s 2U 34;ms 2t 6A 37;Ψ 39 bound AAA and AAG codons, whereas binding of the unmodified ASL Lys3 UUU was barely detectable. The UV hyperchromicity, the circular dichroism, and the structural analyses indicated that Ψ 39 enhanced the thermodynamic stability of the ASL through base stacking while ms 2t 6A 37 restrained the anticodon to adopt an open loop conformation that is required for ribosomal binding. The NMR-restrained molecular-dynamics-derived solution structure revealed that the modifications provided an open, ordered loop for codon binding. The crystal structures of the hASL Lys3 UUU-mcm 5s 2U 34;ms 2t 6A 37;Ψ 39 bound to the 30S ribosomal subunit with each codon in the A site showed that the modified nucleotides mcm 5s 2U 34 and ms 2t 6A 37 participate in the stability of the anticodon–codon interaction. Importantly, the mcm 5s 2U 34·G 3 wobble base pair is in the Watson–Crick geometry, requiring unusual hydrogen bonding to G in which mcm 5s 2U 34 must shift from the keto to the enol form. The results unambiguously demonstrate that modifications pre-structure the anticodon as a key prerequisite for efficient and accurate recognition of cognate and wobble codons.« less

Codon Optimization to Enhance Expression Yields Insights into Chloroplast Translation1[OPEN

PubMed Central

Chan, Hui-Ting; Williams-Carrier, Rosalind; Barkan, Alice

2016-01-01

Codon optimization based on psbA genes from 133 plant species eliminated 105 (human clotting factor VIII heavy chain [FVIII HC]) and 59 (polio VIRAL CAPSID PROTEIN1 [VP1]) rare codons; replacement with only the most highly preferred codons decreased transgene expression (77- to 111-fold) when compared with the codon usage hierarchy of the psbA genes. Targeted proteomic quantification by parallel reaction monitoring analysis showed 4.9- to 7.1-fold or 22.5- to 28.1-fold increase in FVIII or VP1 codon-optimized genes when normalized with stable isotope-labeled standard peptides (or housekeeping protein peptides), but quantitation using western blots showed 6.3- to 8-fold or 91- to 125-fold increase of transgene expression from the same batch of materials, due to limitations in quantitative protein transfer, denaturation, solubility, or stability. Parallel reaction monitoring, to our knowledge validated here for the first time for in planta quantitation of biopharmaceuticals, is especially useful for insoluble or multimeric proteins required for oral drug delivery. Northern blots confirmed that the increase of codon-optimized protein synthesis is at the translational level rather than any impact on transcript abundance. Ribosome footprints did not increase proportionately with VP1 translation or even decreased after FVIII codon optimization but is useful in diagnosing additional rate-limiting steps. A major ribosome pause at CTC leucine codons in the native gene of FVIII HC was eliminated upon codon optimization. Ribosome stalls observed at clusters of serine codons in the codon-optimized VP1 gene provide an opportunity for further optimization. In addition to increasing our understanding of chloroplast translation, these new tools should help to advance this concept toward human clinical studies. PMID:27465114

Genomic localization of the Z/EG transgene in the mouse genome.

PubMed

Colombo, Sophie; Kumasaka, Mayuko; Lobe, Corrinne; Larue, Lionel

2010-02-01

The Z/EG transgenic mouse line, produced by Novak et al., displays tissue-specific EGFP expression after Cre-mediated recombination. The autofluorescence of EGFP allows the visualization of cells of interest displaying Cre recombination. The initial construct was designed such that cells without Cre recombination express the beta-galactosidase marker, facilitating counterselection. We used inverse PCR to identify the site of integration of the Z/EG transgene, to improve the efficiency of homozygous Z/EG mouse production. Recombined cells produced large amounts of EGFP protein, resulting in higher levels of fluorescence and therefore greater contrast with nonrecombined cells. We mapped the transgene to the G1 region of chromosome 5. This random insertion was found to have occurred 230-bp upstream from the start codon of the Rasa4 gene. The insertion of the Z/EG transgene in the C57BL/6 genetic background had no effect on Rasa4 expression. Homozygous Z/EG mice therefore had no obvious phenotype. (c) 2009 Wiley-Liss, Inc.

Plastid, nuclear and reverse transcriptase sequences in the mitochondrial genome of Oenothera: is genetic information transferred between organelles via RNA?

PubMed Central

Schuster, W; Brennicke, A

1987-01-01

We describe an open reading frame (ORF) with high homology to reverse transcriptase in the mitochondrial genome of Oenothera. This ORF displays all the characteristics of an active plant mitochondrial gene with a possible ribosome binding site and 39% T in the third codon position. It is located between a sequence fragment from the plastid genome and one of nuclear origin downstream from the gene encoding subunit 5 of the NADH dehydrogenase. The nuclear derived sequence consists of 528 nucleotides from the small ribosomal RNA and contains an expansion segment unique to nuclear rRNAs. The plastid sequence contains part of the ribosomal protein S4 and the complete tRNA(Ser). The observation that only transcribed sequences have been found i more than one subcellular compartment in higher plants suggests that interorganellar transfer of genetic information may occur via RNA and subsequent local reverse transcription and genomic integration. PMID:14650433

Problem-Solving Test: The Effect of Synonymous Codons on Gene Expression

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2009-01-01

Terms to be familiar with before you start to solve the test: the genetic code, codon, degenerate codons, protein synthesis, aminoacyl-tRNA, anticodon, antiparallel orientation, wobble, unambiguous codons, ribosomes, initiation, elongation and termination of translation, peptidyl transferase, translocation, degenerate oligonucleotides, green…

Codon usage regulates protein structure and function by affecting translation elongation speed in Drosophila cells.

PubMed

Zhao, Fangzhou; Yu, Chien-Hung; Liu, Yi

2017-08-21

Codon usage biases are found in all eukaryotic and prokaryotic genomes and have been proposed to regulate different aspects of translation process. Codon optimality has been shown to regulate translation elongation speed in fungal systems, but its effect on translation elongation speed in animal systems is not clear. In this study, we used a Drosophila cell-free translation system to directly compare the velocity of mRNA translation elongation. Our results demonstrate that optimal synonymous codons speed up translation elongation while non-optimal codons slow down translation. In addition, codon usage regulates ribosome movement and stalling on mRNA during translation. Finally, we show that codon usage affects protein structure and function in vitro and in Drosophila cells. Together, these results suggest that the effect of codon usage on translation elongation speed is a conserved mechanism from fungi to animals that can affect protein folding in eukaryotic organisms. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Synonymous codon choices in the extremely GC-poor genome of Plasmodium falciparum: compositional constraints and translational selection.

PubMed

Musto, H; Romero, H; Zavala, A; Jabbari, K; Bernardi, G

1999-07-01

We have analyzed the patterns of synonymous codon preferences of the nuclear genes of Plasmodium falciparum, a unicellular parasite characterized by an extremely GC-poor genome. When all genes are considered, codon usage is strongly biased toward A and T in third codon positions, as expected, but multivariate statistical analysis detects a major trend among genes. At one end genes display codon choices determined mainly by the extreme genome composition of this parasite, and very probably their expression level is low. At the other end a few genes exhibit an increased relative usage of a particular subset of codons, many of which are C-ending. Since the majority of these few genes is putatively highly expressed, we postulate that the increased C-ending codons are translationally optimal. In conclusion, while codon usage of the majority of P. falciparum genes is determined mainly by compositional constraints, a small number of genes exhibit translational selection.

Most Used Codons per Amino Acid and per Genome in the Code of Man Compared to Other Organisms According to the Rotating Circular Genetic Code

PubMed Central

Castro-Chavez, Fernando

2011-01-01

My previous theoretical research shows that the rotating circular genetic code is a viable tool to make easier to distinguish the rules of variation applied to the amino acid exchange; it presents a precise and positional bio-mathematical balance of codons, according to the amino acids they codify. Here, I demonstrate that when using the conventional or classic circular genetic code, a clearer pattern for the human codon usage per amino acid and per genome emerges. The most used human codons per amino acid were the ones ending with the three hydrogen bond nucleotides: C for 12 amino acids and G for the remaining 8, plus one codon for arginine ending in A that was used approximately with the same frequency than the one ending in G for this same amino acid (plus *). The most used codons in man fall almost all the time at the rightmost position, clockwise, ending either in C or in G within the circular genetic code. The human codon usage per genome is compared to other organisms such as fruit flies (Drosophila melanogaster), squid (Loligo pealei), and many others. The biosemiotic codon usage of each genomic population or ‘Theme’ is equated to a ‘molecular language’. The C/U choice or difference, and the G/A difference in the third nucleotide of the most used codons per amino acid are illustrated by comparing the most used codons per genome in humans and squids. The human distribution in the third position of most used codons is a 12-8-2, C-G-A, nucleotide ending signature, while the squid distribution in the third position of most used codons was an odd, or uneven, distribution in the third position of its most used codons: 13-6-3, U-A-G, as its nucleotide ending signature. These findings may help to design computational tools to compare human genomes, to determine the exchangeability between compatible codons and amino acids, and for the early detection of incompatible changes leading to hereditary diseases. PMID:22997484

Vertebrate codon bias indicates a highly GC-rich ancestral genome.

PubMed

Nabiyouni, Maryam; Prakash, Ashwin; Fedorov, Alexei

2013-04-25

Two factors are thought to have contributed to the origin of codon usage bias in eukaryotes: 1) genome-wide mutational forces that shape overall GC-content and create context-dependent nucleotide bias, and 2) positive selection for codons that maximize efficient and accurate translation. Particularly in vertebrates, these two explanations contradict each other and cloud the origin of codon bias in the taxon. On the one hand, mutational forces fail to explain GC-richness (~60%) of third codon positions, given the GC-poor overall genomic composition among vertebrates (~40%). On the other hand, positive selection cannot easily explain strict regularities in codon preferences. Large-scale bioinformatic assessment, of nucleotide composition of coding and non-coding sequences in vertebrates and other taxa, suggests a simple possible resolution for this contradiction. Specifically, we propose that the last common vertebrate ancestor had a GC-rich genome (~65% GC). The data suggest that whole-genome mutational bias is the major driving force for generating codon bias. As the bias becomes prominent, it begins to affect translation and can result in positive selection for optimal codons. The positive selection can, in turn, significantly modulate codon preferences. Copyright © 2013 Elsevier B.V. All rights reserved.

The Relation of Codon Bias to Tissue-Specific Gene Expression in Arabidopsis thaliana

PubMed Central

Camiolo, Salvatore; Farina, Lorenzo; Porceddu, Andrea

2012-01-01

The codon composition of coding sequences plays an important role in the regulation of gene expression. Herein, we report systematic differences in the usage of synonymous codons among Arabidopsis thaliana genes that are expressed specifically in distinct tissues. Although we observed that both regionally and transcriptionally associated mutational biases were associated significantly with codon bias, they could not explain the observed differences fully. Similarly, given that transcript abundances did not account for the differences in codon usage, it is unlikely that selection for translational efficiency can account exclusively for the observed codon bias. Thus, we considered the possible evolution of codon bias as an adaptive response to the different abundances of tRNAs in different tissues. Our analysis demonstrated that in some cases, codon usage in genes that were expressed in a broad range of tissues was influenced primarily by the tissue in which the gene was expressed maximally. On the basis of this finding we propose that genes that are expressed in certain tissues might show a tissue-specific compositional signature in relation to codon usage. These findings might have implications for the design of transgenes in relation to optimizing their expression. PMID:22865738

Analysis of the synonymous codon usage bias in recently emerged enterovirus D68 strains.

PubMed

Karniychuk, Uladzimir U

2016-09-02

Understanding the codon usage pattern of a pathogen and relationship between pathogen and host's codon usage patterns has fundamental and applied interests. Enterovirus D68 (EV-D68) is an emerging pathogen with a potentially high public health significance. In the present study, the synonymous codon usage bias of 27 recently emerged, and historical EV-D68 strains was analyzed. In contrast to previously studied enteroviruses (enterovirus 71 and poliovirus), EV-D68 and human host have a high discrepancy between favored codons. Analysis of viral synonymous codon usage bias metrics, viral nucleotide/dinucleotide compositional parameters, and viral protein properties showed that mutational pressure is more involved in shaping the synonymous codon usage bias of EV-D68 than translation selection. Computation of codon adaptation indices allowed to estimate expression potential of the EV-D68 genome in several commonly used laboratory animals. This approach requires experimental validation and may provide an auxiliary tool for the rational selection of laboratory animals to model emerging viral diseases. Enterovirus D68 genome compositional and codon usage data can be useful for further pathogenesis, animal model, and vaccine design studies. Copyright © 2016 Elsevier B.V. All rights reserved.

Differences in codon bias cannot explain differences in translational power among microbes.

PubMed

Dethlefsen, Les; Schmidt, Thomas M

2005-01-06

Translational power is the cellular rate of protein synthesis normalized to the biomass invested in translational machinery. Published data suggest a previously unrecognized pattern: translational power is higher among rapidly growing microbes, and lower among slowly growing microbes. One factor known to affect translational power is biased use of synonymous codons. The correlation within an organism between expression level and degree of codon bias among genes of Escherichia coli and other bacteria capable of rapid growth is commonly attributed to selection for high translational power. Conversely, the absence of such a correlation in some slowly growing microbes has been interpreted as the absence of selection for translational power. Because codon bias caused by translational selection varies between rapidly growing and slowly growing microbes, we investigated whether observed differences in translational power among microbes could be explained entirely by differences in the degree of codon bias. Although the data are not available to estimate the effect of codon bias in other species, we developed an empirically-based mathematical model to compare the translation rate of E. coli to the translation rate of a hypothetical strain which differs from E. coli only by lacking codon bias. Our reanalysis of data from the scientific literature suggests that translational power can differ by a factor of 5 or more between E. coli and slowly growing microbial species. Using empirical codon-specific in vivo translation rates for 29 codons, and several scenarios for extrapolating from these data to estimates over all codons, we find that codon bias cannot account for more than a doubling of the translation rate in E. coli, even with unrealistic simplifying assumptions that exaggerate the effect of codon bias. With more realistic assumptions, our best estimate is that codon bias accelerates translation in E. coli by no more than 60% in comparison to microbes with very little codon bias. While codon bias confers a substantial benefit of faster translation and hence greater translational power, the magnitude of this effect is insufficient to explain observed differences in translational power among bacterial and archaeal species, particularly the differences between slowly growing and rapidly growing species. Hence, large differences in translational power suggest that the translational apparatus itself differs among microbes in ways that influence translational performance.

Site-specific creation of uridine from cytidine in apolipoprotein B mRNA editing.

PubMed Central

Hodges, P E; Navaratnam, N; Greeve, J C; Scott, J

1991-01-01

Human apolipoprotein (apo) B mRNA is edited in a tissue specific reaction, to convert glutamine codon 2153 (CAA) to a stop translation codon. The RNA editing product templates and hybridises as uridine, but the chemical nature of this reaction and the physical identity of the product are unknown. After editing in vitro of [32P] labelled RNA, we are able to demonstrate the production of uridine from cytidine; [alpha 32P] cytidine triphosphate incorporated into RNA gave rise to [32P] uridine monophosphate after editing in vitro, hydrolysis with nuclease P1 and thin layer chromatography using two separation systems. By cleaving the RNA into ribonuclease T1 fragments, we show that uridine is produced only at the authentic editing site and is produced in quantities that parallel an independent primer extension assay for editing. We conclude that apo B mRNA editing specifically creates a uridine from a cytidine. These observations are inconsistent with the incorporation of a uridine nucleotide by any polymerase, which would replace the alpha-phosphate and so rule out a model of endonucleolytic excision and repair as the mechanism for the production of uridine. Although transamination and transglycosylation remain to be formally excluded as reaction mechanisms our results argue strongly in favour of the apo B mRNA editing enzyme as a site-specific cytidine deaminase. Images PMID:2030940

Analysis of adaptive evolution in Lyssavirus genomes reveals pervasive diversifying selection during species diversification.

PubMed

Voloch, Carolina M; Capellão, Renata T; Mello, Beatriz; Schrago, Carlos G

2014-11-19

Lyssavirus is a diverse genus of viruses that infect a variety of mammalian hosts, typically causing encephalitis. The evolution of this lineage, particularly the rabies virus, has been a focus of research because of the extensive occurrence of cross-species transmission, and the distinctive geographical patterns present throughout the diversification of these viruses. Although numerous studies have examined pattern-related questions concerning Lyssavirus evolution, analyses of the evolutionary processes acting on Lyssavirus diversification are scarce. To clarify the relevance of positive natural selection in Lyssavirus diversification, we conducted a comprehensive scan for episodic diversifying selection across all lineages and codon sites of the five coding regions in lyssavirus genomes. Although the genomes of these viruses are generally conserved, the glycoprotein (G), RNA-dependent RNA polymerase (L) and polymerase (P) genes were frequently targets of adaptive evolution during the diversification of the genus. Adaptive evolution is particularly manifest in the glycoprotein gene, which was inferred to have experienced the highest density of positively selected codon sites along branches. Substitutions in the L gene were found to be associated with the early diversification of phylogroups. A comparison between the number of positively selected sites inferred along the branches of RABV population branches and Lyssavirus intespecies branches suggested that the occurrence of positive selection was similar on the five coding regions of the genome in both groups.

Analysis of Adaptive Evolution in Lyssavirus Genomes Reveals Pervasive Diversifying Selection during Species Diversification

PubMed Central

Voloch, Carolina M.; Capellão, Renata T.; Mello, Beatriz; Schrago, Carlos G.

2014-01-01

Lyssavirus is a diverse genus of viruses that infect a variety of mammalian hosts, typically causing encephalitis. The evolution of this lineage, particularly the rabies virus, has been a focus of research because of the extensive occurrence of cross-species transmission, and the distinctive geographical patterns present throughout the diversification of these viruses. Although numerous studies have examined pattern-related questions concerning Lyssavirus evolution, analyses of the evolutionary processes acting on Lyssavirus diversification are scarce. To clarify the relevance of positive natural selection in Lyssavirus diversification, we conducted a comprehensive scan for episodic diversifying selection across all lineages and codon sites of the five coding regions in lyssavirus genomes. Although the genomes of these viruses are generally conserved, the glycoprotein (G), RNA-dependent RNA polymerase (L) and polymerase (P) genes were frequently targets of adaptive evolution during the diversification of the genus. Adaptive evolution is particularly manifest in the glycoprotein gene, which was inferred to have experienced the highest density of positively selected codon sites along branches. Substitutions in the L gene were found to be associated with the early diversification of phylogroups. A comparison between the number of positively selected sites inferred along the branches of RABV population branches and Lyssavirus intespecies branches suggested that the occurrence of positive selection was similar on the five coding regions of the genome in both groups. PMID:25415197

Analysis of codon usage bias of envelope glycoprotein genes in nuclear polyhedrosis virus (NPV) and its relation to evolution.

PubMed

Zhao, Yongchao; Zheng, Hao; Xu, Anying; Yan, Donghua; Jiang, Zijian; Qi, Qi; Sun, Jingchen

2016-08-24

Analysis of codon usage bias is an extremely versatile method using in furthering understanding of the genetic and evolutionary paths of species. Codon usage bias of envelope glycoprotein genes in nuclear polyhedrosis virus (NPV) has remained largely unexplored at present. Hence, the codon usage bias of NPV envelope glycoprotein was analyzed here to reveal the genetic and evolutionary relationships between different viral species in baculovirus genus. A total of 9236 codons from 18 different species of NPV of the baculovirus genera were used to perform this analysis. Glycoprotein of NPV exhibits weaker codon usage bias. Neutrality plot analysis and correlation analysis of effective number of codons (ENC) values indicate that natural selection is the main factor influencing codon usage bias, and that the impact of mutation pressure is relatively smaller. Another cluster analysis shows that the kinship or evolutionary relationships of these viral species can be divided into two broad categories despite all of these 18 species are from the same baculovirus genus. There are many elements that can affect codon bias, such as the composition of amino acids, mutation pressure, natural selection, gene expression level, and etc. In the meantime, cluster analysis also illustrates that codon usage bias of virus envelope glycoprotein can serve as an effective means of evolutionary classification in baculovirus genus.

HCV IRES domain IIb affects the configuration of coding RNA in the 40S subunit's decoding groove

PubMed Central

Filbin, Megan E.; Kieft, Jeffrey S.

2011-01-01

Hepatitis C virus (HCV) uses a structured internal ribosome entry site (IRES) RNA to recruit the translation machinery to the viral RNA and begin protein synthesis without the ribosomal scanning process required for canonical translation initiation. Different IRES structural domains are used in this process, which begins with direct binding of the 40S ribosomal subunit to the IRES RNA and involves specific manipulation of the translational machinery. We have found that upon initial 40S subunit binding, the stem–loop domain of the IRES that contains the start codon unwinds and adopts a stable configuration within the subunit's decoding groove. This configuration depends on the sequence and structure of a different stem–loop domain (domain IIb) located far from the start codon in sequence, but spatially proximal in the IRES•40S complex. Mutation of domain IIb results in misconfiguration of the HCV RNA in the decoding groove that includes changes in the placement of the AUG start codon, and a substantial decrease in the ability of the IRES to initiate translation. Our results show that two distal regions of the IRES are structurally communicating at the initial step of 40S subunit binding and suggest that this is an important step in driving protein synthesis. PMID:21606179

HCV IRES domain IIb affects the configuration of coding RNA in the 40S subunit's decoding groove.

PubMed

Filbin, Megan E; Kieft, Jeffrey S

2011-07-01

Hepatitis C virus (HCV) uses a structured internal ribosome entry site (IRES) RNA to recruit the translation machinery to the viral RNA and begin protein synthesis without the ribosomal scanning process required for canonical translation initiation. Different IRES structural domains are used in this process, which begins with direct binding of the 40S ribosomal subunit to the IRES RNA and involves specific manipulation of the translational machinery. We have found that upon initial 40S subunit binding, the stem-loop domain of the IRES that contains the start codon unwinds and adopts a stable configuration within the subunit's decoding groove. This configuration depends on the sequence and structure of a different stem-loop domain (domain IIb) located far from the start codon in sequence, but spatially proximal in the IRES•40S complex. Mutation of domain IIb results in misconfiguration of the HCV RNA in the decoding groove that includes changes in the placement of the AUG start codon, and a substantial decrease in the ability of the IRES to initiate translation. Our results show that two distal regions of the IRES are structurally communicating at the initial step of 40S subunit binding and suggest that this is an important step in driving protein synthesis.

The CUG-initiated larger form coat protein of Chinese wheat mosaic virus binds to the cysteine-rich RNA silencing suppressor.

PubMed

Sun, Liying; Andika, Ida Bagus; Shen, Jiangfeng; Yang, Di; Ratti, Claudio; Chen, Jianping

2013-10-01

Some viruses use alternative translation initiation at non-AUG codons as a strategy to produce multiple proteins during gene expression. Here we show that, using this strategy, Chinese wheat mosaic virus (CWMV; Furovirus) expresses a larger form of coat protein (N-ext/CP) in infected plants. Site-directed mutagenesis and transient expression analysis confirmed that CWMV N-ext/CP is initiated at an upstream in-frame CUG codon at nucleotide position 207-209 of RNA 2, which adds a 39 amino acid (aa) N-terminal extension to the major CP. Interestingly, in planta and in vitro analyses indicated that CWMV N-ext/CP but not CP interacts with the CWMV cysteine-rich protein (CRP), an RNA silencing suppressor. We further determined that the N-terminal 39 aa extension, particularly the 10 aa region immediately upstream of the major CP coding region is responsible for the interaction of N-ext/CP with CRP. In an Agrobacterium co-infiltration assay, co-expression with N-ext/CP did not affect CRP silencing suppression activity. Thus the alternative translation initiation at a CUG codon provides the CWMV N-ext/CP with the ability to bind to the viral silencing suppressor. Copyright © 2013 Elsevier B.V. All rights reserved.

Evolutionary and genetic analysis of the VP2 gene of canine parvovirus.

PubMed

Li, Gairu; Ji, Senlin; Zhai, Xiaofeng; Zhang, Yuxiang; Liu, Jie; Zhu, Mengyan; Zhou, Jiyong; Su, Shuo

2017-07-17

Canine parvovirus (CPV) type 2 emerged in 1978 in the USA and quickly spread among dog populations all over the world with high morbidity. Although CPV is a DNA virus, its genomic substitution rate is similar to some RNA viruses. Therefore, it is important to trace the evolution of CPV to monitor the appearance of mutations that might affect vaccine effectiveness. Our analysis shows that the VP2 genes of CPV isolated from 1979 to 2016 are divided into six groups: GI, GII, GIII, GIV, GV, and GVI. Amino acid mutation analysis revealed several undiscovered important mutation sites: F267Y, Y324I, and T440A. Of note, the evolutionary rate of the CPV VP2 gene from Asia and Europe decreased. Codon usage analysis showed that the VP2 gene of CPV exhibits high bias with an ENC ranging from 34.93 to 36.7. Furthermore, we demonstrate that natural selection plays a major role compared to mutation pressure driving CPV evolution. There are few studies on the codon usage of CPV. Here, we comprehensively studied the genetic evolution, codon usage pattern, and evolutionary characterization of the VP2 gene of CPV. The novel findings revealing the evolutionary process of CPV will greatly serve future CPV research.

Foamy virus reverse transcriptase is expressed independently from the Gag protein.

PubMed Central

Enssle, J; Jordan, I; Mauer, B; Rethwilm, A

1996-01-01

In the foamy virus (FV) subgroup of retroviruses the pol genes are located in the +1 reading frame relative to the gag genes and possess potential ATG initiation codons in their 5' regions. This genome organization suggests either a + 1 ribosomal frameshift to generate a Gag-Pol fusion protein, similar to all other retroviruses studied so far, or new initiation of Pol translation, as used by pararetroviruses, to express the Pol protein. By using a genetic approach we have ruled out the former possibility and provide evidence for the latter. Two down-mutations (M53 and M54) of the pol ATG codon were found to abolish replication and Pol protein expression of the human FV isolate. The introduction of a new ATG in mutation M55, 3' to the down-mutated ATG of mutation M53, restored replication competence, indicating that the pol ATG functions as a translational initiation codon. Two nonsense mutants (M56 and M57), which functionally separated gag and pol with respect to potential frame-shifting sites, were also replication-competent, providing further genetic evidence that FVs express the Pol protein independently from Gag. Our results show that during a particular step of the replication cycle, FVs differ fundamentally from all other retroviruses. Images Fig. 3 PMID:8633029

Codon usage analysis of photolyase encoding genes of cyanobacteria inhabiting diverse habitats.

PubMed

Rajneesh; Pathak, Jainendra; Kannaujiya, Vinod K; Singh, Shailendra P; Sinha, Rajeshwar P

2017-07-01

Nucleotide and amino acid compositions were studied to determine the genomic and structural relationship of photolyase gene in freshwater, marine and hot spring cyanobacteria. Among three habitats, photolyase encoding genes from hot spring cyanobacteria were found to have highest GC content. The genomic GC content was found to influence the codon usage and amino acid variability in photolyases. The third position of codon was found to have more effect on amino acid variability in photolyases than the first and second positions of codon. The variation of amino acids Ala, Asp, Glu, Gly, His, Leu, Pro, Gln, Arg and Val in photolyases of three different habitats was found to be controlled by first position of codon (G1C1). However, second position (G2C2) of codon regulates variation of Ala, Cys, Gly, Pro, Arg, Ser, Thr and Tyr contents in photolyases. Third position (G3C3) of codon controls incorporation of amino acids such as Ala, Phe, Gly, Leu, Gln, Pro, Arg, Ser, Thr and Tyr in photolyases from three habitats. Photolyase encoding genes of hot spring cyanobacteria have 85% codons with G or C at third position, whereas marine and freshwater cyanobacteria showed 82 and 60% codons, respectively, with G or C at third position. Principal component analysis (PCA) showed that GC content has a profound effect in separating the genes along the first major axis according to their RSCU (relative synonymous codon usage) values, and neutrality analysis indicated that mutational pressure has resulted in codon bias in photolyase genes of cyanobacteria.

Rare Autosomal Recessive Cardiac Valvular Form of Ehlers-Danlos Syndrome Results from Mutations in the COL1A2 Gene That Activate the Nonsense-Mediated RNA Decay Pathway

PubMed Central

Schwarze, Ulrike; Hata, Ryu-Ichiro; McKusick, Victor A.; Shinkai, Hiroshi; Hoyme, H. Eugene; Pyeritz, Reed E.; Byers, Peter H.

2004-01-01

Splice site mutations in the COL1A2 gene of type I collagen can give rise to forms of Ehlers-Danlos syndrome (EDS) because of partial or complete skipping of exon 6, as well as to mild, moderate, or lethal forms of osteogenesis imperfecta as a consequence of skipping of other exons. We identified three unrelated individuals with a rare recessively inherited form of EDS (characterized by joint hypermobility, skin hyperextensibility, and cardiac valvular defects); in two of them, COL1A2 messenger RNA (mRNA) instability results from compound heterozygosity for splice site mutations in the COL1A2 gene, and, in the third, it results from homozygosity for a nonsense codon. The splice site mutations led to use of cryptic splice donor sites, creation of a downstream premature termination codon, and extremely unstable mRNA. In the wild-type allele, the two introns (IVS11 and IVS24) in which these mutations occurred were usually spliced slowly in relation to their respective immediate upstream introns. In the mutant alleles, the upstream intron was removed, so that exon skipping could not occur. In the context of the mutation in IVS24, computer-generated folding of a short stretch of mRNA surrounding the mutation site demonstrated realignment of the relationships between the donor and acceptor sites that could facilitate use of a cryptic donor site. These findings suggest that the order of intron removal is an important variable in prediction of mutation outcome at splice sites and that folding of the nascent mRNA could be one element that contributes to determination of order of splicing. The complete absence of proα2(I) chains has the surprising effect of producing cardiac valvular disease without bone involvement. PMID:15077201

Rare autosomal recessive cardiac valvular form of Ehlers-Danlos syndrome results from mutations in the COL1A2 gene that activate the nonsense-mediated RNA decay pathway.

PubMed

Schwarze, Ulrike; Hata, Ryu-Ichiro; McKusick, Victor A; Shinkai, Hiroshi; Hoyme, H Eugene; Pyeritz, Reed E; Byers, Peter H

2004-05-01

Splice site mutations in the COL1A2 gene of type I collagen can give rise to forms of Ehlers-Danlos syndrome (EDS) because of partial or complete skipping of exon 6, as well as to mild, moderate, or lethal forms of osteogenesis imperfecta as a consequence of skipping of other exons. We identified three unrelated individuals with a rare recessively inherited form of EDS (characterized by joint hypermobility, skin hyperextensibility, and cardiac valvular defects); in two of them, COL1A2 messenger RNA (mRNA) instability results from compound heterozygosity for splice site mutations in the COL1A2 gene, and, in the third, it results from homozygosity for a nonsense codon. The splice site mutations led to use of cryptic splice donor sites, creation of a downstream premature termination codon, and extremely unstable mRNA. In the wild-type allele, the two introns (IVS11 and IVS24) in which these mutations occurred were usually spliced slowly in relation to their respective immediate upstream introns. In the mutant alleles, the upstream intron was removed, so that exon skipping could not occur. In the context of the mutation in IVS24, computer-generated folding of a short stretch of mRNA surrounding the mutation site demonstrated realignment of the relationships between the donor and acceptor sites that could facilitate use of a cryptic donor site. These findings suggest that the order of intron removal is an important variable in prediction of mutation outcome at splice sites and that folding of the nascent mRNA could be one element that contributes to determination of order of splicing. The complete absence of pro alpha 2(I) chains has the surprising effect of producing cardiac valvular disease without bone involvement.

Genes encoding intrinsic disorder in Eukaryota have high GC content

PubMed Central

Peng, Zhenling; Uversky, Vladimir N.

2016-01-01

ABSTRACT We analyze a correlation between the GC content in genes of 12 eukaryotic species and the level of intrinsic disorder in their corresponding proteins. Comprehensive computational analysis has revealed that the disordered regions in eukaryotes are encoded by the GC-enriched gene regions and that this enrichment is correlated with the amount of disorder and is present across proteins and species characterized by varying amounts of disorder. The GC enrichment is a result of higher rate of amino acid coded by GC-rich codons in the disordered regions. Individual amino acids have the same GC-content profile between different species. Eukaryotic proteins with the disordered regions encoded by the GC-enriched gene segments carry out important biological functions including interactions with RNAs, DNAs, nucleotides, binding of calcium and metal ions, are involved in transcription, transport, cell division and certain signaling pathways, and are localized primarily in nucleus, cytosol and cytoplasm. We also investigate a possible relationship between GC content, intrinsic disorder and protein evolution. Analysis of a devised “age” of amino acids, their disorder-promoting capacity and the GC-enrichment of their codons suggests that the early amino acids are mostly disorder-promoting and their codons are GC-rich while most of late amino acids are mostly order-promoting. PMID:28232902

Stringent Nucleotide Recognition by the Ribosome at the Middle Codon Position.

PubMed

Liu, Wei; Shin, Dongwon; Ng, Martin; Sanbonmatsu, Karissa Y; Tor, Yitzhak; Cooperman, Barry S

2017-08-29

Accurate translation of the genetic code depends on mRNA:tRNA codon:anticodon base pairing. Here we exploit an emissive, isosteric adenosine surrogate that allows direct measurement of the kinetics of codon:anticodon University of California base formation during protein synthesis. Our results suggest that codon:anticodon base pairing is subject to tighter constraints at the middle position than at the 5'- and 3'-positions, and further suggest a sequential mechanism of formation of the three base pairs in the codon:anticodon helix.

Genetic variability and natural selection at the ligand domain of the Duffy binding protein in brazilian Plasmodium vivax populations

PubMed Central

2010-01-01

Background Plasmodium vivax malaria is a major public health challenge in Latin America, Asia and Oceania, with 130-435 million clinical cases per year worldwide. Invasion of host blood cells by P. vivax mainly depends on a type I membrane protein called Duffy binding protein (PvDBP). The erythrocyte-binding motif of PvDBP is a 170 amino-acid stretch located in its cysteine-rich region II (PvDBPII), which is the most variable segment of the protein. Methods To test whether diversifying natural selection has shaped the nucleotide diversity of PvDBPII in Brazilian populations, this region was sequenced in 122 isolates from six different geographic areas. A Bayesian method was applied to test for the action of natural selection under a population genetic model that incorporates recombination. The analysis was integrated with a structural model of PvDBPII, and T- and B-cell epitopes were localized on the 3-D structure. Results The results suggest that: (i) recombination plays an important role in determining the haplotype structure of PvDBPII, and (ii) PvDBPII appears to contain neutrally evolving codons as well as codons evolving under natural selection. Diversifying selection preferentially acts on sites identified as epitopes, particularly on amino acid residues 417, 419, and 424, which show strong linkage disequilibrium. Conclusions This study shows that some polymorphisms of PvDBPII are present near the erythrocyte-binding domain and might serve to elude antibodies that inhibit cell invasion. Therefore, these polymorphisms should be taken into account when designing vaccines aimed at eliciting antibodies to inhibit erythrocyte invasion. PMID:21092207

Dysfunctional growth hormone receptor in a strain of sex-linked dwarf chicken: evidence for a mutation in the intracellular domain.

PubMed

Agarwal, S K; Cogburn, L A; Burnside, J

1994-09-01

The sex-linked dwarf (dwdw) chicken represents a valuable animal model for studying GH insensitivity and the consequence of mutations in the GH receptor (GHR) gene. We have recently reported undetectable hepatic GH-binding activity and an aberrantly sized transcript in a strain of dwdw chickens obtained from Arbor Acre Farms, Inc. (Glastonbury, CT, USA). Southern blot analysis of the chicken GHR (cGHR) gene revealed a restriction-fragment length polymorphism in HindIII and EcoRI digests of genomic DNA in this strain of dwdw chicken. In order to localize the molecular mutation, we analysed the gene structure and determined the complete sequence of the 3' untranslated region (3' UTR) of the normal cGHR. With the use of this information, we located a large deletion in the 3' end of the cGHR gene of the Connecticut (CT) strain of dwdw chicken. This deletion (1773 bp) contained 27 highly conserved amino acids of the 3' end of the coding region, the in-frame stop codon, a less frequently used poly(A) signal that is normally found 445 bp downstream of the stop codon, and a large portion of the 3' UTR. Because of this deletion, 27 novel amino acids were substituted and the open reading frame was extended for an additional 26 amino acids before reaching the transcriptional termination site. The predicted amino acid sequence of the novel carboxyl-terminus of the dwdw cGHR is largely hydrophobic with a polylysine tail, whereas the carboxyl-terminus of the wild-type (DwDw) cGHR is composed of hydrophilic amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutations in the Small GTPase Gene RAB39B Are Responsible for X-linked Mental Retardation Associated with Autism, Epilepsy, and Macrocephaly

PubMed Central

Giannandrea, Maila; Bianchi, Veronica; Mignogna, Maria Lidia; Sirri, Alessandra; Carrabino, Salvatore; D'Elia, Errico; Vecellio, Matteo; Russo, Silvia; Cogliati, Francesca; Larizza, Lidia; Ropers, Hans-Hilger; Tzschach, Andreas; Kalscheuer, Vera; Oehl-Jaschkowitz, Barbara; Skinner, Cindy; Schwartz, Charles E.; Gecz, Jozef; Van Esch, Hilde; Raynaud, Martine; Chelly, Jamel; de Brouwer, Arjan P.M.; Toniolo, Daniela; D'Adamo, Patrizia

2010-01-01

Human Mental Retardation (MR) is a common and highly heterogeneous pediatric disorder affecting around 3% of the general population; at least 215 X-linked MR (XLMR) conditions have been described, and mutations have been identified in 83 different genes, encoding proteins with a variety of function, such as chromatin remodeling, synaptic function, and intracellular trafficking. The small GTPases of the RAB family, which play an essential role in intracellular vesicular trafficking, have been shown to be involved in MR. We report here the identification of mutations in the small GTPase RAB39B gene in two male patients. One mutation in family X (D-23) introduced a stop codon seven amino acids after the start codon (c.21C > A; p.Y7X). A second mutation, in the MRX72 family, altered the 5′ splice site (c.215+1G > A) and normal splicing. Neither instance produced a protein. Mutations segregate with the disease in the families, and in some family members intellectual disabilities were associated with autism spectrum disorder, epileptic seizures, and macrocephaly. We show that RAB39B, a novel RAB GTPase of unknown function, is a neuronal-specific protein that is localized to the Golgi compartment. Its downregulation leads to an alteration in the number and morphology of neurite growth cones and a significant reduction in presynaptic buttons, suggesting that RAB39B is required for synapse formation and maintenance. Our results demonstrate developmental and functional neuronal alteration as a consequence of downregulation of RAB39B and emphasize the critical role of vesicular trafficking in the development of neurons and human intellectual abilities. PMID:20159109

Balanced Codon Usage Optimizes Eukaryotic Translational Efficiency

PubMed Central

Qian, Wenfeng; Yang, Jian-Rong; Pearson, Nathaniel M.; Maclean, Calum; Zhang, Jianzhi

2012-01-01

Cellular efficiency in protein translation is an important fitness determinant in rapidly growing organisms. It is widely believed that synonymous codons are translated with unequal speeds and that translational efficiency is maximized by the exclusive use of rapidly translated codons. Here we estimate the in vivo translational speeds of all sense codons from the budding yeast Saccharomyces cerevisiae. Surprisingly, preferentially used codons are not translated faster than unpreferred ones. We hypothesize that this phenomenon is a result of codon usage in proportion to cognate tRNA concentrations, the optimal strategy in enhancing translational efficiency under tRNA shortage. Our predicted codon–tRNA balance is indeed observed from all model eukaryotes examined, and its impact on translational efficiency is further validated experimentally. Our study reveals a previously unsuspected mechanism by which unequal codon usage increases translational efficiency, demonstrates widespread natural selection for translational efficiency, and offers new strategies to improve synthetic biology. PMID:22479199

Tail-extension following the termination codon is critical for release of the nascent chain from membrane-bound ribosomes in a reticulocyte lysate cell-free system.

PubMed

Takahara, Michiyo; Sakaue, Haruka; Onishi, Yukiko; Yamagishi, Marifu; Kida, Yuichiro; Sakaguchi, Masao

2013-01-11

Nascent chain release from membrane-bound ribosomes by the termination codon was investigated using a cell-free translation system from rabbit supplemented with rough microsomal membrane vesicles. Chain release was extremely slow when mRNA ended with only the termination codon. Tail extension after the termination codon enhanced the release of the nascent chain. Release reached plateau levels with tail extension of 10 bases. This requirement was observed with all termination codons: TAA, TGA and TAG. Rapid release was also achieved by puromycin even in the absence of the extension. Efficient translation termination cannot be achieved in the presence of only a termination codon on the mRNA. Tail extension might be required for correct positioning of the termination codon in the ribosome and/or efficient recognition by release factors. Copyright © 2012. Published by Elsevier Inc.

A common periodic table of codons and amino acids.

PubMed

Biro, J C; Benyó, B; Sansom, C; Szlávecz, A; Fördös, G; Micsik, T; Benyó, Z

2003-06-27

A periodic table of codons has been designed where the codons are in regular locations. The table has four fields (16 places in each) one with each of the four nucleotides (A, U, G, C) in the central codon position. Thus, AAA (lysine), UUU (phenylalanine), GGG (glycine), and CCC (proline) were placed into the corners of the fields as the main codons (and amino acids) of the fields. They were connected to each other by six axes. The resulting nucleic acid periodic table showed perfect axial symmetry for codons. The corresponding amino acid table also displaced periodicity regarding the biochemical properties (charge and hydropathy) of the 20 amino acids and the position of the stop signals. The table emphasizes the importance of the central nucleotide in the codons and predicts that purines control the charge while pyrimidines determine the polarity of the amino acids. This prediction was experimentally tested.

Codon usage and amino acid usage influence genes expression level.

PubMed

Paul, Prosenjit; Malakar, Arup Kumar; Chakraborty, Supriyo

2018-02-01

Highly expressed genes in any species differ in the usage frequency of synonymous codons. The relative recurrence of an event of the favored codon pair (amino acid pairs) varies between gene and genomes due to varying gene expression and different base composition. Here we propose a new measure for predicting the gene expression level, i.e., codon plus amino bias index (CABI). Our approach is based on the relative bias of the favored codon pair inclination among the genes, illustrated by analyzing the CABI score of the Medicago truncatula genes. CABI showed strong correlation with all other widely used measures (CAI, RCBS, SCUO) for gene expression analysis. Surprisingly, CABI outperforms all other measures by showing better correlation with the wet-lab data. This emphasizes the importance of the neighboring codons of the favored codon in a synonymous group while estimating the expression level of a gene.

Absence of opioid stress-induced analgesia in mice lacking beta-endorphin by site-directed mutagenesis.

PubMed Central

Rubinstein, M; Mogil, J S; Japón, M; Chan, E C; Allen, R G; Low, M J

1996-01-01

A physiological role for beta-endorphin in endogenous pain inhibition was investigated by targeted mutagenesis of the proopiomelanocortin gene in mouse embryonic stem cells. The tyrosine codon at position 179 of the proopiomelanocortin gene was converted to a premature translational stop codon. The resulting transgenic mice display no overt developmental or behavioral alterations and have a normally functioning hypothalamic-pituitary-adrenal axis. Homozygous transgenic mice with a selective deficiency of beta-endorphin exhibit normal analgesia in response to morphine, indicating the presence of functional mu-opiate receptors. However, these mice lack the opioid (naloxone reversible) analgesia induced by mild swim stress. Mutant mice also display significantly greater nonopioid analgesia in response to cold water swim stress compared with controls and display paradoxical naloxone-induced analgesia. These changes may reflect compensatory upregulation of alternative pain inhibitory mechanisms. Images Fig. 1 Fig. 2 PMID:8633004

A more accurate detection of codon 72 polymorphism and LOH of the TP53 gene.

PubMed

Baccouche, Sami; Mabrouk, Imed; Said, Salem; Mosbah, Ali; Jlidi, Rachid; Gargouri, Ali

2003-01-10

The polymorphism at codon 72 of the TP53 gene has been extensively studied for its involvement in cancerogenesis and loss of heterozygosity (LOH) detection. Usually, the exon 4 of the TP53 gene is amplified by polymerase chain reaction (PCR) on DNA extracted from blood and tumor tissues, then digested by AccII. In the case of heterozygosity, the comparison of AccII profile from blood and tumor DNA PCR products allowed the identification of a potential LOH in the TP53 locus. This method can be hindered by a partial AccII digestion and/or DNA contamination of non-tumor cells. To circumvent these problems, we have developed a new approach by using the AccII restriction site between exon 4 and exon 6. The PCR amplification of exon 4-6, followed by AccII digestion allowed us to detect without ambiguity any LOH case.

Puromycin and Methotrexate Resistance Cassettes and Optimized cre-recombinase Expression Plasmids for use in Yeast

PubMed Central

MacDonald, Chris; Piper, Robert C.

2015-01-01

Here we expand the set of tools for genetically manipulating Saccharomyces cerevisiae. We show that puromycin-resistance can be achieved in yeast through expression of a bacterial puromycin-resistance gene optimized to the yeast codon bias, which in turn serves as an easy to use dominant genetic marker suitable for gene disruption. We have constructed a similar DNA cassette expressing yeast codon-optimized mutant human dihydrofolate reductase (DHFR) that confers resistance to methotrexate and can also be used as a dominant selectable marker. Both of these drug-resistant marker cassettes are flanked by loxP sites allowing for their excision from the genome following expression of cre-recombinase. Finally, we have created a series of plasmids for low-level constitutive expression of cre-recombinase in yeast that allows for efficient excision of loxP-flanked markers. PMID:25688547

Large-Scale Genomic Analysis of Codon Usage in Dengue Virus and Evaluation of Its Phylogenetic Dependence

PubMed Central

Lara-Ramírez, Edgar E.; Salazar, Ma Isabel; López-López, María de Jesús; Salas-Benito, Juan Santiago; Sánchez-Varela, Alejandro

2014-01-01

The increasing number of dengue virus (DENV) genome sequences available allows identifying the contributing factors to DENV evolution. In the present study, the codon usage in serotypes 1–4 (DENV1–4) has been explored for 3047 sequenced genomes using different statistics methods. The correlation analysis of total GC content (GC) with GC content at the three nucleotide positions of codons (GC1, GC2, and GC3) as well as the effective number of codons (ENC, ENCp) versus GC3 plots revealed mutational bias and purifying selection pressures as the major forces influencing the codon usage, but with distinct pressure on specific nucleotide position in the codon. The correspondence analysis (CA) and clustering analysis on relative synonymous codon usage (RSCU) within each serotype showed similar clustering patterns to the phylogenetic analysis of nucleotide sequences for DENV1–4. These clustering patterns are strongly related to the virus geographic origin. The phylogenetic dependence analysis also suggests that stabilizing selection acts on the codon usage bias. Our analysis of a large scale reveals new feature on DENV genomic evolution. PMID:25136631

Di-codon Usage for Gene Classification

NASA Astrophysics Data System (ADS)

Nguyen, Minh N.; Ma, Jianmin; Fogel, Gary B.; Rajapakse, Jagath C.

Classification of genes into biologically related groups facilitates inference of their functions. Codon usage bias has been described previously as a potential feature for gene classification. In this paper, we demonstrate that di-codon usage can further improve classification of genes. By using both codon and di-codon features, we achieve near perfect accuracies for the classification of HLA molecules into major classes and sub-classes. The method is illustrated on 1,841 HLA sequences which are classified into two major classes, HLA-I and HLA-II. Major classes are further classified into sub-groups. A binary SVM using di-codon usage patterns achieved 99.95% accuracy in the classification of HLA genes into major HLA classes; and multi-class SVM achieved accuracy rates of 99.82% and 99.03% for sub-class classification of HLA-I and HLA-II genes, respectively. Furthermore, by combining codon and di-codon usages, the prediction accuracies reached 100%, 99.82%, and 99.84% for HLA major class classification, and for sub-class classification of HLA-I and HLA-II genes, respectively.

tRNA1Ser(G34) with the anticodon GGA can recognize not only UCC and UCU codons but also UCA and UCG codons.

PubMed

Yamada, Yuko; Matsugi, Jitsuhiro; Ishikura, Hisayuki

2003-04-15

The tRNA1Ser (anticodon VGA, V=uridin-5-oxyacetic acid) is essential for translation of the UCA codon in Escherichia coli. Here, we studied the translational abilities of serine tRNA derivatives, which have different bases from wild type at the first positions of their anticodons, using synthetic mRNAs containing the UCN (N=A, G, C, or U) codon. The tRNA1Ser(G34) having the anticodon GGA was able to read not only UCC and UCU codons but also UCA and UCG codons. This means that the formation of G-A or G-G pair allowed at the wobble position and these base pairs are noncanonical. The translational efficiency of the tRNA1Ser(G34) for UCA or UCG codon depends on the 2'-O-methylation of the C32 (Cm). The 2'-O-methylation of C32 may give rise to the space necessary for G-A or G-G base pair formation between the first position of anticodon and the third position of codon.

Comparative evolutionary genomics of Corynebacterium with special reference to codon and amino acid usage diversities.

PubMed

Pal, Shilpee; Sarkar, Indrani; Roy, Ayan; Mohapatra, Pradeep K Das; Mondal, Keshab C; Sen, Arnab

2018-02-01

The present study has been aimed to the comparative analysis of high GC composition containing Corynebacterium genomes and their evolutionary study by exploring codon and amino acid usage patterns. Phylogenetic study by MLSA approach, indel analysis and BLAST matrix differentiated Corynebacterium species in pathogenic and non-pathogenic clusters. Correspondence analysis on synonymous codon usage reveals that, gene length, optimal codon frequencies and tRNA abundance affect the gene expression of Corynebacterium. Most of the optimal codons as well as translationally optimal codons are C ending i.e. RNY (R-purine, N-any nucleotide base, and Y-pyrimidine) and reveal translational selection pressure on codon bias of Corynebacterium. Amino acid usage is affected by hydrophobicity, aromaticity, protein energy cost, etc. Highly expressed genes followed the cost minimization hypothesis and are less diverged at their synonymous positions of codons. Functional analysis of core genes shows significant difference in pathogenic and non-pathogenic Corynebacterium. The study reveals close relationship between non-pathogenic and opportunistic pathogenic Corynebaterium as well as between molecular evolution and survival niches of the organism.

Codon usage bias and tRNA over-expression in Buchnera aphidicola after aromatic amino acid nutritional stress on its host Acyrthosiphon pisum.

PubMed

Charles, Hubert; Calevro, Federica; Vinuelas, José; Fayard, Jean-Michel; Rahbe, Yvan

2006-01-01

Codon usage bias and relative abundances of tRNA isoacceptors were analysed in the obligate intracellular symbiotic bacterium, Buchnera aphidicola from the aphid Acyrthosiphon pisum, using a dedicated 35mer oligonucleotide microarray. Buchnera is archetypal of organisms living with minimal metabolic requirements and presents a reduced genome with high-evolutionary rate. Codonusage in Buchnera has been overcome by the high mutational bias towards AT bases. However, several lines of evidence for codon usage selection are given here. A significant correlation was found between tRNA relative abundances and codon composition of Buchnera genes. A significant codon usage bias was found for the choice of rare codons in Buchnera: C-ending codons are preferred in highly expressed genes, whereas G-ending codons are avoided. This bias is not explained by GC skew in the bacteria and might correspond to a selection for perfect matching between codon-anticodon pairs for some essential amino acids in Buchnera proteins. Nutritional stress applied to the aphid host induced a significant overexpression of most of the tRNA isoacceptors in bacteria. Although, molecular regulation of the tRNA operons in Buchnera was not investigated, a correlation between relative expression levels and organization in transcription unit was found in the genome of Buchnera.

Codon optimization of the adenoviral fiber negatively impacts structural protein expression and viral fitness

NASA Astrophysics Data System (ADS)

Villanueva, Eneko; Martí-Solano, Maria; Fillat, Cristina

2016-06-01

Codon usage adaptation of lytic viruses to their hosts is determinant for viral fitness. In this work, we analyzed the codon usage of adenoviral proteins by principal component analysis and assessed their codon adaptation to the host. We observed a general clustering of adenoviral proteins according to their function. However, there was a significant variation in the codon preference between the host-interacting fiber protein and the rest of structural late phase proteins, with a non-optimal codon usage of the fiber. To understand the impact of codon bias in the fiber, we optimized the Adenovirus-5 fiber to the codon usage of the hexon structural protein. The optimized fiber displayed increased expression in a non-viral context. However, infection with adenoviruses containing the optimized fiber resulted in decreased expression of the fiber and of wild-type structural proteins. Consequently, this led to a drastic reduction in viral release. The insertion of an exogenous optimized protein as a late gene in the adenovirus with the optimized fiber further interfered with viral fitness. These results highlight the importance of balancing codon usage in viral proteins to adequately exploit cellular resources for efficient infection and open new opportunities to regulate viral fitness for virotherapy and vaccine development.

ChloroMitoCU: Codon patterns across organelle genomes for functional genomics and evolutionary applications.

PubMed

Sablok, Gaurav; Chen, Ting-Wen; Lee, Chi-Ching; Yang, Chi; Gan, Ruei-Chi; Wegrzyn, Jill L; Porta, Nicola L; Nayak, Kinshuk C; Huang, Po-Jung; Varotto, Claudio; Tang, Petrus

2017-06-01

Organelle genomes are widely thought to have arisen from reduction events involving cyanobacterial and archaeal genomes, in the case of chloroplasts, or α-proteobacterial genomes, in the case of mitochondria. Heterogeneity in base composition and codon preference has long been the subject of investigation of topics ranging from phylogenetic distortion to the design of overexpression cassettes for transgenic expression. From the overexpression point of view, it is critical to systematically analyze the codon usage patterns of the organelle genomes. In light of the importance of codon usage patterns in the development of hyper-expression organelle transgenics, we present ChloroMitoCU, the first-ever curated, web-based reference catalog of the codon usage patterns in organelle genomes. ChloroMitoCU contains the pre-compiled codon usage patterns of 328 chloroplast genomes (29,960 CDS) and 3,502 mitochondrial genomes (49,066 CDS), enabling genome-wide exploration and comparative analysis of codon usage patterns across species. ChloroMitoCU allows the phylogenetic comparison of codon usage patterns across organelle genomes, the prediction of codon usage patterns based on user-submitted transcripts or assembled organelle genes, and comparative analysis with the pre-compiled patterns across species of interest. ChloroMitoCU can increase our understanding of the biased patterns of codon usage in organelle genomes across multiple clades. ChloroMitoCU can be accessed at: http://chloromitocu.cgu.edu.tw/. © The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Efficient Reassignment of a Frequent Serine Codon in Wild-Type Escherichia coli.

PubMed

Ho, Joanne M; Reynolds, Noah M; Rivera, Keith; Connolly, Morgan; Guo, Li-Tao; Ling, Jiqiang; Pappin, Darryl J; Church, George M; Söll, Dieter

2016-02-19

Expansion of the genetic code through engineering the translation machinery has greatly increased the chemical repertoire of the proteome. This has been accomplished mainly by read-through of UAG or UGA stop codons by the noncanonical aminoacyl-tRNA of choice. While stop codon read-through involves competition with the translation release factors, sense codon reassignment entails competition with a large pool of endogenous tRNAs. We used an engineered pyrrolysyl-tRNA synthetase to incorporate 3-iodo-l-phenylalanine (3-I-Phe) at a number of different serine and leucine codons in wild-type Escherichia coli. Quantitative LC-MS/MS measurements of amino acid incorporation yields carried out in a selected reaction monitoring experiment revealed that the 3-I-Phe abundance at the Ser208AGU codon in superfolder GFP was 65 ± 17%. This method also allowed quantification of other amino acids (serine, 33 ± 17%; phenylalanine, 1 ± 1%; threonine, 1 ± 1%) that compete with 3-I-Phe at both the aminoacylation and decoding steps of translation for incorporation at the same codon position. Reassignments of different serine (AGU, AGC, UCG) and leucine (CUG) codons with the matching tRNA(Pyl) anticodon variants were met with varying success, and our findings provide a guideline for the choice of sense codons to be reassigned. Our results indicate that the 3-iodo-l-phenylalanyl-tRNA synthetase (IFRS)/tRNA(Pyl) pair can efficiently outcompete the cellular machinery to reassign select sense codons in wild-type E. coli.

Drosophila Muller F Elements Maintain a Distinct Set of Genomic Properties Over 40 Million Years of Evolution

PubMed Central

Leung, Wilson; Shaffer, Christopher D.; Reed, Laura K.; Smith, Sheryl T.; Barshop, William; Dirkes, William; Dothager, Matthew; Lee, Paul; Wong, Jeannette; Xiong, David; Yuan, Han; Bedard, James E. J.; Machone, Joshua F.; Patterson, Seantay D.; Price, Amber L.; Turner, Bryce A.; Robic, Srebrenka; Luippold, Erin K.; McCartha, Shannon R.; Walji, Tezin A.; Walker, Chelsea A.; Saville, Kenneth; Abrams, Marita K.; Armstrong, Andrew R.; Armstrong, William; Bailey, Robert J.; Barberi, Chelsea R.; Beck, Lauren R.; Blaker, Amanda L.; Blunden, Christopher E.; Brand, Jordan P.; Brock, Ethan J.; Brooks, Dana W.; Brown, Marie; Butzler, Sarah C.; Clark, Eric M.; Clark, Nicole B.; Collins, Ashley A.; Cotteleer, Rebecca J.; Cullimore, Peterson R.; Dawson, Seth G.; Docking, Carter T.; Dorsett, Sasha L.; Dougherty, Grace A.; Downey, Kaitlyn A.; Drake, Andrew P.; Earl, Erica K.; Floyd, Trevor G.; Forsyth, Joshua D.; Foust, Jonathan D.; Franchi, Spencer L.; Geary, James F.; Hanson, Cynthia K.; Harding, Taylor S.; Harris, Cameron B.; Heckman, Jonathan M.; Holderness, Heather L.; Howey, Nicole A.; Jacobs, Dontae A.; Jewell, Elizabeth S.; Kaisler, Maria; Karaska, Elizabeth A.; Kehoe, James L.; Koaches, Hannah C.; Koehler, Jessica; Koenig, Dana; Kujawski, Alexander J.; Kus, Jordan E.; Lammers, Jennifer A.; Leads, Rachel R.; Leatherman, Emily C.; Lippert, Rachel N.; Messenger, Gregory S.; Morrow, Adam T.; Newcomb, Victoria; Plasman, Haley J.; Potocny, Stephanie J.; Powers, Michelle K.; Reem, Rachel M.; Rennhack, Jonathan P.; Reynolds, Katherine R.; Reynolds, Lyndsey A.; Rhee, Dong K.; Rivard, Allyson B.; Ronk, Adam J.; Rooney, Meghan B.; Rubin, Lainey S.; Salbert, Luke R.; Saluja, Rasleen K.; Schauder, Taylor; Schneiter, Allison R.; Schulz, Robert W.; Smith, Karl E.; Spencer, Sarah; Swanson, Bryant R.; Tache, Melissa A.; Tewilliager, Ashley A.; Tilot, Amanda K.; VanEck, Eve; Villerot, Matthew M.; Vylonis, Megan B.; Watson, David T.; Wurzler, Juliana A.; Wysocki, Lauren M.; Yalamanchili, Monica; Zaborowicz, Matthew A.; Emerson, Julia A.; Ortiz, Carlos; Deuschle, Frederic J.; DiLorenzo, Lauren A.; Goeller, Katie L.; Macchi, Christopher R.; Muller, Sarah E.; Pasierb, Brittany D.; Sable, Joseph E.; Tucci, Jessica M.; Tynon, Marykathryn; Dunbar, David A.; Beken, Levent H.; Conturso, Alaina C.; Danner, Benjamin L.; DeMichele, Gabriella A.; Gonzales, Justin A.; Hammond, Maureen S.; Kelley, Colleen V.; Kelly, Elisabeth A.; Kulich, Danielle; Mageeney, Catherine M.; McCabe, Nikie L.; Newman, Alyssa M.; Spaeder, Lindsay A.; Tumminello, Richard A.; Revie, Dennis; Benson, Jonathon M.; Cristostomo, Michael C.; DaSilva, Paolo A.; Harker, Katherine S.; Jarrell, Jenifer N.; Jimenez, Luis A.; Katz, Brandon M.; Kennedy, William R.; Kolibas, Kimberly S.; LeBlanc, Mark T.; Nguyen, Trung T.; Nicolas, Daniel S.; Patao, Melissa D.; Patao, Shane M.; Rupley, Bryan J.; Sessions, Bridget J.; Weaver, Jennifer A.; Goodman, Anya L.; Alvendia, Erica L.; Baldassari, Shana M.; Brown, Ashley S.; Chase, Ian O.; Chen, Maida; Chiang, Scott; Cromwell, Avery B.; Custer, Ashley F.; DiTommaso, Tia M.; El-Adaimi, Jad; Goscinski, Nora C.; Grove, Ryan A.; Gutierrez, Nestor; Harnoto, Raechel S.; Hedeen, Heather; Hong, Emily L.; Hopkins, Barbara L.; Huerta, Vilma F.; Khoshabian, Colin; LaForge, Kristin M.; Lee, Cassidy T.; Lewis, Benjamin M.; Lydon, Anniken M.; Maniaci, Brian J.; Mitchell, Ryan D.; Morlock, Elaine V.; Morris, William M.; Naik, Priyanka; Olson, Nicole C.; Osterloh, Jeannette M.; Perez, Marcos A.; Presley, Jonathan D.; Randazzo, Matt J.; Regan, Melanie K.; Rossi, Franca G.; Smith, Melanie A.; Soliterman, Eugenia A.; Sparks, Ciani J.; Tran, Danny L.; Wan, Tiffany; Welker, Anne A.; Wong, Jeremy N.; Sreenivasan, Aparna; Youngblom, Jim; Adams, Andrew; Alldredge, Justin; Bryant, Ashley; Carranza, David; Cifelli, Alyssa; Coulson, Kevin; Debow, Calise; Delacruz, Noelle; Emerson, Charlene; Farrar, Cassandra; Foret, Don; Garibay, Edgar; Gooch, John; Heslop, Michelle; Kaur, Sukhjit; Khan, Ambreen; Kim, Van; Lamb, Travis; Lindbeck, Peter; Lucas, Gabi; Macias, Elizabeth; Martiniuc, Daniela; Mayorga, Lissett; Medina, Joseph; Membreno, Nelson; Messiah, Shady; Neufeld, Lacey; Nguyen, San Francisco; Nichols, Zachary; Odisho, George; Peterson, Daymon; Rodela, Laura; Rodriguez, Priscilla; Rodriguez, Vanessa; Ruiz, Jorge; Sherrill, Will; Silva, Valeria; Sparks, Jeri; Statton, Geeta; Townsend, Ashley; Valdez, Isabel; Waters, Mary; Westphal, Kyle; Winkler, Stacey; Zumkehr, Joannee; DeJong, Randall J.; Hoogewerf, Arlene J.; Ackerman, Cheri M.; Armistead, Isaac O.; Baatenburg, Lara; Borr, Matthew J.; Brouwer, Lindsay K.; Burkhart, Brandon J.; Bushhouse, Kelsey T.; Cesko, Lejla; Choi, Tiffany Y. Y.; Cohen, Heather; Damsteegt, Amanda M.; Darusz, Jess M.; Dauphin, Cory M.; Davis, Yelena P.; Diekema, Emily J.; Drewry, Melissa; Eisen, Michelle E. 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Fosselman, Daniel S.; French, Kristin M.; Gill, Ishwar S.; Havill, Jessen T.; Johnson, Jaelyn L.; Keny, Lauren J.; Kerber, John M.; Klett, Bethany M.; Kufel, Christina N.; May, Francis J.; Mecoli, Jonathan P.; Merry, Callie R.; Meyer, Lauren R.; Miller, Emily G.; Mullen, Gregory J.; Palozola, Katherine C.; Pfeil, Jacob J.; Thomas, Jessica G.; Verbofsky, Evan M.; Spana, Eric P.; Agarwalla, Anant; Chapman, Julia; Chlebina, Ben; Chong, Insun; Falk, I.N.; Fitzgibbons, John D.; Friedman, Harrison; Ighile, Osagie; Kim, Andrew J.; Knouse, Kristin A.; Kung, Faith; Mammo, Danny; Ng, Chun Leung; Nikam, Vinayak S.; Norton, Diana; Pham, Philip; Polk, Jessica W.; Prasad, Shreya; Rankin, Helen; Ratliff, Camille D.; Scala, Victoria; Schwartz, Nicholas U.; Shuen, Jessica A.; Xu, Amy; Xu, Thomas Q.; Zhang, Yi; Rosenwald, Anne G.; Burg, Martin G.; Adams, Stephanie J.; Baker, Morgan; Botsford, Bobbi; Brinkley, Briana; Brown, Carter; Emiah, Shadie; Enoch, Erica; Gier, Chad; Greenwell, Alyson; Hoogenboom, Lindsay; 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Minnick, Phillip J.; Musick, Adam E.; Newhart, William H.; Noynaert, Joseph W.; Ogden, Bradley J.; Sandusky, Michael W.; Schmuecker, Samantha M.; Shipman, Anna L.; Smith, Anna L.; Thomsen, Kristen M.; Unzicker, Matthew R.; Vernon, William B.; Winn, Wesley W.; Woyski, Dustin S.; Zhu, Xiao; Du, Chunguang; Ament, Caitlin; Aso, Soham; Bisogno, Laura Simone; Caronna, Jason; Fefelova, Nadezhda; Lopez, Lenin; Malkowitz, Lorraine; Marra, Jonathan; Menillo, Daniella; Obiorah, Ifeanyi; Onsarigo, Eric Nyabeta; Primus, Shekerah; Soos, Mahdi; Tare, Archana; Zidan, Ameer; Jones, Christopher J.; Aronhalt, Todd; Bellush, James M.; Burke, Christa; DeFazio, Steve; Does, Benjamin R.; Johnson, Todd D.; Keysock, Nicholas; Knudsen, Nelson H.; Messler, James; Myirski, Kevin; Rekai, Jade Lea; Rempe, Ryan Michael; Salgado, Michael S.; Stagaard, Erica; Starcher, Justin R.; Waggoner, Andrew W.; Yemelyanova, Anastasia K.; Hark, Amy T.; Bertolet, Anne; Kuschner, Cyrus E.; Parry, Kesley; Quach, Michael; Shantzer, Lindsey; 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Gloria C.; Augustine, LaJerald; Davis-Reyes, Brionna; Echendu, Vivienne; Hales, Jasmine; Ibarra, Sharon; Johnson, Lauriaun; Ovu, Steven; Braverman, John M.; Bahr, Thomas J.; Caesar, Nicole M.; Campana, Christopher; Cassidy, Daniel W.; Cognetti, Peter A.; English, Johnathan D.; Fadus, Matthew C.; Fick, Cameron N.; Freda, Philip J.; Hennessy, Bryan M.; Hockenberger, Kelsey; Jones, Jennifer K.; King, Jessica E.; Knob, Christopher R.; Kraftmann, Karen J.; Li, Linghui; Lupey, Lena N.; Minniti, Carl J.; Minton, Thomas F.; Moran, Joseph V.; Mudumbi, Krishna; Nordman, Elizabeth C.; Puetz, William J.; Robinson, Lauren M.; Rose, Thomas J.; Sweeney, Edward P.; Timko, Ashley S.; Paetkau, Don W.; Eisler, Heather L.; Aldrup, Megan E.; Bodenberg, Jessica M.; Cole, Mara G.; Deranek, Kelly M.; DeShetler, Megan; Dowd, Rose M.; Eckardt, Alexandra K.; Ehret, Sharon C.; Fese, Jessica; Garrett, Amanda D.; Kammrath, Anna; Kappes, Michelle L.; Light, Morgan R.; Meier, Anne C.; O’Rouke, Allison; Perella, Mallory; Ramsey, Kimberley; Ramthun, Jennifer R.; Reilly, Mary T.; Robinett, Deirdre; Rossi, Nadine L.; Schueler, Mary Grace; Shoemaker, Emma; Starkey, Kristin M.; Vetor, Ashley; Vrable, Abby; Chandrasekaran, Vidya; Beck, Christopher; Hatfield, Kristen R.; Herrick, Douglas A.; Khoury, Christopher B.; Lea, Charlotte; Louie, Christopher A.; Lowell, Shannon M.; Reynolds, Thomas J.; Schibler, Jeanine; Scoma, Alexandra H.; Smith-Gee, Maxwell T.; Tuberty, Sarah; Smith, Christopher D.; Lopilato, Jane E.; Hauke, Jeanette; Roecklein-Canfield, Jennifer A.; Corrielus, Maureen; Gilman, Hannah; Intriago, Stephanie; Maffa, Amanda; Rauf, Sabya A.; Thistle, Katrina; Trieu, Melissa; Winters, Jenifer; Yang, Bib; Hauser, Charles R.; Abusheikh, Tariq; Ashrawi, Yara; Benitez, Pedro; Boudreaux, Lauren R.; Bourland, Megan; Chavez, Miranda; Cruz, Samantha; Elliott, GiNell; Farek, Jesse R.; Flohr, Sarah; Flores, Amanda H.; Friedrichs, Chelsey; Fusco, Zach; Goodwin, Zane; Helmreich, Eric; Kiley, John; Knepper, John Mark; Langner, Christine; Martinez, Megan; Mendoza, Carlos; Naik, Monal; Ochoa, Andrea; Ragland, Nicolas; Raimey, England; Rathore, Sunil; Reza, Evangelina; Sadovsky, Griffin; Seydoux, Marie-Isabelle B.; Smith, Jonathan E.; Unruh, Anna K.; Velasquez, Vicente; Wolski, Matthew W.; Gosser, Yuying; Govind, Shubha; Clarke-Medley, Nicole; Guadron, Leslie; Lau, Dawn; Lu, Alvin; Mazzeo, Cheryl; Meghdari, Mariam; Ng, Simon; Pamnani, Brad; Plante, Olivia; Shum, Yuki Kwan Wa; Song, Roy; Johnson, Diana E.; Abdelnabi, Mai; Archambault, Alexi; Chamma, Norma; Gaur, Shailly; Hammett, Deborah; Kandahari, Adrese; Khayrullina, Guzal; Kumar, Sonali; Lawrence, Samantha; Madden, Nigel; Mandelbaum, Max; Milnthorp, Heather; Mohini, Shiv; Patel, Roshni; Peacock, Sarah J.; Perling, Emily; Quintana, Amber; Rahimi, Michael; Ramirez, Kristen; Singhal, Rishi; Weeks, Corinne; Wong, Tiffany; Gillis, Aubree T.; Moore, Zachary D.; Savell, Christopher D.; Watson, Reece; Mel, Stephanie F.; Anilkumar, Arjun A.; Bilinski, Paul; Castillo, Rostislav; Closser, Michael; Cruz, Nathalia M.; Dai, Tiffany; Garbagnati, Giancarlo F.; Horton, Lanor S.; Kim, Dongyeon; Lau, Joyce H.; Liu, James Z.; Mach, Sandy D.; Phan, Thu A.; Ren, Yi; Stapleton, Kenneth E.; Strelitz, Jean M.; Sunjed, Ray; Stamm, Joyce; Anderson, Morgan C.; Bonifield, Bethany Grace; Coomes, Daniel; Dillman, Adam; Durchholz, Elaine J.; Fafara-Thompson, Antoinette E.; Gross, Meleah J.; Gygi, Amber M.; Jackson, Lesley E.; Johnson, Amy; Kocsisova, Zuzana; Manghelli, Joshua L.; McNeil, Kylie; Murillo, Michael; Naylor, Kierstin L.; Neely, Jessica; Ogawa, Emmy E.; Rich, Ashley; Rogers, Anna; Spencer, J. 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2015-01-01

The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25–50%) than euchromatic reference regions (3–11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11–27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4–3.6 vs. 8.4–8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu. PMID:25740935

KRAS exon 2 codon 13 mutation is associated with a better prognosis than codon 12 mutation following lung metastasectomy in colorectal cancer

PubMed Central

Renaud, Stéphane; Guerrera, Francesco; Seitlinger, Joseph; Costardi, Lorena; Schaeffer, Mickaël; Romain, Benoit; Mossetti, Claudio; Claire-Voegeli, Anne; Filosso, Pier Luigi; Legrain, Michèle; Ruffini, Enrico; Falcoz, Pierre-Emmanuel; Oliaro, Alberto; Massard, Gilbert

2017-01-01

Introduction The utilization of molecular markers as routinely used biomarkers is steadily increasing. We aimed to evaluate the potential different prognostic values of KRAS exon 2 codons 12 and 13 after lung metastasectomy in colorectal cancer (CRC). Results KRAS codon 12 mutations were observed in 116 patients (77%), whereas codon 13 mutations were observed in 34 patients (23%). KRAS codon 13 mutations were associated with both longer time to pulmonary recurrence (TTPR) (median TTPR: 78 months (95% CI: 50.61–82.56) vs 56 months (95% CI: 68.71–127.51), P = 0.008) and improved overall survival (OS) (median OS: 82 months vs 54 months (95% CI: 48.93–59.07), P = 0.009). Multivariate analysis confirmed that codon 13 mutations were associated with better outcomes (TTPR: HR: 0.40 (95% CI: 0.17–0.93), P = 0.033); OS: HR: 0.39 (95% CI: 0.14–1.07), P = 0.07). Otherwise, no significant difference in OS (P = 0.78) or TTPR (P = 0.72) based on the type of amino-acid substitutions was observed among KRAS codon 12 mutations. Materials and Methods We retrospectively reviewed data from 525 patients who underwent a lung metastasectomy for CRC in two departments of thoracic surgery from 1998 to 2015 and focused on 150 patients that had KRAS exon 2 codon 12/13 mutations. Conclusions KRAS exon 2 codon 13 mutations, compared to codon 12 mutations, seem to be associated with better outcomes following lung metastasectomy in CRC. Prospective multicenter studies are necessary to fully understand the prognostic value of KRAS mutations in the lung metastases of CRC. PMID:27911859

Bicluster Pattern of Codon Context Usages between Flavivirus and Vector Mosquito Aedes aegypti: Relevance to Infection and Transcriptional Response of Mosquito Genes

PubMed Central

Behura, Susanta K.; Severson, David W.

2014-01-01

The mosquito Aedes aegypti is the primary vector of dengue virus (DENV) infection in most of the subtropical and tropical countries. Besides DENV, yellow fever virus (YFV) is also transmitted by A. aegypti. Susceptibility of A. aegypti to West Nile virus (WNV) has also been confirmed. Although studies have indicated correlation of codon bias between flaviviridae and their animal/insect hosts, it is not clear if codon sequences have any relation to susceptibility of A. aegypti to DENV, YFV and WNV. In the current study, usages of codon context sequences (codon pairs for neighboring amino acids) of the vector (A. aegypti) genome as well as the flaviviral genomes are investigated. We used bioinformatics methods to quantify codon context bias in a genome-wide manner of A. aegypti as well as DENV, WNV and YFV sequences. Mutual information statistics was applied to perform bicluster analysis of codon context bias between vector and flaviviral sequences. Functional relevance of the bicluster pattern was inferred from published microarray data. Our study shows that codon context bias of DENV, WNV and YFV sequences varies in a bicluster manner with that of specific sets of genes of A. aegypti. Many of these mosquito genes are known to be differentially expressed in response to flaviviral infection suggesting that codon context sequences of A. aegypti and the flaviviruses may play a role in the susceptible interaction between flaviviruses and this mosquito. The bias inusages of codon context sequences likely has a functional association with susceptibility of A. aegypti to flaviviral infection. The results from this study will allow us to conduct hypothesis driven tests to examine the role of codon contexts bias in evolution of vector-virus interactions at the molecular level. PMID:24838953

Nonstructural proteins nsP3 and nsP4 of Ross River and O'Nyong-nyong viruses: sequence and comparison with those of other alphaviruses.

PubMed

Strauss, E G; Levinson, R; Rice, C M; Dalrymple, J; Strauss, J H

1988-05-01

We have sequenced the nsP3 and nsP4 region of two alphaviruses, Ross River virus and O'Nyong-nyong virus, in order to examine these viruses for the presence or absence of an opal termination codon present between nsP3 and nsP4 in many alphaviruses. We found that Ross River virus possesses an in-phase opal termination codon between nsP3 and nsP4, whereas in O'Nyong-nyong virus this termination codon is replaced by an arginine codon. Previous studies have shown that two other alphaviruses, Sindbis virus and Middelburg virus, possess an opal termination codon separating nsP3 and nsP4 [E.G. Strauss, C.M. Rice, and J.H. Strauss (1983), Proc. Natl. Acad. Sci. USA 80, 5271-5275], whereas Semliki Forest virus possesses an arginine codon in lieu of the opal codon [K. Takkinen (1986), Nucleic Acids Res. 14, 5667-5682]. Thus, of the five alphaviruses examined to date, three possess the opal codon and two do not. Production of nsP4 requires readthrough of the opal codon in those alphaviruses that possess this termination codon and the function of the termination codon may be to regulate the amount of nsP4 produced. It is an open question then as to whether alphaviruses with no termination codon use other mechanisms to regulate the activity of this gene. The nsP4s of these five alphaviruses are highly conserved, sharing 71-76% amino acid sequence similarity, and all five contain the Gly-Asp-Asp motif found in many RNA virus replicases. The nsP3s are somewhat less conserved, sharing 52-73% amino acid sequence similarity throughout most of the protein, but each possesses a nonconserved C-terminal domain of 134 to 246 amino acids of unknown function.

Non-uniqueness of factors constraint on the codon usage in Bombyx mori.

PubMed

Jia, Xian; Liu, Shuyu; Zheng, Hao; Li, Bo; Qi, Qi; Wei, Lei; Zhao, Taiyi; He, Jian; Sun, Jingchen

2015-05-06

The analysis of codon usage is a good way to understand the genetic and evolutionary characteristics of an organism. However, there are only a few reports related with the codon usage of the domesticated silkworm, Bombyx mori (B. mori). Hence, the codon usage of B. mori was analyzed here to reveal the constraint factors and it could be helpful to improve the bioreactor based on B. mori. A total of 1,097 annotated mRNA sequences from B. mori were analyzed, revealing there is only a weak codon bias. It also shows that the gene expression level is related to the GC content, and the amino acids with higher general average hydropathicity (GRAVY) and aromaticity (Aromo). And the genes on the primary axis are strongly positively correlated with the GC content, and GC3s. Meanwhile, the effective number of codons (ENc) is strongly correlated with codon adaptation index (CAI), gene length, and Aromo values. However, the ENc values are correlated with the second axis, which indicates that the codon usage in B. mori is affected by not only mutation pressure and natural selection, but also nucleotide composition and the gene expression level. It is also associated with Aromo values, and gene length. Additionally, B. mori has a greater relative discrepancy in codon preferences with Drosophila melanogaster (D. melanogaster) or Saccharomyces cerevisiae (S. cerevisiae) than with Arabidopsis thaliana (A. thaliana), Escherichia coli (E. coli), or Caenorhabditis elegans (C. elegans). The codon usage bias in B. mori is relatively weak, and many influence factors are found here, such as nucleotide composition, mutation pressure, natural selection, and expression level. Additionally, it is also associated with Aromo values, and gene length. Among them, natural selection might play a major role. Moreover, the "optimal codons" of B. mori are all encoded by G and C, which provides useful information for enhancing the gene expression in B. mori through codon optimization.

Benchmarking Various Green Fluorescent Protein Variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for Live Cell Imaging

PubMed Central

Overkamp, Wout; Beilharz, Katrin; Detert Oude Weme, Ruud; Solopova, Ana; Karsens, Harma; Kovács, Ákos T.; Kok, Jan

2013-01-01

Green fluorescent protein (GFP) offers efficient ways of visualizing promoter activity and protein localization in vivo, and many different variants are currently available to study bacterial cell biology. Which of these variants is best suited for a certain bacterial strain, goal, or experimental condition is not clear. Here, we have designed and constructed two “superfolder” GFPs with codon adaptation specifically for Bacillus subtilis and Streptococcus pneumoniae and have benchmarked them against five other previously available variants of GFP in B. subtilis, S. pneumoniae, and Lactococcus lactis, using promoter-gfp fusions. Surprisingly, the best-performing GFP under our experimental conditions in B. subtilis was the one codon optimized for S. pneumoniae and vice versa. The data and tools described in this study will be useful for cell biology studies in low-GC-rich Gram-positive bacteria. PMID:23956387

Identification and characterization of a catechol-o-methyltransferase cDNA in the catfish Heteropneustes fossilis: Tissue, sex and seasonal variations, and effects of gonadotropin and 2-hydroxyestradiol-17β on mRNA expression.

PubMed

Chaube, R; Rawat, A; Inbaraj, R M; Bobe, J; Guiguen, Y; Fostier, A; Joy, K P

2017-05-15

Catechol-O-methyltransferase (COMT) is involved in the methylation and inactivation of endogenous and xenobiotic catechol compounds, and serves as a common biochemical link in the catecholamine and catecholestrogen metabolism. Studies on cloning, sequencing and function characterization comt gene in lower vertebrates like fish are fewer. In the present study, a full-length comt cDNA of 1442bp with an open-reading frame (ORF) of 792bp, and start codon (ATG) at nucleotide 162 and stop codon (TAG) at nucleotide 953 was isolated and characterized in the stinging catfish Heteropneustes fossilis (accession No. KT597925). The ORF codes for a protein of 263 amino acid residues, which is also validated by the catfish transcriptome data analysis. The catfish Comt shared conserved putative structural regions important for S-adenosyl methionine (AdoMet)- and catechol-binding, transmembrane regions, two glycosylation sites (N-65 and N-91) at the N-terminus and two phosphorylation sites (Ser-235 and Thr-240) at the C-terminus. The gene was expressed in all tissues examined and the expression showed significant sex dimorphic distribution with high levels in females. The transcript was abundant in the liver, brain and gonads and low in muscles. The transcripts showed significant seasonal variations in the brain and ovary, increased progressively to the peak levels in spawning phase and then declined. The brain and ovarian comt mRNA levels showed periovulatory changes after in vivo and in vitro human chorionic gonadotropin (hCG) treatments with high fold increases at 16 and 24h in the brain and at 16h in the ovary. The catecholestrogen 2-hydroxyE 2 up regulated ovarian comt expression in vitro with the highest fold increase at 16h. The mRNA and protein was localized in the follicular layer of the vitellogenic follicles and in the cytoplasm of primary follicles. The data were discussed in relation to catecholamine and catecholestrogen-mediated functions in the brain and ovary of the stinging catfish. Copyright © 2016 Elsevier Inc. All rights reserved.

Stringent Nucleotide Recognition by the Ribosome at the Middle Codon Position

PubMed Central

Liu, Wei; Shin, Dongwon; Ng, Martin; Sanbonmatsu, Karissa Y.; Tor, Yitzhak; Cooperman, Barry S.

2017-01-01

Accurate translation of the genetic code depends on mRNA:tRNA codon:anticodon base pairing. Here we exploit an emissive, isosteric adenosine surrogate that allows direct measurement of the kinetics of codon:anticodon base formation during protein synthesis. Our results suggest that codon:anticodon base pairing is subject to tighter constraints at the middle position than at the 5′- and 3′-positions, and further suggest a sequential mechanism of formation of the three base pairs in the codon:anticodon helix. PMID:28850078

The prediction of human exons by oligonucleotide composition and discriminant analysis of spliceable open reading frames

DOE Office of Scientific and Technical Information (OSTI.GOV)

Solovyev, V.V.; Salamov, A.A.; Lawrence, C.B.

1994-12-31

Discriminant analysis is applied to the problem of recognition 5`-, internal and 3`-exons in human DNA sequences. Specific recognition functions were developed for revealing exons of particular types. The method based on a splice site prediction algorithm that uses the linear Fisher discriminant to combine the information about significant triplet frequencies of various functional parts of splice site regions and preferences of oligonucleotide in protein coding and nation regions. The accuracy of our splice site recognition function is about 97%. A discriminant function for 5`-exon prediction includes hexanucleotide composition of upstream region, triplet composition around the ATG codon, ORF codingmore » potential, donor splice site potential and composition of downstream introit region. For internal exon prediction, we combine in a discriminant function the characteristics describing the 5`- intron region, donor splice site, coding region, acceptor splice site and Y-intron region for each open reading frame flanked by GT and AG base pairs. The accuracy of precise internal exon recognition on a test set of 451 exon and 246693 pseudoexon sequences is 77% with a specificity of 79% and a level of pseudoexon ORF prediction of 99.96%. The recognition quality computed at the level of individual nucleotides is 89%, for exon sequences and 98% for intron sequences. A discriminant function for 3`-exon prediction includes octanucleolide composition of upstream nation region, triplet composition around the stop codon, ORF coding potential, acceptor splice site potential and hexanucleotide composition of downstream region. We unite these three discriminant functions in exon predicting program FEX (find exons). FEX exactly predicts 70% of 1016 exons from the test of 181 complete genes with specificity 73%, and 89% exons are exactly or partially predicted. On the average, 85% of nucleotides were predicted accurately with specificity 91%.« less

Evaluation of dihydrofolate reductase and dihydropteroate synthetase genotypes that confer resistance to sulphadoxine-pyrimethamine in Plasmodium falciparum in Haiti

PubMed Central

2012-01-01

Background Malaria caused by Plasmodium falciparum infects roughly 30,000 individuals in Haiti each year. Haiti has used chloroquine (CQ) as a first-line treatment for malaria for many years and as a result there are concerns that malaria parasites may develop resistance to CQ over time. Therefore it is important to prepare for alternative malaria treatment options should CQ resistance develop. In many other malaria-endemic regions, antifolates, particularly pyrimethamine (PYR) and sulphadoxine (SDX) treatment combination (SP), have been used as an alternative when CQ resistance has developed. This study evaluated mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes that confer PYR and SDX resistance, respectively, in P. falciparum to provide baseline data in Haiti. This study is the first comprehensive study to examine PYR and SDX resistance genotypes in P. falciparum in Haiti. Methods DNA was extracted from dried blood spots and genotyped for PYR and SDX resistance mutations in P. falciparum using PCR and DNA sequencing methods. Sixty-one samples were genotyped for PYR resistance in codons 51, 59, 108 and 164 of the dhfr gene and 58 samples were genotyped for SDX resistance codons 436, 437, 540 of the dhps gene in P. falciparum. Results Thirty-three percent (20/61) of the samples carried a mutation at codon 108 (S108N) of the dhfr gene. No mutations in dhfr at codons 51, 59, 164 were observed in any of the samples. In addition, no mutations were observed in dhps at the three codons (436, 437, 540) examined. No significant difference was observed between samples collected in urban vs rural sites (Welch’s T-test p-value = 0.53 and permutations p-value = 0.59). Conclusion This study has shown the presence of the S108N mutation in P. falciparum that confers low-level PYR resistance in Haiti. However, the absence of SDX resistance mutations suggests that SP resistance may not be present in Haiti. These results have important implications for ongoing discussions on alternative malaria treatment options in Haiti. PMID:22889367

Identification of pH-sensitive regions in the mouse prion by the cysteine-scanning spin-labeling ESR technique

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watanabe, Yasuko; Inanami, Osamu; Horiuchi, Motohiro

2006-11-24

We analyzed the pH-induced mobility changes in moPrP{sup C} {alpha}-helix and {beta}-sheets by cysteine-scanning site-directed spin labeling (SDSL) with ESR. Nine amino acid residues of {alpha}-helix1 (H1, codon 143-151), four amino acid residues of {beta}-sheet1 (S1, codon 127-130), and four amino acid residues of {beta}-sheet2 (S2, codon 160-163) were substituted for by cysteine residues. These recombinant mouse PrP{sup C} (moPrP{sup C}) mutants were reacted with a methane thiosulfonate sulfhydryl-specific spin labeling reagent (MTSSL). The 1/{delta}H of the central ({sup 14}N hyperfine) component (M{sub I} = 0) in the ESR spectrum of spin-labeled moPrP{sup C} was measured as a mobility parametermore » of nitroxide residues (R1). The mobilities of E145R1 and Y149R1 at pH 7.4, which was identified as a tertiary contact site by a previous NMR study of moPrP, were lower than those of D143R1, R147R1, and R150R1 reported on the helix surface. Thus, the mobility in the H1 region in the neutral solution was observed with the periodicity associated with a helical structure. On the other hand, the values in the S2 region, known to be located in the buried side, were lower than those in the S1 region located in the surface side. These results indicated that the mobility parameter of the nitroxide label was well correlated with the 3D structure of moPrP. Furthermore, the present study clearly demonstrated three pH-sensitive sites in moPrP, i.e. (1) the N-terminal tertiary contact site of H1 (2) the C-terminal end of H1, and (3) the S2 region. In particular, among these pH-sensitive sites, the N-terminal tertiary contact region of H1 was found to be the most pH-sensitive one and was easily converted to a flexible structure by a slight decrease of pH in the solution. These data provided molecular evidence to explain the cellular mechanism for conversion from PrP{sup C} to PrP{sup Sc} in acidic organelles such as the endosome.« less

Abundant off-target edits from site-directed RNA editing can be reduced by nuclear localization of the editing enzyme.

PubMed

Vallecillo-Viejo, Isabel C; Liscovitch-Brauer, Noa; Montiel-Gonzalez, Maria Fernanda; Eisenberg, Eli; Rosenthal, Joshua J C

2018-01-02

Site-directed RNA editing (SDRE) is a general strategy for making targeted base changes in RNA molecules. Although the approach is relatively new, several groups, including our own, have been working on its development. The basic strategy has been to couple the catalytic domain of an adenosine (A) to inosine (I) RNA editing enzyme to a guide RNA that is used for targeting. Although highly efficient on-target editing has been reported, off-target events have not been rigorously quantified. In this report we target premature termination codons (PTCs) in messages encoding both a fluorescent reporter protein and the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein transiently transfected into human epithelial cells. We demonstrate that while on-target editing is efficient, off-target editing is extensive, both within the targeted message and across the entire transcriptome of the transfected cells. By redirecting the editing enzymes from the cytoplasm to the nucleus, off-target editing is reduced without compromising the on-target editing efficiency. The addition of the E488Q mutation to the editing enzymes, a common strategy for increasing on-target editing efficiency, causes a tremendous increase in off-target editing. These results underscore the need to reduce promiscuity in current approaches to SDRE.

A Major Controversy in Codon-Anticodon Adaptation Resolved by a New Codon Usage Index

PubMed Central

Xia, Xuhua

2015-01-01

Two alternative hypotheses attribute different benefits to codon-anticodon adaptation. The first assumes that protein production is rate limited by both initiation and elongation and that codon-anticodon adaptation would result in higher elongation efficiency and more efficient and accurate protein production, especially for highly expressed genes. The second claims that protein production is rate limited only by initiation efficiency but that improved codon adaptation and, consequently, increased elongation efficiency have the benefit of increasing ribosomal availability for global translation. To test these hypotheses, a recent study engineered a synthetic library of 154 genes, all encoding the same protein but differing in degrees of codon adaptation, to quantify the effect of differential codon adaptation on protein production in Escherichia coli. The surprising conclusion that “codon bias did not correlate with gene expression” and that “translation initiation, not elongation, is rate-limiting for gene expression” contradicts the conclusion reached by many other empirical studies. In this paper, I resolve the contradiction by reanalyzing the data from the 154 sequences. I demonstrate that translation elongation accounts for about 17% of total variation in protein production and that the previous conclusion is due to the use of a codon adaptation index (CAI) that does not account for the mutation bias in characterizing codon adaptation. The effect of translation elongation becomes undetectable only when translation initiation is unrealistically slow. A new index of translation elongation ITE is formulated to facilitate studies on the efficiency and evolution of the translation machinery. PMID:25480780

Novel expression of the stanniocalcin gene in fish.

PubMed

McCudden, C R; Kogon, M R; DiMattia, G E; Wagner, G F

2001-10-01

It is currently accepted that the fish stanniocalcin (STC) gene is expressed exclusively in the corpuscles of Stannius (CS), unique endocrine glands on the kidneys of bony fishes. In this study, we have re-examined the pattern of fish STC gene expression in the light of the recent evidence for widespread expression of the gene in mammals. Surprisingly, we found by Northern blotting that the fish gene was also expressed in the kidneys and gonads, in addition to the CS glands. Moreover, Southern blotting of RT-PCR products revealed STC mRNA transcripts in all tissues assayed, including brain, heart, gill, muscle and intestine. In situ hybridization studies using digoxigenin-labeled riboprobes localized STC mRNA to chondrocytes, and both mature and developing nephritic tubules. Immunocytochemical staining indicated that the STC protein was widespread in cells of the gill, kidney, brain, eye, pseudobranch and skin. We also characterized the salmon STC gene, establishing that it was comprised of five exons as opposed to four in mammals. A single transcription start site was identified by primer extension 99 bp upstream of the start codon. This is the first evidence of STC gene expression in fish tissues other than the CS glands and suggests that, as in mammals, fish STC operates via both local and endocrine pathways.

Mapping Structurally Defined Guanine Oxidation Products along DNA Duplexes: Influence of Local Sequence Context and Endogenous Cytosine Methylation

PubMed Central

2015-01-01

DNA oxidation by reactive oxygen species is nonrandom, potentially leading to accumulation of nucleobase damage and mutations at specific sites within the genome. We now present the first quantitative data for sequence-dependent formation of structurally defined oxidative nucleobase adducts along p53 gene-derived DNA duplexes using a novel isotope labeling-based approach. Our results reveal that local nucleobase sequence context differentially alters the yields of 2,2,4-triamino-2H-oxal-5-one (Z) and 8-oxo-7,8-dihydro-2′-deoxyguanosine (OG) in double stranded DNA. While both lesions are overproduced within endogenously methylated MeCG dinucleotides and at 5′ Gs in runs of several guanines, the formation of Z (but not OG) is strongly preferred at solvent-exposed guanine nucleobases at duplex ends. Targeted oxidation of MeCG sequences may be caused by a lowered ionization potential of guanine bases paired with MeC and the preferential intercalation of riboflavin photosensitizer adjacent to MeC:G base pairs. Importantly, some of the most frequently oxidized positions coincide with the known p53 lung cancer mutational “hotspots” at codons 245 (GGC), 248 (CGG), and 158 (CGC) respectively, supporting a possible role of oxidative degradation of DNA in the initiation of lung cancer. PMID:24571128

Fluorescent protein tagging of endogenous protein in brain neurons using CRISPR/Cas9-mediated knock-in and in utero electroporation techniques

PubMed Central

Uemura, Takeshi; Mori, Takuma; Kurihara, Taiga; Kawase, Shiori; Koike, Rie; Satoga, Michiru; Cao, Xueshan; Li, Xue; Yanagawa, Toru; Sakurai, Takayuki; Shindo, Takayuki; Tabuchi, Katsuhiko

2016-01-01

Genome editing is a powerful technique for studying gene functions. CRISPR/Cas9-mediated gene knock-in has recently been applied to various cells and organisms. Here, we successfully knocked in an EGFP coding sequence at the site immediately after the first ATG codon of the β-actin gene in neurons in the brain by the combined use of the CRISPR/Cas9 system and in utero electroporation technique, resulting in the expression of the EGFP-tagged β-actin protein in cortical layer 2/3 pyramidal neurons. We detected EGFP fluorescence signals in the soma and neurites of EGFP knock-in neurons. These signals were particularly abundant in the head of dendritic spines, corresponding to the localization of the endogenous β-actin protein. EGFP knock-in neurons showed no detectable changes in spine density and basic electrophysiological properties. In contrast, exogenously overexpressed EGFP-β-actin showed increased spine density and EPSC frequency, and changed resting membrane potential. Thus, our technique provides a potential tool to elucidate the localization of various endogenous proteins in neurons by epitope tagging without altering neuronal and synaptic functions. This technique can be also useful for introducing a specific mutation into genes to study the function of proteins and genomic elements in brain neurons. PMID:27782168

Fluorescent protein tagging of endogenous protein in brain neurons using CRISPR/Cas9-mediated knock-in and in utero electroporation techniques.

PubMed

Uemura, Takeshi; Mori, Takuma; Kurihara, Taiga; Kawase, Shiori; Koike, Rie; Satoga, Michiru; Cao, Xueshan; Li, Xue; Yanagawa, Toru; Sakurai, Takayuki; Shindo, Takayuki; Tabuchi, Katsuhiko

2016-10-26

Genome editing is a powerful technique for studying gene functions. CRISPR/Cas9-mediated gene knock-in has recently been applied to various cells and organisms. Here, we successfully knocked in an EGFP coding sequence at the site immediately after the first ATG codon of the β-actin gene in neurons in the brain by the combined use of the CRISPR/Cas9 system and in utero electroporation technique, resulting in the expression of the EGFP-tagged β-actin protein in cortical layer 2/3 pyramidal neurons. We detected EGFP fluorescence signals in the soma and neurites of EGFP knock-in neurons. These signals were particularly abundant in the head of dendritic spines, corresponding to the localization of the endogenous β-actin protein. EGFP knock-in neurons showed no detectable changes in spine density and basic electrophysiological properties. In contrast, exogenously overexpressed EGFP-β-actin showed increased spine density and EPSC frequency, and changed resting membrane potential. Thus, our technique provides a potential tool to elucidate the localization of various endogenous proteins in neurons by epitope tagging without altering neuronal and synaptic functions. This technique can be also useful for introducing a specific mutation into genes to study the function of proteins and genomic elements in brain neurons.

Characterization of codon usage pattern and influencing factors in Japanese encephalitis virus.

PubMed

Singh, Niraj K; Tyagi, Anuj; Kaur, Rajinder; Verma, Ramneek; Gupta, Praveen K

2016-08-02

Recently, several outbreaks of Japanese encephalitis (JE), caused by Japanese encephalitis virus (JEV), have been reported and it has become cause of concern across the world. In this study, detailed analysis of JEV codon usage pattern was performed. The relative synonymous codon usage (RSCU) values along with mean effective number of codons (ENC) value of 55.30 indicated the presence of low codon usages bias in JEV. The effect of mutational pressure on codon usage bias was confirmed by significant correlations of A3s, U3s, G3s, C3s, GC3s, ENC values, with overall nucleotide contents (A%, U%, G%, C%, and GC%). The correlation analysis of A3s, U3s, G3s, C3s, GC3s, with axis values of correspondence analysis (CoA) further confirmed the role of mutational pressure. However, the correlation analysis of Gravy values and Aroma values with A3s, U3s, G3s, C3s, and GC3s, indicated the presence of natural selection on codon usage bias in addition to mutational pressure. The natural selection was further confirmed by codon adaptation index (CAI) analysis. Additionally, relative dinucleotide frequencies, geographical distribution, and evolutionary processes also influenced the codon usage pattern to some extent. Copyright © 2016 Elsevier B.V. All rights reserved.

Whole Genome Sequencing Based Characterization of Extensively Drug-Resistant Mycobacterium tuberculosis Isolates from Pakistan

PubMed Central

Ali, Asho; Hasan, Zahra; McNerney, Ruth; Mallard, Kim; Hill-Cawthorne, Grant; Coll, Francesc; Nair, Mridul; Pain, Arnab; Clark, Taane G.; Hasan, Rumina

2015-01-01

Improved molecular diagnostic methods for detection drug resistance in Mycobacterium tuberculosis (MTB) strains are required. Resistance to first- and second- line anti-tuberculous drugs has been associated with single nucleotide polymorphisms (SNPs) in particular genes. However, these SNPs can vary between MTB lineages therefore local data is required to describe different strain populations. We used whole genome sequencing (WGS) to characterize 37 extensively drug-resistant (XDR) MTB isolates from Pakistan and investigated 40 genes associated with drug resistance. Rifampicin resistance was attributable to SNPs in the rpoB hot-spot region. Isoniazid resistance was most commonly associated with the katG codon 315 (92%) mutation followed by inhA S94A (8%) however, one strain did not have SNPs in katG, inhA or oxyR-ahpC. All strains were pyrazimamide resistant but only 43% had pncA SNPs. Ethambutol resistant strains predominantly had embB codon 306 (62%) mutations, but additional SNPs at embB codons 406, 378 and 328 were also present. Fluoroquinolone resistance was associated with gyrA 91–94 codons in 81% of strains; four strains had only gyrB mutations, while others did not have SNPs in either gyrA or gyrB. Streptomycin resistant strains had mutations in ribosomal RNA genes; rpsL codon 43 (42%); rrs 500 region (16%), and gidB (34%) while six strains did not have mutations in any of these genes. Amikacin/kanamycin/capreomycin resistance was associated with SNPs in rrs at nt1401 (78%) and nt1484 (3%), except in seven (19%) strains. We estimate that if only the common hot-spot region targets of current commercial assays were used, the concordance between phenotypic and genotypic testing for these XDR strains would vary between rifampicin (100%), isoniazid (92%), flouroquinolones (81%), aminoglycoside (78%) and ethambutol (62%); while pncA sequencing would provide genotypic resistance in less than half the isolates. This work highlights the importance of expanded targets for drug resistance detection in MTB isolates. PMID:25719196

Identification of a PTEN mutation with reduced protein stability, phosphatase activity, and nuclear localization in Hong Kong patients with autistic features, neurodevelopmental delays, and macrocephaly.

PubMed

Wong, Chi Wai; Or, Penelope Mei Yu; Wang, Yubing; Li, Lisha; Li, Jing; Yan, Mingfei; Cao, Ye; Luk, Ho Ming; Tong, Tony Ming For; Leslie, Nick R; Lo, Ivan Fai-Man; Choy, Kwong Wai; Chan, Andrew Man Lok

2018-04-02

PTEN is a tumor suppressor gene inactivated in over 30% of human cancers. It encodes a lipid phosphatase that serves as a gatekeeper of the phosphoinositide 3-kinase signaling pathway. Germline mutation frequently occurs in this gene in patients diagnosed with PTEN Hamartoma Tumor Syndrome (PHTS). PHTS individuals are characterized by macrocephaly, benign growth of multiple tissues and increased tumor risk. In addition, autistic phenotypes are found in 10-20% of individuals carrying the germline PTEN mutation with macrocephaly. In this report, 13 suspected PHTS patients were screened for mutation in the PTEN gene. A missense variant (c. 302T > C) substituting the isoleucine at codon 101 to a threonine, a single nucleotide insertion (c. 327-328insC) causing a frame shift mutation and termination at codon 109, and a nonsense variant (c. 1003C > T) truncated the protein at codon 335 were identified. The I101T mutation significantly reduced PTEN protein expression levels by 2.5- to 4.0-fold. Mechanistically, I101T reduced the protein half-life of PTEN possibly due to enhanced polyubiquitination at Lysine 13. However, the I101T mutant retained almost 30% of the lipid phosphatase activity of the wild-type protein. Finally, the I101T mutant has reduced phosphorylation at a PTEN auto-dephosphorylation site at Threonine 366 and a lowered ratio of nuclear to cytosolic protein level. These partial losses of multiple PTEN biochemical functions may contribute to the tissue overgrowth and autistic features of this PHTS patient. Autism Res 2018. © 2018 The Authors Autism Research published by International Society for Autism Research and Wiley Periodicals, Inc. The genetics of autism spectrum disorders is highly complex with individual risk influenced by both genetic and environmental factors. Mutation in the human PTEN gene confers a high risk of developing autistic behavior. This report revealed that PTEN mutations occurred in 23% of a selected group of Hong Kong patients harboring autistic features with gross overgrowth symptoms. Detailed characterization of a PTEN mutation revealed reduced protein stability as one of the underlying mechanisms responsible for reduced PTEN activity. © 2018 The Authors Autism Research published by International Society for Autism Research and Wiley Periodicals, Inc.

tRNA-mediated codon-biased translation in mycobacterial hypoxic persistence

NASA Astrophysics Data System (ADS)

Chionh, Yok Hian; McBee, Megan; Babu, I. Ramesh; Hia, Fabian; Lin, Wenwei; Zhao, Wei; Cao, Jianshu; Dziergowska, Agnieszka; Malkiewicz, Andrzej; Begley, Thomas J.; Alonso, Sylvie; Dedon, Peter C.

2016-11-01

Microbial pathogens adapt to the stress of infection by regulating transcription, translation and protein modification. We report that changes in gene expression in hypoxia-induced non-replicating persistence in mycobacteria--which models tuberculous granulomas--are partly determined by a mechanism of tRNA reprogramming and codon-biased translation. Mycobacterium bovis BCG responded to each stage of hypoxia and aerobic resuscitation by uniquely reprogramming 40 modified ribonucleosides in tRNA, which correlate with selective translation of mRNAs from families of codon-biased persistence genes. For example, early hypoxia increases wobble cmo5U in tRNAThr(UGU), which parallels translation of transcripts enriched in its cognate codon, ACG, including the DosR master regulator of hypoxic bacteriostasis. Codon re-engineering of dosR exaggerates hypoxia-induced changes in codon-biased DosR translation, with altered dosR expression revealing unanticipated effects on bacterial survival during hypoxia. These results reveal a coordinated system of tRNA modifications and translation of codon-biased transcripts that enhance expression of stress response proteins in mycobacteria.

tRNA-mediated codon-biased translation in mycobacterial hypoxic persistence

PubMed Central

Chionh, Yok Hian; McBee, Megan; Babu, I. Ramesh; Hia, Fabian; Lin, Wenwei; Zhao, Wei; Cao, Jianshu; Dziergowska, Agnieszka; Malkiewicz, Andrzej; Begley, Thomas J.; Alonso, Sylvie; Dedon, Peter C.

2016-01-01

Microbial pathogens adapt to the stress of infection by regulating transcription, translation and protein modification. We report that changes in gene expression in hypoxia-induced non-replicating persistence in mycobacteria—which models tuberculous granulomas—are partly determined by a mechanism of tRNA reprogramming and codon-biased translation. Mycobacterium bovis BCG responded to each stage of hypoxia and aerobic resuscitation by uniquely reprogramming 40 modified ribonucleosides in tRNA, which correlate with selective translation of mRNAs from families of codon-biased persistence genes. For example, early hypoxia increases wobble cmo5U in tRNAThr(UGU), which parallels translation of transcripts enriched in its cognate codon, ACG, including the DosR master regulator of hypoxic bacteriostasis. Codon re-engineering of dosR exaggerates hypoxia-induced changes in codon-biased DosR translation, with altered dosR expression revealing unanticipated effects on bacterial survival during hypoxia. These results reveal a coordinated system of tRNA modifications and translation of codon-biased transcripts that enhance expression of stress response proteins in mycobacteria. PMID:27834374

Codon optimization underpins generalist parasitism in fungi

PubMed Central

Badet, Thomas; Peyraud, Remi; Mbengue, Malick; Navaud, Olivier; Derbyshire, Mark; Oliver, Richard P; Barbacci, Adelin; Raffaele, Sylvain

2017-01-01

The range of hosts that parasites can infect is a key determinant of the emergence and spread of disease. Yet, the impact of host range variation on the evolution of parasite genomes remains unknown. Here, we show that codon optimization underlies genome adaptation in broad host range parasites. We found that the longer proteins encoded by broad host range fungi likely increase natural selection on codon optimization in these species. Accordingly, codon optimization correlates with host range across the fungal kingdom. At the species level, biased patterns of synonymous substitutions underpin increased codon optimization in a generalist but not a specialist fungal pathogen. Virulence genes were consistently enriched in highly codon-optimized genes of generalist but not specialist species. We conclude that codon optimization is related to the capacity of parasites to colonize multiple hosts. Our results link genome evolution and translational regulation to the long-term persistence of generalist parasitism. DOI: http://dx.doi.org/10.7554/eLife.22472.001 PMID:28157073

Ribosomes slide on lysine-encoding homopolymeric A stretches

PubMed Central

Koutmou, Kristin S; Schuller, Anthony P; Brunelle, Julie L; Radhakrishnan, Aditya; Djuranovic, Sergej; Green, Rachel

2015-01-01

Protein output from synonymous codons is thought to be equivalent if appropriate tRNAs are sufficiently abundant. Here we show that mRNAs encoding iterated lysine codons, AAA or AAG, differentially impact protein synthesis: insertion of iterated AAA codons into an ORF diminishes protein expression more than insertion of synonymous AAG codons. Kinetic studies in E. coli reveal that differential protein production results from pausing on consecutive AAA-lysines followed by ribosome sliding on homopolymeric A sequence. Translation in a cell-free expression system demonstrates that diminished output from AAA-codon-containing reporters results from premature translation termination on out of frame stop codons following ribosome sliding. In eukaryotes, these premature termination events target the mRNAs for Nonsense-Mediated-Decay (NMD). The finding that ribosomes slide on homopolymeric A sequences explains bioinformatic analyses indicating that consecutive AAA codons are under-represented in gene-coding sequences. Ribosome ‘sliding’ represents an unexpected type of ribosome movement possible during translation. DOI: http://dx.doi.org/10.7554/eLife.05534.001 PMID:25695637

Synonymous codon changes in the oncogenes of the cottontail rabbit papillomavirus lead to increased oncogenicity and immunogenicity of the virus

PubMed Central

Cladel, Nancy M.; Budgeon, Lynn R.; Hu, Jiafen; Balogh, Karla K.; Christensen, Neil D.

2013-01-01

Papillomaviruses use rare codons with respect to the host. The reasons for this are incompletely understood but among the hypotheses is the concept that rare codons result in low protein production and this allows the virus to escape immune surveillance. We changed rare codons in the oncogenes E6 and E7 of the cottontail rabbit papillomavirus to make them more mammalian-like and tested the mutant genomes in our in vivo animal model. While the amino acid sequences of the proteins remained unchanged, the oncogenic potential of some of the altered genomes increased dramatically. In addition, increased immunogenicity, as measured by spontaneous regression, was observed as the numbers of codon changes increased. This work suggests that codon usage may modify protein production in ways that influence disease outcome and that evaluation of synonymous codons should be included in the analysis of genetic variants of infectious agents and their association with disease. PMID:23433866

Cryptic splice site in the complementary DNA of glucocerebrosidase causes inefficient expression.

PubMed

Bukovac, Scott W; Bagshaw, Richard D; Rigat, Brigitte A; Callahan, John W; Clarke, Joe T R; Mahuran, Don J

2008-10-15

The low levels of human lysosomal glucocerebrosidase activity expressed in transiently transfected Chinese hamster ovary (CHO) cells were investigated. Reverse transcription PCR (RT-PCR) demonstrated that a significant portion of the transcribed RNA was misspliced owing to the presence of a cryptic splice site in the complementary DNA (cDNA). Missplicing results in the deletion of 179 bp of coding sequence and a premature stop codon. A repaired cDNA was constructed abolishing the splice site without changing the amino acid sequence. The level of glucocerebrosidase expression was increased sixfold. These data demonstrate that for maximum expression of any cDNA construct, the transcription products should be examined.

EUGENE'HOM: A generic similarity-based gene finder using multiple homologous sequences.

PubMed

Foissac, Sylvain; Bardou, Philippe; Moisan, Annick; Cros, Marie-Josée; Schiex, Thomas

2003-07-01

EUGENE'HOM is a gene prediction software for eukaryotic organisms based on comparative analysis. EUGENE'HOM is able to take into account multiple homologous sequences from more or less closely related organisms. It integrates the results of TBLASTX analysis, splice site and start codon prediction and a robust coding/non-coding probabilistic model which allows EUGENE'HOM to handle sequences from a variety of organisms. The current target of EUGENE'HOM is plant sequences. The EUGENE'HOM web site is available at http://genopole.toulouse.inra.fr/bioinfo/eugene/EuGeneHom/cgi-bin/EuGeneHom.pl.

Molecular analysis of beta-globin gene mutations among Thai beta-thalassemia children: results from a single center study

PubMed Central

Boonyawat, Boonchai; Monsereenusorn, Chalinee; Traivaree, Chanchai

2014-01-01

Background Beta-thalassemia is one of the most common genetic disorders in Thailand. Clinical phenotype ranges from silent carrier to clinically manifested conditions including severe beta-thalassemia major and mild beta-thalassemia intermedia. Objective This study aimed to characterize the spectrum of beta-globin gene mutations in pediatric patients who were followed-up in Phramongkutklao Hospital. Patients and methods Eighty unrelated beta-thalassemia patients were enrolled in this study including 57 with beta-thalassemia/hemoglobin E, eight with homozygous beta-thalassemia, and 15 with heterozygous beta-thalassemia. Mutation analysis was performed by multiplex amplification refractory mutation system (M-ARMS), direct DNA sequencing of beta-globin gene, and gap polymerase chain reaction for 3.4 kb deletion detection, respectively. Results A total of 13 different beta-thalassemia mutations were identified among 88 alleles. The most common mutation was codon 41/42 (-TCTT) (37.5%), followed by codon 17 (A>T) (26.1%), IVS-I-5 (G>C) (8%), IVS-II-654 (C>T) (6.8%), IVS-I-1 (G>T) (4.5%), and codon 71/72 (+A) (2.3%), and all these six common mutations (85.2%) were detected by M-ARMS. Six uncommon mutations (10.2%) were identified by DNA sequencing including 4.5% for codon 35 (C>A) and 1.1% initiation codon mutation (ATG>AGG), codon 15 (G>A), codon 19 (A>G), codon 27/28 (+C), and codon 123/124/125 (-ACCCCACC), respectively. The 3.4 kb deletion was detected at 4.5%. The most common genotype of beta-thalassemia major patients was codon 41/42 (-TCTT)/codon 26 (G>A) or betaE accounting for 40%. Conclusion All of the beta-thalassemia alleles have been characterized by a combination of techniques including M-ARMS, DNA sequencing, and gap polymerase chain reaction for 3.4 kb deletion detection. Thirteen mutations account for 100% of the beta-thalassemia genes among the pediatric patients in our study. PMID:25525381

Evolutionary characterization of Tembusu virus infection through identification of codon usage patterns.

PubMed

Zhou, Hao; Yan, Bing; Chen, Shun; Wang, Mingshu; Jia, Renyong; Cheng, Anchun

2015-10-01

Tembusu virus (TMUV) is a single-stranded, positive-sense RNA virus. As reported, TMUV infection has resulted in significant poultry losses, and the virus may also pose a threat to public health. To characterize TMUV evolutionarily and to understand the factors accounting for codon usage properties, we performed, for the first time, a comprehensive analysis of codon usage bias for the genomes of 60 TMUV strains. The most recently published TMUV strains were found to be widely distributed in coastal cities of southeastern China. Codon preference among TMUV genomes exhibits a low bias (effective number of codons (ENC)=53.287) and is maintained at a stable level. ENC-GC3 plots and the high correlation between composition constraints and principal component factor analysis of codon usage demonstrated that mutation pressure dominates over natural selection pressure in shaping the TMUV coding sequence composition. The high correlation between the major components of the codon usage pattern and hydrophobicity (Gravy) or aromaticity (Aromo) was obvious, indicating that properties of viral proteins also account for the observed variation in TMUV codon usage. Principal component analysis (PCA) showed that CQW1 isolated from Chongqing may have evolved from GX2013H or GX2013G isolated from Guangxi, thus indicating that TMUV likely disseminated from southeastern China to the mainland. Moreover, the preferred codons encoding eight amino acids were consistent with the optimal codons for human cells, indicating that TMUV may pose a threat to public health due to possible cross-species transmission (birds to birds or birds to humans). The results of this study not only have theoretical value for uncovering the characteristics of synonymous codon usage patterns in TMUV genomes but also have significant meaning with regard to the molecular evolutionary tendencies of TMUV. Copyright © 2015 Elsevier B.V. All rights reserved.

Estimation of fine-scale recombination intensity variation in the white-echinus interval of D. melanogaster

PubMed Central

Singh, Nadia D.; Aquadro, Charles F.; Clark, Andrew G.

2009-01-01

Accurate assessment of local recombination rate variation is crucial for understanding the recombination process and for determining the impact of natural selection on linked sites. In Drosophila, local recombination intensity has been estimated primarily by statistical approaches, estimating the local slope of the relationship between the physical and genetic maps. However, these estimates are limited in resolution, and as a result, the physical scale at which recombination intensity varies in Drosophila is largely unknown. While there is some evidence suggesting as much as a 40-fold variation in crossover rate at a local scale in D. pseudoobscura, little is known about the fine-scale structure of recombination rate variation in D. melanogaster. Here, we experimentally examine the fine-scale distribution of crossover events in a 1.2 Mb region on the D. melanogaster X chromosome using a classic genetic mapping approach. Our results show that crossover frequency is significantly heterogeneous within this region, varying ~ 3.5 fold. Simulations suggest that this degree of heterogeneity is sufficient to affect levels of standing nucleotide diversity, although the magnitude of this effect is small. We recover no statistical association between empirical estimates of nucleotide diversity and recombination intensity, which is likely due to the limited number of loci sampled in our population genetic dataset. However, codon bias is significantly negatively correlated with fine-scale recombination intensity estimates, as expected. Our results shed light on the relevant physical scale to consider in evolutionary analyses relating to recombination rate, and highlight the motivations to increase the resolution of the recombination map in Drosophila. PMID:19504037

Numeral series hidden in the distribution of atomic mass of amino acids to codon domains in the genetic code.

PubMed

Wohlin, Åsa

2015-03-21

The distribution of codons in the nearly universal genetic code is a long discussed issue. At the atomic level, the numeral series 2x(2) (x=5-0) lies behind electron shells and orbitals. Numeral series appear in formulas for spectral lines of hydrogen. The question here was if some similar scheme could be found in the genetic code. A table of 24 codons was constructed (synonyms counted as one) for 20 amino acids, four of which have two different codons. An atomic mass analysis was performed, built on common isotopes. It was found that a numeral series 5 to 0 with exponent 2/3 times 10(2) revealed detailed congruency with codon-grouped amino acid side-chains, simultaneously with the division on atom kinds, further with main 3rd base groups, backbone chains and with codon-grouped amino acids in relation to their origin from glycolysis or the citrate cycle. Hence, it is proposed that this series in a dynamic way may have guided the selection of amino acids into codon domains. Series with simpler exponents also showed noteworthy correlations with the atomic mass distribution on main codon domains; especially the 2x(2)-series times a factor 16 appeared as a conceivable underlying level, both for the atomic mass and charge distribution. Furthermore, it was found that atomic mass transformations between numeral systems, possibly interpretable as dimension degree steps, connected the atomic mass of codon bases with codon-grouped amino acids and with the exponent 2/3-series in several astonishing ways. Thus, it is suggested that they may be part of a deeper reference system. Copyright © 2015 The Author. Published by Elsevier Ltd.. All rights reserved.

Compositional pressure and translational selection determine codon usage in the extremely GC-poor unicellular eukaryote Entamoeba histolytica.

PubMed

Romero, H; Zavala, A; Musto, H

2000-01-25

It is widely accepted that the compositional pressure is the only factor shaping codon usage in unicellular species displaying extremely biased genomic compositions. This seems to be the case in the prokaryotes Mycoplasma capricolum, Rickettsia prowasekii and Borrelia burgdorferi (GC-poor), and in Micrococcus luteus (GC-rich). However, in the GC-poor unicellular eukaryotes Dictyostelium discoideum and Plasmodium falciparum, there is evidence that selection, acting at the level of translation, influences codon choices. This is a twofold intriguing finding, since (1) the genomic GC levels of the above mentioned eukaryotes are lower than the GC% of any studied bacteria, and (2) bacteria usually have larger effective population sizes than eukaryotes, and hence natural selection is expected to overcome more efficiently the randomizing effects of genetic drift among prokaryotes than among eukaryotes. In order to gain a new insight about this problem, we analysed the patterns of codon preferences of the nuclear genes of Entamoeba histolytica, a unicellular eukaryote characterised by an extremely AT-rich genome (GC = 25%). The overall codon usage is strongly biased towards A and T in the third codon positions, and among the presumed highly expressed sequences, there is an increased relative usage of a subset of codons, many of which are C-ending. Since an increase in C in third codon positions is 'against' the compositional bias, we conclude that codon usage in E. histolytica, as happens in D. discoideum and P. falciparum, is the result of an equilibrium between compositional pressure and selection. These findings raise the question of why strongly compositionally biased eukaryotic cells may be more sensitive to the (presumed) slight differences among synonymous codons than compositionally biased bacteria.

Transcriptional regulation of the human mitochondrial peptide deformylase (PDF).

PubMed

Pereira-Castro, Isabel; Costa, Luís Teixeira da; Amorim, António; Azevedo, Luisa

2012-05-18

The last years of research have been particularly dynamic in establishing the importance of peptide deformylase (PDF), a protein of the N-terminal methionine excision (NME) pathway that removes formyl-methionine from mitochondrial-encoded proteins. The genomic sequence of the human PDF gene is shared with the COG8 gene, which encodes a component of the oligomeric golgi complex, a very unusual case in Eukaryotic genomes. Since PDF is crucial in maintaining mitochondrial function and given the atypical short distance between the end of COG8 coding sequence and the PDF initiation codon, we investigated whether the regulation of the human PDF is affected by the COG8 overlapping partner. Our data reveals that PDF has several transcription start sites, the most important of which only 18 bp from the initiation codon. Furthermore, luciferase-activation assays using differently-sized fragments defined a 97 bp minimal promoter region for human PDF, which is capable of very strong transcriptional activity. This fragment contains a potential Sp1 binding site highly conserved in mammalian species. We show that this binding site, whose mutation significantly reduces transcription activation, is a target for the Sp1 transcription factor, and possibly of other members of the Sp family. Importantly, the entire minimal promoter region is located after the end of COG8's coding region, strongly suggesting that the human PDF preserves an independent regulation from its overlapping partner. Copyright © 2012 Elsevier Inc. All rights reserved.

Patterns and effects of GC3 heterogeneity and parsimony informative sites on the phylogenetic tree of genes.

PubMed

Ma, Shuai; Wu, Qi; Hu, Yibo; Wei, Fuwen

2018-05-20

The explosive growth in genomic data has provided novel insights into the conflicting signals hidden in phylogenetic trees. Although some studies have explored the effects of the GC content and parsimony informative sites (PIS) on the phylogenetic tree, the effect of the heterogeneity of the GC content at the first/second/third codon position on parsimony informative sites (GC1/2/3 PIS ) among different species and the effect of PIS on phylogenetic tree construction remain largely unexplored. Here, we used two different mammal genomic datasets to explore the patterns of GC1/2/3 PIS heterogeneity and the effect of PIS on the phylogenetic tree of genes: (i) all GC1/2/3 PIS have obvious heterogeneity between different mammals, and the levels of heterogeneity are GC3 PIS  > GC2 PIS  > GC1 PIS ; (ii) the number of PIS is positively correlated with the metrics of "good" gene tree topologies, and excluding the third codon position (C3) decreases the quality of gene trees by removing too many PIS. These results provide novel insights into the heterogeneity pattern of GC1/2/3 PIS in mammals and the relationship between GC3/PIS and gene trees. Additionally, it is necessary to carefully consider whether to exclude C3 to improve the quality of gene trees, especially in the super-tree method. Copyright © 2018 Elsevier B.V. All rights reserved.

Species Based Synonymous Codon Usage in Fusion Protein Gene of Newcastle Disease Virus

PubMed Central

Kumar, Chandra Shekhar; Kumar, Sachin

2014-01-01

Newcastle disease is highly pathogenic to poultry and many other avian species. However, the Newcastle disease virus (NDV) has also been reported from many non-avian species. The NDV fusion protein (F) is a major determinant of its pathogenicity and virulence. The functionalities of F gene have been explored for the development of vaccine and diagnostics against NDV. Although the F protein is well studied but the codon usage and its nucleotide composition from NDV isolated from different species have not yet been explored. In present study, we have analyzed the factors responsible for the determination of codon usage in NDV isolated from four major avian host species. The F gene of NDV is analyzed for its base composition and its correlation with the bias in codon usage. Our result showed that random mutational pressure is responsible for codon usage bias in F protein of NDV isolates. Aromaticity, GC3s, and aliphatic index were not found responsible for species based synonymous codon usage bias in F gene of NDV. Moreover, the low amount of codon usage bias and expression level was further confirmed by a low CAI value. The phylogenetic analysis of isolates was found in corroboration with the relatedness of species based on codon usage bias. The relationship between the host species and the NDV isolates from the host does not represent a significant correlation in our study. The present study provides a basic understanding of the mechanism involved in codon usage among species. PMID:25479071

Purification and DNA binding properties of the blaI gene product, repressor for the beta-lactamase gene, blaP, of Bacillus licheniformis.

PubMed Central

Grossman, M J; Lampen, J O

1987-01-01

The location of the repressor gene, blaI, for the beta-lactamase gene blaP of Bacillus licheniformis 749, on the 5' side of blaP, was confirmed by sequencing the bla region of the constitutive mutant 749/C. An amber stop codon, likely to result in a nonfunctional truncated repressor, was found at codon 32 of the 128 codon blaI open reading frame (ORF) located 5' to blaP. In order to study the DNA binding activity of the repressor, the structural gene for blaI, from strain 749, with its ribosome binding site was expressed using a two plasmid T7 RNA polymerase/promotor system (S. Tabor and C. C. Richardson. Proc. Natl. Acad. Sci. 82, 1074-1078 (1985). Heat induction of this system in Escherichia coli K38 resulted in the production of BlaI as 5-10% of the soluble cell protein. Repressor protein was then purified by ammonium sulfate fractionation and cation exchange chromatography. The sequence of the N-terminal 28 amino acid residues was determined and was as predicted from the DNA. Binding of BlaI to DNA was detected by the slower migration of protein DNA complexes during polyacrylamide gel electrophoresis. BlaI was shown to selectively bind DNA fragments carrying the promoter regions of blaI and blaP. Images PMID:3498148

Human immunodeficiency virus type 1 pol gene mutations which cause decreased susceptibility to 2',3'-dideoxycytidine.

PubMed Central

Fitzgibbon, J E; Howell, R M; Haberzettl, C A; Sperber, S J; Gocke, D J; Dubin, D T

1992-01-01

To investigate whether human immunodeficiency virus type 1 pol gene mutations are selected during prolonged 2',3'-dideoxycytidine (ddC) therapy, we used the polymerase chain reaction to amplify a portion of the reverse transcriptase segment of the pol gene from the peripheral blood mononuclear cell DNA of a patient with AIDS before and after an 80-week course of ddC therapy. The consensus sequence from the second sample contained a unique double mutation (ACT to GAT) in the codon for reverse transcriptase amino acid 69, causing substitution of aspartic acid (Asp) for the wild-type threonine (Thr). A mutation (ACA to ATA) also occurred in the codon for position 165, causing substitution of isoleucine (Ile) for Thr. The GAT (Asp) codon was introduced into the pol gene of a molecular clone of human immunodeficiency virus via site-directed mutagenesis. Following transfection, mutant and wild-type viruses were tested for susceptibility to ddC by a plaque reduction assay. The mutant virus was fivefold less susceptible to ddC than the wild type; cross-resistance to 3'-azido-3'-deoxythymidine or 2'3'-dideoxyinosine was not found. The Ile-165 mutation did not confer additional ddC resistance. The Asp-69 substitution may have contributed to the generation of resistant virus in this patient. Images PMID:1317143

Two groups of phenylalanine biosynthetic operon leader peptides genes: a high level of apparently incidental frameshifting in decoding Escherichia coli pheL

PubMed Central

Gurvich, Olga L.; Näsvall, S. Joakim; Baranov, Pavel V.; Björk, Glenn R.; Atkins, John F.

2011-01-01

The bacterial pheL gene encodes the leader peptide for the phenylalanine biosynthetic operon. Translation of pheL mRNA controls transcription attenuation and, consequently, expression of the downstream pheA gene. Fifty-three unique pheL genes have been identified in sequenced genomes of the gamma subdivision. There are two groups of pheL genes, both of which are short and contain a run(s) of phenylalanine codons at an internal position. One group is somewhat diverse and features different termination and 5′-flanking codons. The other group, mostly restricted to Enterobacteria and including Escherichia coli pheL, has a conserved nucleotide sequence that ends with UUC_CCC_UGA. When these three codons in E. coli pheL mRNA are in the ribosomal E-, P- and A-sites, there is an unusually high level, 15%, of +1 ribosomal frameshifting due to features of the nascent peptide sequence that include the penultimate phenylalanine. This level increases to 60% with a natural, heterologous, nascent peptide stimulator. Nevertheless, studies with different tRNAPro mutants in Salmonella enterica suggest that frameshifting at the end of pheL does not influence expression of the downstream pheA. This finding of incidental, rather than utilized, frameshifting is cautionary for other studies of programmed frameshifting. PMID:21177642

Codon usage bias and tRNA over-expression in Buchnera aphidicola after aromatic amino acid nutritional stress on its host Acyrthosiphon pisum

PubMed Central

Charles, Hubert; Calevro, Federica; Vinuelas, José; Fayard, Jean-Michel; Rahbe, Yvan

2006-01-01

Codon usage bias and relative abundances of tRNA isoacceptors were analysed in the obligate intracellular symbiotic bacterium, Buchnera aphidicola from the aphid Acyrthosiphon pisum, using a dedicated 35mer oligonucleotide microarray. Buchnera is archetypal of organisms living with minimal metabolic requirements and presents a reduced genome with high-evolutionary rate. Codonusage in Buchnera has been overcome by the high mutational bias towards AT bases. However, several lines of evidence for codon usage selection are given here. A significant correlation was found between tRNA relative abundances and codon composition of Buchnera genes. A significant codon usage bias was found for the choice of rare codons in Buchnera: C-ending codons are preferred in highly expressed genes, whereas G-ending codons are avoided. This bias is not explained by GC skew in the bacteria and might correspond to a selection for perfect matching between codon–anticodon pairs for some essential amino acids in Buchnera proteins. Nutritional stress applied to the aphid host induced a significant overexpression of most of the tRNA isoacceptors in bacteria. Although, molecular regulation of the tRNA operons in Buchnera was not investigated, a correlation between relative expression levels and organization in transcription unit was found in the genome of Buchnera. PMID:16963497

Three stages during the evolution of the genetic code. [Abstract only

NASA Technical Reports Server (NTRS)

Baumann, U.; Oro, J.

1994-01-01

A diversification of the genetic code based on the number of codons available for the proteinous amino acids is established. Three groups of amino acids during evolution of the code are distinguished. On the basis of their chemical complexity and a small codon number those amino acids emerging later in a translation process are derived. Both criteria indicate that His, Phe, Tyr, Cys and either Lys or Asn were introduced in the second stage, whereas the number of codons alone gives evidence that Trp and Met were introduced in the third stage. The amino acids of stage one use purines rich codons, thus purines have been retained in their third codon position. All the amino acids introduced in the second stage, in contrast, use pyrimidines in this codon position. A low abundance of pyrimidines during early translation is derived. This assumption is supported by experiments on non enzymatic replication and interactions of DNA hairpin loops with a complementary strand. A back extrapolation concludes a high purine content of the first nucleic acids which gradually decreased during their evolution. Amino acids independently available form prebiotic synthesis were thus correlated to purine rich codons. Conclusions on prebiotic replication are discussed also in the light of recent codon usage data.

Relative codon adaptation: a generic codon bias index for prediction of gene expression.

PubMed

Fox, Jesse M; Erill, Ivan

2010-06-01

The development of codon bias indices (CBIs) remains an active field of research due to their myriad applications in computational biology. Recently, the relative codon usage bias (RCBS) was introduced as a novel CBI able to estimate codon bias without using a reference set. The results of this new index when applied to Escherichia coli and Saccharomyces cerevisiae led the authors of the original publications to conclude that natural selection favours higher expression and enhanced codon usage optimization in short genes. Here, we show that this conclusion was flawed and based on the systematic oversight of an intrinsic bias for short sequences in the RCBS index and of biases in the small data sets used for validation in E. coli. Furthermore, we reveal that how the RCBS can be corrected to produce useful results and how its underlying principle, which we here term relative codon adaptation (RCA), can be made into a powerful reference-set-based index that directly takes into account the genomic base composition. Finally, we show that RCA outperforms the codon adaptation index (CAI) as a predictor of gene expression when operating on the CAI reference set and that this improvement is significantly larger when analysing genomes with high mutational bias.

Osteogenesis imperfecta type I: Molecular heterogeneity for COL1A1 null alleles of type I collagen

DOE Office of Scientific and Technical Information (OSTI.GOV)

Willing, M.C.; Deschenes, S.P.; Pitts, S.H.

Osteogenesis imperfecta (OI) type I is the mildest form of inherited brittle-bone disease. Dermal fibroblasts from most affected individuals produce about half the usual amount of type I procollagen, as a result of a COL1A1 {open_quotes}null{close_quotes} allele. Using PCR amplification of genomic DNA from affected individuals, followed by denaturing gradient gel electrophoresis (DGGE) and SSCP, we identified seven different COL1A1 gene mutations in eight unrelated families with OI type I. Three families have single nucleotide substitutions that alter 5{prime} donor splice sites; two of these unrelated families have the same mutation. One family has a point mutation, in an exon,more » that creates a premature termination codon, and four have small deletions or insertions, within exons, that create translational frameshifts and new termination codons downstream of the mutation sites. Each mutation leads to both marked reduction in steady-state levels of mRNA from the mutant allele and a quantitative decrease in type I procollagen production. Our data demonstrate that different molecular mechanisms that have the same effect on type I collagen production result in the same clinical phenotype. 58 refs., 4 figs., 1 tab.« less

Identification of Bombyx mori bidensovirus VD1-ORF4 reveals a novel protein associated with viral structural component.

PubMed

Li, Guohui; Hu, Zhaoyang; Guo, Xuli; Li, Guangtian; Tang, Qi; Wang, Peng; Chen, Keping; Yao, Qin

2013-06-01

Bombyx mori bidensovirus (BmBDV) VD1-ORF4 (open reading frame 4, ORF4) consists of 3,318 nucleotides, which codes for a predicted 1,105-amino acid protein containing a conserved DNA polymerase motif. However, its functions in viral propagation remain unknown. In the current study, the transcription of VD1-ORF4 was examined from 6 to 96 h postinfection (p.i.) by RT-PCR, 5'-RACE revealed the transcription initiation site of BmBDV ORF4 to be -16 nucleotides upstream from the start codon, and 3'-RACE revealed the transcription termination site of VD1-ORF4 to be +7 nucleotides downstream from termination codon. Three different proteins were examined in the extracts of BmBDV-infected silkworms midguts by Western blot using raised antibodies against VD1-ORF4 deduced amino acid, and a specific protein band about 53 kDa was further detected in purified virions using the same antibodies. Taken together, BmBDV VD1-ORF4 codes for three or more proteins during the viral life cycle, one of which is a 53 kDa protein and confirmed to be a component of BmBDV virion.

Association between mismatch repair gene MSH3 codons 1036 and 222 polymorphisms and sporadic prostate cancer in the Iranian population.

PubMed

Jafary, Fariba; Salehi, Mansoor; Sedghi, Maryam; Nouri, Nayereh; Jafary, Farzaneh; Sadeghi, Farzaneh; Motamedi, Shima; Talebi, Maede

2012-01-01

The mismatch repair system (MMR) is a post-replicative DNA repair mechanism whose defects can lead to cancer. The MSH3 protein is an essential component of the system. We postulated that MSH3 gene polymorphisms might therefore be associated with prostate cancer (PC). We studied MSH3 codon 222 and MSH3 codon 1036 polymorphisms in a group of Iranian sporadic PC patients. A total of 60 controls and 18 patients were assessed using the polymerase chain reaction and single strand conformational polymorphism. For comparing the genotype frequencies of patients and controls the chi-square test was applied. The obtained result indicated that there was significantly association between G/A genotype of MSH3 codon 222 and G/G genotype of MSH3 codon 1036 with an increased PC risk (P=0.012 and P=0.02 respectively). Our results demonstrated that MSH3 codon 222 and MSH3 codon 1036 polymorphisms may be risk factors for sporadic prostate cancer in the Iranian population.

Relationship between mRNA secondary structure and sequence variability in Chloroplast genes: possible life history implications.

PubMed

Krishnan, Neeraja M; Seligmann, Hervé; Rao, Basuthkar J

2008-01-28

Synonymous sites are freer to vary because of redundancy in genetic code. Messenger RNA secondary structure restricts this freedom, as revealed by previous findings in mitochondrial genes that mutations at third codon position nucleotides in helices are more selected against than those in loops. This motivated us to explore the constraints imposed by mRNA secondary structure on evolutionary variability at all codon positions in general, in chloroplast systems. We found that the evolutionary variability and intrinsic secondary structure stability of these sequences share an inverse relationship. Simulations of most likely single nucleotide evolution in Psilotum nudum and Nephroselmis olivacea mRNAs, indicate that helix-forming propensities of mutated mRNAs are greater than those of the natural mRNAs for short sequences and vice-versa for long sequences. Moreover, helix-forming propensity estimated by the percentage of total mRNA in helices increases gradually with mRNA length, saturating beyond 1000 nucleotides. Protection levels of functionally important sites vary across plants and proteins: r-strategists minimize mutation costs in large genes; K-strategists do the opposite. Mrna length presumably predisposes shorter mRNAs to evolve under different constraints than longer mRNAs. The positive correlation between secondary structure protection and functional importance of sites suggests that some sites might be conserved due to packing-protection constraints at the nucleic acid level in addition to protein level constraints. Consequently, nucleic acid secondary structure a priori biases mutations. The converse (exposure of conserved sites) apparently occurs in a smaller number of cases, indicating a different evolutionary adaptive strategy in these plants. The differences between the protection levels of functionally important sites for r- and K-strategists reflect their respective molecular adaptive strategies. These converge with increasing domestication levels of K-strategists, perhaps because domestication increases reproductive output.

Reducing codon redundancy and screening effort of combinatorial protein libraries created by saturation mutagenesis.

PubMed

Kille, Sabrina; Acevedo-Rocha, Carlos G; Parra, Loreto P; Zhang, Zhi-Gang; Opperman, Diederik J; Reetz, Manfred T; Acevedo, Juan Pablo

2013-02-15

Saturation mutagenesis probes define sections of the vast protein sequence space. However, even if randomization is limited this way, the combinatorial numbers problem is severe. Because diversity is created at the codon level, codon redundancy is a crucial factor determining the necessary effort for library screening. Additionally, due to the probabilistic nature of the sampling process, oversampling is required to ensure library completeness as well as a high probability to encounter all unique variants. Our trick employs a special mixture of three primers, creating a degeneracy of 22 unique codons coding for the 20 canonical amino acids. Therefore, codon redundancy and subsequent screening effort is significantly reduced, and a balanced distribution of codon per amino acid is achieved, as demonstrated exemplarily for a library of cyclohexanone monooxygenase. We show that this strategy is suitable for any saturation mutagenesis methodology to generate less-redundant libraries.

Diverse expression levels of two codon-optimized genes that encode human papilloma virus type 16 major protein L1 in Hansenula polymorpha.

PubMed

Liu, Cunbao; Yang, Xu; Yao, Yufeng; Huang, Weiwei; Sun, Wenjia; Ma, Yanbing

2014-05-01

Two versions of an optimized gene that encodes human papilloma virus type 16 major protein L1 were designed according to the codon usage frequency of Pichia pastoris. Y16 was highly expressed in both P. pastoris and Hansenula polymorpha. M16 expression was as efficient as that of Y16 in P. pastoris, but merely detectable in H. polymorpha even though transcription levels of M16 and Y16 were similar. H. polymorpha had a unique codon usage frequency that contains many more rare codons than Saccharomyces cerevisiae or P. pastoris. These findings indicate that even codon-optimized genes that are expressed well in S. cerevisiae and P. pastoris may be inefficiently expressed in H. polymorpha; thus rare codons must be avoided when universal optimized gene versions are designed to facilitate expression in a variety of yeast expression systems, especially H. polymorpha is involved.

ClubSub-P: Cluster-Based Subcellular Localization Prediction for Gram-Negative Bacteria and Archaea

PubMed Central

Paramasivam, Nagarajan; Linke, Dirk

2011-01-01

The subcellular localization (SCL) of proteins provides important clues to their function in a cell. In our efforts to predict useful vaccine targets against Gram-negative bacteria, we noticed that misannotated start codons frequently lead to wrongly assigned SCLs. This and other problems in SCL prediction, such as the relatively high false-positive and false-negative rates of some tools, can be avoided by applying multiple prediction tools to groups of homologous proteins. Here we present ClubSub-P, an online database that combines existing SCL prediction tools into a consensus pipeline from more than 600 proteomes of fully sequenced microorganisms. On top of the consensus prediction at the level of single sequences, the tool uses clusters of homologous proteins from Gram-negative bacteria and from Archaea to eliminate false-positive and false-negative predictions. ClubSub-P can assign the SCL of proteins from Gram-negative bacteria and Archaea with high precision. The database is searchable, and can easily be expanded using either new bacterial genomes or new prediction tools as they become available. This will further improve the performance of the SCL prediction, as well as the detection of misannotated start codons and other annotation errors. ClubSub-P is available online at http://toolkit.tuebingen.mpg.de/clubsubp/ PMID:22073040

Codon usage bias and phylogenetic analysis of mitochondrial ND1 gene in pisces, aves, and mammals.

PubMed

Uddin, Arif; Choudhury, Monisha Nath; Chakraborty, Supriyo

2018-01-01

The mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1 (MT-ND1) gene is a subunit of the respiratory chain complex I and involved in the first step of the electron transport chain of oxidative phosphorylation (OXPHOS). To understand the pattern of compositional properties, codon usage and expression level of mitochondrial ND1 genes in pisces, aves, and mammals, we used bioinformatic approaches as no work was reported earlier. In this study, a perl script was used for calculating nucleotide contents and different codon usage bias parameters. The codon usage bias of MT-ND1 was low but the expression level was high as revealed from high ENC and CAI value. Correspondence analysis (COA) suggests that the pattern of codon usage for MT-ND1 gene is not same across species and that compositional constraint played an important role in codon usage pattern of this gene among pisces, aves, and mammals. From the regression equation of GC12 on GC3, it can be inferred that the natural selection might have played a dominant role while mutation pressure played a minor role in influencing the codon usage patterns. Further, ND1 gene has a discrepancy with cytochrome B (CYB) gene in preference of codons as evident from COA. The codon usage bias was low. It is influenced by nucleotide composition, natural selection, mutation pressure, length (number) of amino acids, and relative dinucleotide composition. This study helps in understanding the molecular biology, genetics, evolution of MT-ND1 gene, and also for designing a synthetic gene.

Codon usage and expression level of human mitochondrial 13 protein coding genes across six continents.

PubMed

Chakraborty, Supriyo; Uddin, Arif; Mazumder, Tarikul Huda; Choudhury, Monisha Nath; Malakar, Arup Kumar; Paul, Prosenjit; Halder, Binata; Deka, Himangshu; Mazumder, Gulshana Akthar; Barbhuiya, Riazul Ahmed; Barbhuiya, Masuk Ahmed; Devi, Warepam Jesmi

2017-12-02

The study of codon usage coupled with phylogenetic analysis is an important tool to understand the genetic and evolutionary relationship of a gene. The 13 protein coding genes of human mitochondria are involved in electron transport chain for the generation of energy currency (ATP). However, no work has yet been reported on the codon usage of the mitochondrial protein coding genes across six continents. To understand the patterns of codon usage in mitochondrial genes across six different continents, we used bioinformatic analyses to analyze the protein coding genes. The codon usage bias was low as revealed from high ENC value. Correlation between codon usage and GC3 suggested that all the codons ending with G/C were positively correlated with GC3 but vice versa for A/T ending codons with the exception of ND4L and ND5 genes. Neutrality plot revealed that for the genes ATP6, COI, COIII, CYB, ND4 and ND4L, natural selection might have played a major role while mutation pressure might have played a dominant role in the codon usage bias of ATP8, COII, ND1, ND2, ND3, ND5 and ND6 genes. Phylogenetic analysis indicated that evolutionary relationships in each of 13 protein coding genes of human mitochondria were different across six continents and further suggested that geographical distance was an important factor for the origin and evolution of 13 protein coding genes of human mitochondria. Copyright © 2017 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

JCoDA: a tool for detecting evolutionary selection.

PubMed

Steinway, Steven N; Dannenfelser, Ruth; Laucius, Christopher D; Hayes, James E; Nayak, Sudhir

2010-05-27

The incorporation of annotated sequence information from multiple related species in commonly used databases (Ensembl, Flybase, Saccharomyces Genome Database, Wormbase, etc.) has increased dramatically over the last few years. This influx of information has provided a considerable amount of raw material for evaluation of evolutionary relationships. To aid in the process, we have developed JCoDA (Java Codon Delimited Alignment) as a simple-to-use visualization tool for the detection of site specific and regional positive/negative evolutionary selection amongst homologous coding sequences. JCoDA accepts user-inputted unaligned or pre-aligned coding sequences, performs a codon-delimited alignment using ClustalW, and determines the dN/dS calculations using PAML (Phylogenetic Analysis Using Maximum Likelihood, yn00 and codeml) in order to identify regions and sites under evolutionary selection. The JCoDA package includes a graphical interface for Phylip (Phylogeny Inference Package) to generate phylogenetic trees, manages formatting of all required file types, and streamlines passage of information between underlying programs. The raw data are output to user configurable graphs with sliding window options for straightforward visualization of pairwise or gene family comparisons. Additionally, codon-delimited alignments are output in a variety of common formats and all dN/dS calculations can be output in comma-separated value (CSV) format for downstream analysis. To illustrate the types of analyses that are facilitated by JCoDA, we have taken advantage of the well studied sex determination pathway in nematodes as well as the extensive sequence information available to identify genes under positive selection, examples of regional positive selection, and differences in selection based on the role of genes in the sex determination pathway. JCoDA is a configurable, open source, user-friendly visualization tool for performing evolutionary analysis on homologous coding sequences. JCoDA can be used to rapidly screen for genes and regions of genes under selection using PAML. It can be freely downloaded at http://www.tcnj.edu/~nayaklab/jcoda.

JCoDA: a tool for detecting evolutionary selection

PubMed Central

2010-01-01

Background The incorporation of annotated sequence information from multiple related species in commonly used databases (Ensembl, Flybase, Saccharomyces Genome Database, Wormbase, etc.) has increased dramatically over the last few years. This influx of information has provided a considerable amount of raw material for evaluation of evolutionary relationships. To aid in the process, we have developed JCoDA (Java Codon Delimited Alignment) as a simple-to-use visualization tool for the detection of site specific and regional positive/negative evolutionary selection amongst homologous coding sequences. Results JCoDA accepts user-inputted unaligned or pre-aligned coding sequences, performs a codon-delimited alignment using ClustalW, and determines the dN/dS calculations using PAML (Phylogenetic Analysis Using Maximum Likelihood, yn00 and codeml) in order to identify regions and sites under evolutionary selection. The JCoDA package includes a graphical interface for Phylip (Phylogeny Inference Package) to generate phylogenetic trees, manages formatting of all required file types, and streamlines passage of information between underlying programs. The raw data are output to user configurable graphs with sliding window options for straightforward visualization of pairwise or gene family comparisons. Additionally, codon-delimited alignments are output in a variety of common formats and all dN/dS calculations can be output in comma-separated value (CSV) format for downstream analysis. To illustrate the types of analyses that are facilitated by JCoDA, we have taken advantage of the well studied sex determination pathway in nematodes as well as the extensive sequence information available to identify genes under positive selection, examples of regional positive selection, and differences in selection based on the role of genes in the sex determination pathway. Conclusions JCoDA is a configurable, open source, user-friendly visualization tool for performing evolutionary analysis on homologous coding sequences. JCoDA can be used to rapidly screen for genes and regions of genes under selection using PAML. It can be freely downloaded at http://www.tcnj.edu/~nayaklab/jcoda. PMID:20507581

Overcoming codon bias: a method for high-level overexpression of Plasmodium and other AT-rich parasite genes in Escherichia coli.

PubMed

Baca, A M; Hol, W G

2000-02-01

Parasite genes often use codons which are rarely used in the highly expressed genes of Escherichia coli, possibly resulting in translational stalling and lower yields of recombinant protein. We have constructed the "RIG" plasmid to overcome the potential codon-bias problem seen in Plasmodium genes. RIG contains the genes that encode three tRNAs (Arg, Ile, Gly), which recognise rare codons found in parasite genes. When co-transformed into E. coli along with expression plasmids containing parasite genes, RIG can greatly increase levels of overexpressed protein. Codon frequency analysis suggests that RIG may be applied to a variety of protozoan and helminth genes.

A Novel Frameshift Mutation at Codons 138/139 (HBB: c.417_418insT) on the β-Globin Gene Leads to β-Thalassemia.

PubMed

Jiang, Fan; Huang, Lv-Yin; Chen, Gui-Lan; Zhou, Jian-Ying; Xie, Xing-Mei; Li, Dong-Zhi

2017-01-01

We describe a new β-thalassemic mutation in a Chinese subject. This allele develops by insertion of one nucleotide (+T) between codons 138 and 139 in the third exon of the β-globin gene. The mutation causes a frameshift that leads to a termination codon at codon 139. In the heterozygote, this allele has the phenotype of classical β-thalassemia (β-thal) minor.

Codon Optimization of the Human Papillomavirus E7 Oncogene Induces a CD8+ T Cell Response to a Cryptic Epitope Not Harbored by Wild-Type E7

PubMed Central

Lorenz, Felix K. M.; Wilde, Susanne; Voigt, Katrin; Kieback, Elisa; Mosetter, Barbara; Schendel, Dolores J.; Uckert, Wolfgang

2015-01-01

Codon optimization of nucleotide sequences is a widely used method to achieve high levels of transgene expression for basic and clinical research. Until now, immunological side effects have not been described. To trigger T cell responses against human papillomavirus, we incubated T cells with dendritic cells that were pulsed with RNA encoding the codon-optimized E7 oncogene. All T cell receptors isolated from responding T cell clones recognized target cells expressing the codon-optimized E7 gene but not the wild type E7 sequence. Epitope mapping revealed recognition of a cryptic epitope from the +3 alternative reading frame of codon-optimized E7, which is not encoded by the wild type E7 sequence. The introduction of a stop codon into the +3 alternative reading frame protected the transgene product from recognition by T cell receptor gene-modified T cells. This is the first experimental study demonstrating that codon optimization can render a transgene artificially immunogenic through generation of a dominant cryptic epitope. This finding may be of great importance for the clinical field of gene therapy to avoid rejection of gene-corrected cells and for the design of DNA- and RNA-based vaccines, where codon optimization may artificially add a strong immunogenic component to the vaccine. PMID:25799237

Differential Properties of Cytomegalovirus pUL97 Kinase Isoforms Affect Viral Replication and Maribavir Susceptibility

PubMed Central

Webel, Rike; Hakki, Morgan; Prichard, Mark N.; Rawlinson, William D.; Marschall, Manfred

2014-01-01

ABSTRACT The human cytomegalovirus (HCMV)-encoded kinase pUL97 is required for efficient viral replication. Previous studies described two isoforms of pUL97, the full-length isoform (M1) and a smaller isoform likely resulting from translation initiation at codon 74 (M74). Here, we report the detection of a third pUL97 isoform during viral infection resulting from translation initiation at codon 157 (isoform M157). The consistent expression of isoform M157 as a minor component of pUL97 during infection with clinical and laboratory-adapted HCMV strains was suppressed when codon 157 was mutagenized. Viral mutants expressing specific isoforms were generated to compare their growth and drug susceptibility phenotypes, as well as pUL97 intracellular localization patterns and kinase activities. The exclusive expression of isoform M157 resulted in substantially reduced viral growth and resistance to the pUL97 inhibitor maribavir while retaining susceptibility to ganciclovir. Confocal imaging demonstrated reduced nuclear import of amino-terminal deletion isoforms compared to isoform M1. Isoform M157 showed reduced efficiency of various substrate protein interactions and autophosphorylation, whereas Rb phosphorylation was preserved. These results reveal differential properties of pUL97 isoforms that affect viral replication, with implications for the antiviral efficacy of maribavir. IMPORTANCE The HCMV UL97 kinase performs important functions in viral replication that are targeted by the antiviral drug maribavir. Here, we describe a naturally occurring short isoform of the kinase that when expressed by itself in a recombinant virus results in altered intracellular localization, impaired growth, and high-level resistance to maribavir compared to those of the predominant full-length counterpart. This is another factor to consider in explaining why maribavir appears to have variable antiviral activity in cell culture and in vivo. PMID:24522923

Early Clinical Diagnosis of PC1/3 Deficiency in a Patient With a Novel Homozygous PCSK1 Splice-Site Mutation.

PubMed

Härter, Bettina; Fuchs, Irene; Müller, Thomas; Akbulut, Ulas Emre; Cakir, Murat; Janecke, Andreas R

2016-04-01

Autosomal recessive proprotein convertase 1/3 (PC1/3) deficiency, caused by mutations in the PCSK1 gene, is characterized by severe congenital malabsorptive diarrhea, early-onset obesity, and certain endocrine abnormalities. We suspected PC1/3 deficiency in a 4-month-old girl based on the presence of congenital diarrhea and polyuria. Sequencing the whole coding region and splice sites detected a novel homozygous PCSK1 splice-site mutation, c.544-2A>G, in the patient. The mutation resulted in the skipping of exon 5, the generation of a premature termination codon, and nonsense-mediated PCSK1 messenger ribonucleic acid decay, which was demonstrated in complementary DNA derived from fibroblasts.

MicroRNA Targeting Specificity in Mammals: Determinants Beyond Seed Pairing

PubMed Central

Grimson, Andrew; Farh, Kyle Kai-How; Johnston, Wendy K.; Garrett-Engele, Philip; Lim, Lee P.; Bartel, David P.

2013-01-01

Summary Mammalian microRNAs (miRNAs) pair to 3'UTRs of mRNAs to direct their posttranscriptional repression. Important for target recognition are ~7-nt sites that match the seed region of the miRNA. However, these seed matches are not always sufficient for repression, indicating that other characteristics help specify targeting. By combining computational and experimental approaches, we uncovered five general features of site context that boost site efficacy: AU-rich nucleotide composition near the site, proximity to sites for co-expressed miRNAs (which leads to cooperative action), proximity to residues pairing to miRNA nucleotides 13–16, and positioning within the 3'UTR at least 15 nt from the stop codon and away from the center of long UTRs. A model combining these context determinants quantitatively predicts site performance both for exogenously added miRNAs and for endogenous miRNA-message interactions. Because it predicts site efficacy without recourse to evolutionary conservation, the model also identifies effective nonconserved sites and siRNA off-targets. PMID:17612493

Influence of certain forces on evolution of synonymous codon usage bias in certain species of three basal orders of aquatic insects.

PubMed

Selva Kumar, C; Nair, Rahul R; Sivaramakrishnan, K G; Ganesh, D; Janarthanan, S; Arunachalam, M; Sivaruban, T

2012-12-01

Forces that influence the evolution of synonymous codon usage bias are analyzed in six species of three basal orders of aquatic insects. The rationale behind choosing six species of aquatic insects (three from Ephemeroptera, one from Plecoptera, and two from Odonata) for the present analysis is based on phylogenetic position at the basal clades of the Order Insecta facilitating the understanding of the evolution of codon bias and of factors shaping codon usage patterns in primitive clades of insect lineages and their subtle differences in some of their ecological and environmental requirements in terms of habitat-microhabitat requirements, altitudinal preferences, temperature tolerance ranges, and consequent responses to climate change impacts. The present analysis focuses on open reading frames of the 13 protein-coding genes in the mitochondrial genome of six carefully chosen insect species to get a comprehensive picture of the evolutionary intricacies of codon bias. In all the six species, A and T contents are observed to be significantly higher than G and C, and are used roughly equally. Since transcription hypothesis on codon usage demands A richness and T poorness, it is quite likely that mutation pressure may be the key factor associated with synonymous codon usage (SCU) variations in these species because the mutation hypothesis predicts AT richness and GC poorness in the mitochondrial DNA. Thus, AT-biased mutation pressure seems to be an important factor in framing the SCU variation in all the selected species of aquatic insects, which in turn explains the predominance of A and T ending codons in these species. This study does not find any association between microhabitats and codon usage variations in the mitochondria of selected aquatic insects. However, this study has identified major forces, such as compositional constraints and mutation pressure, which shape patterns of codon usage in mitochondrial genes in the primitive clades of insect lineages.

Role of large thermal fluctuations and magnesium ions in t-RNA selectivity of the ribosome

PubMed Central

Guo, Zuojun; Gibson, Meghan; Sitha, Sanyasi; Chu, Steven; Mohanty, Udayan

2011-01-01

The fidelity of translation selection begins with the base pairing of codon-anticodon complex between the m-RNA and tRNAs. Binding of cognate and near-cognate tRNAs induces 30S subunit of the ribosome to wrap around the ternary complex, EF-Tu(GTP)aa-tRNA. We have proposed that large thermal fluctuations play a crucial role in the selection process. To test this conjecture, we have developed a theoretical technique to determine the probability that the ternary complex, as a result of large thermal fluctuations, forms contacts leading to stabilization of the GTPase activated state. We argue that the configurational searches for such processes are in the tail end of the probability distribution and show that the probability for this event is localized around the most likely configuration. Small variations in the repositioning of cognate relative to near-cognate complexes lead to rate enhancement of the cognate complex. The binding energies of over a dozen unique site-bound magnesium structural motifs are investigated and provide insights into the nature of interaction of divalent metal ions with the ribosome. PMID:21368154

Analysis of the regulatory region of the protease III (ptr) gene of Escherichia coli K-12.

PubMed

Claverie-Martin, F; Diaz-Torres, M R; Kushner, S R

1987-01-01

The ptr gene of Escherichia coli encodes protease III (Mr 110,000) and a 50-kDa polypeptide, both of which are found in the periplasmic space. The gene is physically located between the recC and recB loci on the E. coli chromosome. The nucleotide sequence of a 1167-bp EcoRV-ClaI fragment of chromosomal DNA containing the promoter region and 885 bp of the ptr coding sequence has been determined. S1 nuclease mapping analysis showed that the major 5' end of the ptr mRNA was localized 127 bp upstream from the ATG start codon. The open reading frame (ORF), preceded by a Shine-Dalgarno sequence, extends to the end of the sequenced DNA. Downstream from the -35 and -10 regions is a sequence that strongly fits the consensus sequence of known nitrogen-regulated promoters. A signal peptide of 23 amino acids residues is present at the N terminus of the derived amino acid sequence. The cleavage site as well as the ORF were confirmed by sequencing the N terminus of mature protease III.

Three stages in the evolution of the genetic code

NASA Technical Reports Server (NTRS)

Baumann, U.; Oro, J.

1993-01-01

A diversification of the genetic code based on the number of codons available for the proteinous amino acids is established. Three groups of amino acids during evolution of the code are distinguished. On the basis of their chemical complexity those amino acids emerging later in a translation process are derived. Codon number and chemical complexity indicate that His, Phe, Tyr, Cys and either Lys or Asn were introduced in the second stage, whereas the number of codons alone gives evidence that Trp and Met were introduced in the third stage. The amino acids of stage 1 use purine-rich codons, while all the amino acids introduced in the second stage, in contrast, use pyrimidines in the third position of their codons. A low abundance of pyrimidines during early translation is derived. This assumption is supported by experiments on non-enzymatic replication and interactions of hairpin loops with a complementary strand. A back extrapolation concludes a high purine content of the first nucleic acids, which gradually decreased during their evolution. Amino acids independently available from prebiotic synthesis were thus correlated to purine-rich codons. Implications on the prebiotic replication are discussed also in the light of recent codon usage data.

Sequence analysis of MHC class I α2 from sockeye salmon (Oncorhynchus nerka).

PubMed

McClelland, Erin K; Ming, Tobi J; Tabata, Amy; Miller, Kristina M

2011-09-01

Most studies assessing adaptive MHC diversity in salmon populations have focused on the classical class II DAB or DAA loci, as these have been most amenable to single PCR amplifications due to their relatively low level of sequence divergence. Herein, we report the characterization of the classical class I UBA α2 locus based on collections taken throughout the species range of sockeye salmon (Oncorhynchus nerka). Through use of multiple lineage-specific primer sets, denaturing gradient gel electrophoresis and sequencing, we identified thirty-four alleles from three highly divergent lineages. Sequence identity between lineages ranged from 30.0% to 56.8% but was relatively high within lineages. Allelic identity within the antigen recognition site (ARS) was greater than for the longer sequence. Global positive selection on UBA was seen at the sequence level (dN:dS = 1.012) with four codons under positive selection and 12 codons under negative selection. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

Glyphosate resistance: state of knowledge

PubMed Central

Sammons, Robert Douglas; Gaines, Todd A

2014-01-01

Studies of mechanisms of resistance to glyphosate have increased current understanding of herbicide resistance mechanisms. Thus far, single-codon non-synonymous mutations of EPSPS (5-enolypyruvylshikimate-3-phosphate synthase) have been rare and, relative to other herbicide mode of action target-site mutations, unconventionally weak in magnitude for resistance to glyphosate. However, it is possible that weeds will emerge with non-synonymous mutations of two codons of EPSPS to produce an enzyme endowing greater resistance to glyphosate. Today, target-gene duplication is a common glyphosate resistance mechanism and could become a fundamental process for developing any resistance trait. Based on competition and substrate selectivity studies in several species, rapid vacuole sequestration of glyphosate occurs via a transporter mechanism. Conversely, as the chloroplast requires transporters for uptake of important metabolites, transporters associated with the two plastid membranes may separately, or together, successfully block glyphosate delivery. A model based on finite glyphosate dose and limiting time required for chloroplast loading sets the stage for understanding how uniquely different mechanisms can contribute to overall glyphosate resistance. PMID:25180399

Emergent Rules for Codon Choice Elucidated by Editing Rare Arginine Codons in Escherichia coli

DTIC Science & Technology

2016-09-20

alternative codons are more likely to be viable. To evaluate synonymous and nonsynonymous alternatives to essential AGRs further, we imple- mented a CRISPR ... Crispr -assisted MAGE). First, we designed oligos that changed not only the target AGR codon to NNN but also made several synonymous changes at least 50...nt downstream that would disrupt a 20-bp CRISPR target lo- cus. MAGE was used to replace each AGR with NNN in parallel, and CRISPR /cas9 was used to

Identification and codon reading properties of 5-cyanomethyl uridine, a new modified nucleoside found in the anticodon wobble position of mutant haloarchaeal isoleucine tRNAs

PubMed Central

Mandal, Debabrata; Köhrer, Caroline; Su, Dan; Babu, I. Ramesh; Chan, Clement T.Y.; Liu, Yuchen; Söll, Dieter; Blum, Paul; Kuwahara, Masayasu; Dedon, Peter C.; RajBhandary, Uttam L.

2014-01-01

Most archaea and bacteria use a modified C in the anticodon wobble position of isoleucine tRNA to base pair with A but not with G of the mRNA. This allows the tRNA to read the isoleucine codon AUA without also reading the methionine codon AUG. To understand why a modified C, and not U or modified U, is used to base pair with A, we mutated the C34 in the anticodon of Haloarcula marismortui isoleucine tRNA (tRNA2Ile) to U, expressed the mutant tRNA in Haloferax volcanii, and purified and analyzed the tRNA. Ribosome binding experiments show that although the wild-type tRNA2Ile binds exclusively to the isoleucine codon AUA, the mutant tRNA binds not only to AUA but also to AUU, another isoleucine codon, and to AUG, a methionine codon. The G34 to U mutant in the anticodon of another H. marismortui isoleucine tRNA species showed similar codon binding properties. Binding of the mutant tRNA to AUG could lead to misreading of the AUG codon and insertion of isoleucine in place of methionine. This result would explain why most archaea and bacteria do not normally use U or a modified U in the anticodon wobble position of isoleucine tRNA for reading the codon AUA. Biochemical and mass spectrometric analyses of the mutant tRNAs have led to the discovery of a new modified nucleoside, 5-cyanomethyl U in the anticodon wobble position of the mutant tRNAs. 5-Cyanomethyl U is present in total tRNAs from euryarchaea but not in crenarchaea, eubacteria, or eukaryotes. PMID:24344322

The complete mitochondrial genome of Setaria digitata (Nematoda: Filarioidea): Mitochondrial gene content, arrangement and composition compared with other nematodes.

PubMed

Yatawara, Lalani; Wickramasinghe, Susiji; Rajapakse, R P V J; Agatsuma, Takeshi

2010-09-01

In the present study, we determined the complete mitochondrial (mt) genome sequence (13,839bp) of parasitic nematode Setaria digitata and its structure and organization compared with Onchocerca volvulus, Dirofilaria immitis and Brugia malayi. The mt genome of S. digitata is slightly larger than the mt genomes of other filarial nematodes. S. digitata mt genome contains 36 genes (12 protein-coding genes, 22 transfer RNAs and 2 ribosomal RNAs) that are typically found in metazoans. This genome contains a high A+T (75.1%) content and low G+C content (24.9%). The mt gene order for S. digitata is the same as those for O. volvulus, D. immitis and B. malayi but it is distinctly different from other nematodes compared. The start codons inferred in the mt genome of S. digitata are TTT, ATT, TTG, ATG, GTT and ATA. Interestingly, the initiation codon TTT is unique to S. digitata mt genome and four protein-coding genes use this codon as a translation initiation codon. Five protein-coding genes use TAG as a stop codon whereas three genes use TAA and four genes use T as a termination codon. Out of 64 possible codons, only 57 are used for mitochondrial protein-coding genes of S. digitata. T-rich codons such as TTT (18.9%), GTT (7.9%), TTG (7.8%), TAT (7%), ATT (5.7%), TCT (4.8%) and TTA (4.1%) are used more frequently. This pattern of codon usage reflects the strong bias for T in the mt genome of S. digitata. In conclusion, the present investigation provides new molecular data for future studies of the comparative mitochondrial genomics and systematic of parasitic nematodes of socio-economic importance. 2010 Elsevier B.V. All rights reserved.

Demonstration of GTG as an endogenous initiation codon for a human mRNA transcript revealed by molecular cloning of the serpin endopin 2B.

PubMed

Hwang, Shin-Rong; Garza, Christina Z; Wegrzyn, Jill; Hook, Vivian Y H

2004-08-16

This study demonstrates utilization of the novel GTG initiation codon for translation of a human mRNA transcript that encodes the serpin endopin 2B, a protease inhibitor. Molecular cloning revealed the nucleotide sequence of the human endopin 2B cDNA. Its deduced primary sequence shows high homology to bovine endopin 2A that possesses cross-class protease inhibition of elastase and papain. Notably, the human endopin 2B cDNA sequence revealed GTG as the predicted translation initiation codon; the predicted translation product of 46 kDa endopin 2B was produced by in vitro translation of 35S-endopin 2B with mammalian (rabbit) protein translation components. Importantly, bioinformatic studies demonstrated the presence of the entire human endopin 2B cDNA sequence with GTG as initiation codon within the human genome on chromosome 14. Further evidence for GTG as a functional initiation codon was illustrated by GTG-mediated in vitro translation of the heterologous protein EGFP, and by GTG-mediated expression of EGFP in mammalian PC12 cells. Mutagenesis of GTG to GTC resulted in the absence of EGFP expression in PC12 cells, indicating the function of GTG as an initiation codon. In addition, it was apparent that the GTG initiation codon produces lower levels of translated protein compared to ATG as initiation codon. Significantly, GTG-mediated translation of endopin 2B demonstrates a functional human gene product not previously predicted from initial analyses of the human genome. Further analyses based on GTG as an alternative initiation codon may predict new candidate genes of the human genome.

Selection of the simplest RNA that binds isoleucine

PubMed Central

LOZUPONE, CATHERINE; CHANGAYIL, SHANKAR; MAJERFELD, IRENE; YARUS, MICHAEL

2003-01-01

We have identified the simplest RNA binding site for isoleucine using selection-amplification (SELEX), by shrinking the size of the randomized region until affinity selection is extinguished. Such a protocol can be useful because selection does not necessarily make the simplest active motif most prominent, as is often assumed. We find an isoleucine binding site that behaves exactly as predicted for the site that requires fewest nucleotides. This UAUU motif (16 highly conserved positions; 27 total), is also the most abundant site in successful selections on short random tracts. The UAUU site, now isolated independently at least 63 times, is a small asymmetric internal loop. Conserved loop sequences include isoleucine codon and anticodon triplets, whose nucleotides are required for amino acid binding. This reproducible association between isoleucine and its coding sequences supports the idea that the genetic code is, at least in part, a stereochemical residue of the most easily isolated RNA–amino acid binding structures. PMID:14561881

Systematic bacterialization of yeast genes identifies a near-universally swappable pathway

PubMed Central

Kachroo, Aashiq H; Laurent, Jon M; Akhmetov, Azat; Szilagyi-Jones, Madelyn; McWhite, Claire D; Zhao, Alice; Marcotte, Edward M

2017-01-01

Eukaryotes and prokaryotes last shared a common ancestor ~2 billion years ago, and while many present-day genes in these lineages predate this divergence, the extent to which these genes still perform their ancestral functions is largely unknown. To test principles governing retention of ancient function, we asked if prokaryotic genes could replace their essential eukaryotic orthologs. We systematically replaced essential genes in yeast by their 1:1 orthologs from Escherichia coli. After accounting for mitochondrial localization and alternative start codons, 31 out of 51 bacterial genes tested (61%) could complement a lethal growth defect and replace their yeast orthologs with minimal effects on growth rate. Replaceability was determined on a pathway-by-pathway basis; codon usage, abundance, and sequence similarity contributed predictive power. The heme biosynthesis pathway was particularly amenable to inter-kingdom exchange, with each yeast enzyme replaceable by its bacterial, human, or plant ortholog, suggesting it as a near-universally swappable pathway. DOI: http://dx.doi.org/10.7554/eLife.25093.001 PMID:28661399

Importance of codon usage for the temporal regulation of viral gene expression

PubMed Central

Shin, Young C.; Bischof, Georg F.; Lauer, William A.; Desrosiers, Ronald C.

2015-01-01

The glycoproteins of herpesviruses and of HIV/SIV are made late in the replication cycle and are derived from transcripts that use an unusual codon usage that is quite different from that of the host cell. Here we show that the actions of natural transinducers from these two different families of persistent viruses (Rev of SIV and ORF57 of the rhesus monkey rhadinovirus) are dependent on the nature of the skewed codon usage. In fact, the transinducibility of expression of these glycoproteins by Rev and by ORF57 can be flipped simply by changing the nature of the codon usage. Even expression of a luciferase reporter could be made Rev dependent or ORF57 dependent by distinctive changes to its codon usage. Our findings point to a new general principle in which different families of persisting viruses use a poor codon usage that is skewed in a distinctive way to temporally regulate late expression of structural gene products. PMID:26504241

Comparison of codon usage bias across Leishmania and Trypanosomatids to understand mRNA secondary structure, relative protein abundance and pathway functions.

PubMed

Subramanian, Abhishek; Sarkar, Ram Rup

2015-10-01

Understanding the variations in gene organization and its effect on the phenotype across different Leishmania species, and to study differential clinical manifestations of parasite within the host, we performed large scale analysis of codon usage patterns between Leishmania and other known Trypanosomatid species. We present the causes and consequences of codon usage bias in Leishmania genomes with respect to mutational pressure, translational selection and amino acid composition bias. We establish GC bias at wobble position that governs codon usage bias across Leishmania species, rather than amino acid composition bias. We found that, within Leishmania, homogenous codon context coding for less frequent amino acid pairs and codons avoiding formation of folding structures in mRNA are essentially chosen. We predicted putative differences in global expression between genes belonging to specific pathways across Leishmania. This explains the role of evolution in shaping the otherwise conserved genome to demonstrate species-specific function-level differences for efficient survival. Copyright © 2015 Elsevier Inc. All rights reserved.

Expression of codon-optmized phosphoenolpyruvate carboxylase gene from Glaciecola sp. HTCC2999 in Escherichia coli and its application for C4 chemical production.

PubMed

Park, Soohyun; Pack, Seung Pil; Lee, Jinwon

2012-08-01

We examined the expression of the phosphoenolpyruvate carboxylase (PEPC) gene from marine bacteria in Escherichia coli using codon optimization. The codon-optimized PEPC gene was expressed in the E. coli K-12 strain W3110. SDS-PAGE analysis revealed that the codon-optimized PEPC gene was only expressed in E. coli, and measurement of enzyme activity indicated the highest PEPC activity in the E. coli SGJS112 strain that contained the codon-optimized PEPC gene. In fermentation assays, the E. coli SGJS112 produced the highest yield of oxaloacetate using glucose as the source and produced a 20-times increase in the yield of malate compared to the control. We concluded that the codon optimization enabled E. coli to express the PEPC gene derived from the Glaciecola sp. HTCC2999. Also, the expressed protein exhibited an enzymatic activity similar to that of E. coli PEPC and increased the yield of oxaloacetate and malate in an E. coli system.

Linkage and mutational analysis of familial Alzheimer disease kindreds for the APP gene region

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kamino, K.; Anderson, L.; O'dahl, S.

1992-11-01

A large number of familial Alzheimer disease (FAD) kindreds were examined to determine whether mutations in the amyloid precursor protein (APP) gene could be responsible for the disease. Previous studies have identified three mutations at APP codon 717 which are pathogenic for Alzheimer disease (AD). Samples from affected subjects were examined for mutations in exons 16 and 17 of the APP gene. A combination of direct sequencing and single-strand conformational polymorphism analysis was used. Sporadic AD and normal controls were also examined by the same methods. Five sequence variants were identified. One variant at APP codon 693 resulted in amore » Glu[yields]Gly change. This is the same codon as the hereditary cerebral hemorrhage with amyloidosis-Dutch type Glu[yields]Gln mutation. Another single-base change at APP codon 708 did not alter the amino acid encoded at this site. Two point mutations and a 6-bp deletion were identified in the intronic sequences surrounding exon 17. None of the variants could be unambigously determined to be responsible for FAD. The larger families were also analyzed by testing for linkage of FAD to a highly polymorphic short tandem repeat marker (D21S210) that is tightly linked to APP. Highly negative LOD scores were obtained for the family groups tested, and linkage was formally excluded beyond [theta] = .10 for the Volga German kindreds, [theta] = .20 for early-onset non-Volga Germans, and [theta] = .10 for late-onset families. LOD scores for linkage of FAD to markers centromeric to APP (D21S1/S11, D21S13, and D21S215) were also negative in the three family groups. These studies show that APP mutations account for AD in only a small fraction of FAD kindreds. 49 refs., 6 figs., 4 tabs.« less

EUGÈNE'HOM: a generic similarity-based gene finder using multiple homologous sequences

PubMed Central

Foissac, Sylvain; Bardou, Philippe; Moisan, Annick; Cros, Marie-Josée; Schiex, Thomas

2003-01-01

EUGÈNE'HOM is a gene prediction software for eukaryotic organisms based on comparative analysis. EUGÈNE'HOM is able to take into account multiple homologous sequences from more or less closely related organisms. It integrates the results of TBLASTX analysis, splice site and start codon prediction and a robust coding/non-coding probabilistic model which allows EUGÈNE'HOM to handle sequences from a variety of organisms. The current target of EUGÈNE'HOM is plant sequences. The EUGÈNE'HOM web site is available at http://genopole.toulouse.inra.fr/bioinfo/eugene/EuGeneHom/cgi-bin/EuGeneHom.pl. PMID:12824408

Development of a Novel Technology for Label Free DNA Sequencing

DTIC Science & Technology

2012-05-21

of the C-H bond stretch vibrations in the planes of the corresponding DNA bases , and in the higher-frequency side, sequence-identifier region is...composed of the N-H bond stretch vibrations in the planes of the corresponding DNA bases . In addition, the sequence-identifier dividing region almost...regions are localized at the corresponding DNA bases and exhibit a definable dependence on the sequence form of the codons under study. Final

Stop codon readthrough generates a C-terminally extended variant of the human vitamin D receptor with reduced calcitriol response

PubMed Central

Loughran, Gary; Jungreis, Irwin; Tzani, Ioanna; Power, Michael; Dmitriev, Ruslan I.; Ivanov, Ivaylo P.; Kellis, Manolis; Atkins, John F.

2018-01-01

Although stop codon readthrough is used extensively by viruses to expand their gene expression, verified instances of mammalian readthrough have only recently been uncovered by systems biology and comparative genomics approaches. Previously, our analysis of conserved protein coding signatures that extend beyond annotated stop codons predicted stop codon readthrough of several mammalian genes, all of which have been validated experimentally. Four mRNAs display highly efficient stop codon readthrough, and these mRNAs have a UGA stop codon immediately followed by CUAG (UGA_CUAG) that is conserved throughout vertebrates. Extending on the identification of this readthrough motif, we here investigated stop codon readthrough, using tissue culture reporter assays, for all previously untested human genes containing UGA_CUAG. The readthrough efficiency of the annotated stop codon for the sequence encoding vitamin D receptor (VDR) was 6.7%. It was the highest of those tested but all showed notable levels of readthrough. The VDR is a member of the nuclear receptor superfamily of ligand-inducible transcription factors, and it binds its major ligand, calcitriol, via its C-terminal ligand-binding domain. Readthrough of the annotated VDR mRNA results in a 67 amino acid–long C-terminal extension that generates a VDR proteoform named VDRx. VDRx may form homodimers and heterodimers with VDR but, compared with VDR, VDRx displayed a reduced transcriptional response to calcitriol even in the presence of its partner retinoid X receptor. PMID:29386352

Stop codons in the hepatitis B surface proteins are enriched during antiviral therapy and are associated with host cell apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Colledge, Danielle; Soppe, Sally; Yuen, Lilly

Premature stop codons in the hepatitis B virus (HBV) surface protein can be associated with nucleos(t)ide analogue resistance due to overlap of the HBV surface and polymerase genes. The aim of this study was to determine the effect of the replication of three common surface stop codon variants on the hepatocyte. Cell lines were transfected with infectious HBV clones encoding surface stop codons rtM204I/sW196*, rtA181T/sW172*, rtV191I/sW182*, and a panel of substitutions in the surface proteins. HBsAg was measured by Western blotting. Proliferation and apoptosis were measured using flow cytometry. All three surface stop codon variants were defective in HBsAg secretion.more » Cells transfected with these variants were less proliferative and had higher levels of apoptosis than those transfected with variants that did not encode surface stop codons. The most cytopathic variant was rtM204I/sW196*. Replication of HBV encoding surface stop codons was toxic to the cell and promoted apoptosis, exacerbating disease progression. - Highlights: •Under normal circumstances, HBV replication is not cytopathic. •Premature stop codons in the HBV surface protein can be selected and enriched during nucleos(t)ide analogue therapy. •Replication of these variants can be cytopathic to the cell and promote apoptosis. •Inadequate antiviral therapy may actually promote disease progression.« less

Comparative Genomic Analysis MERS CoV Isolated from Humans and Camels with Special Reference to Virus Encoded Helicase.

PubMed

Alnazawi, Mohamed; Altaher, Abdallah; Kandeel, Mahmoud

2017-01-01

Middle East Respiratory Syndrome Coronavirus (MERS CoV) is a new emerging viral disease characterized by high fatality rate. Understanding MERS CoV genetic aspects and codon usage pattern is important to understand MERS CoV survival, adaptation, evolution, resistance to innate immunity, and help in finding the unique aspects of the virus for future drug discovery experiments. In this work, we provide comprehensive analysis of 238 MERS CoV full genomes comprised of human (hMERS) and camel (cMERS) isolates of the virus. MERS CoV genome shaping seems to be under compositional and mutational bias, as revealed by preference of A/T over G/C nucleotides, preferred codons, nucleotides at the third position of codons (NT3s), relative synonymous codon usage, hydropathicity (Gravy), and aromaticity (Aromo) indices. Effective number of codons (ENc) analysis reveals a general slight codon usage bias. Codon adaptation index reveals incomplete adaptation to host environment. MERS CoV showed high ability to resist the innate immune response by showing lower CpG frequencies. Neutrality evolution analysis revealed a more significant role of mutation pressure in cMERS over hMERS. Correspondence analysis revealed that MERS CoV genomes have three genetic clusters, which were distinct in their codon usage, host, and geographic distribution. Additionally, virtual screening and binding experiments were able to identify three new virus-encoded helicase binding compounds. These compounds can be used for further optimization of inhibitors.

GC-Content of Synonymous Codons Profoundly Influences Amino Acid Usage

PubMed Central

Li, Jing; Zhou, Jun; Wu, Ying; Yang, Sihai; Tian, Dacheng

2015-01-01

Amino acids typically are encoded by multiple synonymous codons that are not used with the same frequency. Codon usage bias has drawn considerable attention, and several explanations have been offered, including variation in GC-content between species. Focusing on a simple parameter—combined GC proportion of all the synonymous codons for a particular amino acid, termed GCsyn—we try to deepen our understanding of the relationship between GC-content and amino acid/codon usage in more details. We analyzed 65 widely distributed representative species and found a close association between GCsyn, GC-content, and amino acids usage. The overall usages of the four amino acids with the greatest GCsyn and the five amino acids with the lowest GCsyn both vary with the regional GC-content, whereas the usage of the remaining 11 amino acids with intermediate GCsyn is less variable. More interesting, we discovered that codon usage frequencies are nearly constant in regions with similar GC-content. We further quantified the effects of regional GC-content variation (low to high) on amino acid usage and found that GC-content determines the usage variation of amino acids, especially those with extremely high GCsyn, which accounts for 76.7% of the changed GC-content for those regions. Our results suggest that GCsyn correlates with GC-content and has impact on codon/amino acid usage. These findings suggest a novel approach to understanding the role of codon and amino acid usage in shaping genomic architecture and evolutionary patterns of organisms. PMID:26248983

Translational control of ribosomal protein S15.

PubMed

Portier, C; Philippe, C; Dondon, L; Grunberg-Manago, M; Ebel, J P; Ehresmann, B; Ehresmann, C

1990-08-27

The expression of ribosomal protein S15 is shown to be translationally and negatively autocontrolled using a fusion within a reporter gene. Isolation and characterization of several deregulated mutants indicate that the regulatory site (the translational operator site) overlaps the ribosome loading site of the S15 messenger. In this region, three domains, each exhibiting a stem-loop structure, were determined using chemical and enzymatic probes. The most downstream hairpin carries the Shine-Dalgarno sequence and the initiation codon. Genetic and structural data derived from mutants constructed by site-directed mutagenesis show that the operator is a dynamic structure, two domains of which can form a pseudoknot. Binding of S15 to these two domains suggests that the pseudoknot could be stabilized by S15. A model is presented in which two alternative structures would explain the molecular basis of the S15 autocontrol.

Labeling proteins on live mammalian cells using click chemistry.

PubMed

Nikić, Ivana; Kang, Jun Hee; Girona, Gemma Estrada; Aramburu, Iker Valle; Lemke, Edward A

2015-05-01

We describe a protocol for the rapid labeling of cell-surface proteins in living mammalian cells using click chemistry. The labeling method is based on strain-promoted alkyne-azide cycloaddition (SPAAC) and strain-promoted inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC) reactions, in which noncanonical amino acids (ncAAs) bearing ring-strained alkynes or alkenes react, respectively, with dyes containing azide or tetrazine groups. To introduce ncAAs site specifically into a protein of interest (POI), we use genetic code expansion technology. The protocol can be described as comprising two steps. In the first step, an Amber stop codon is introduced--by site-directed mutagenesis--at the desired site on the gene encoding the POI. This plasmid is then transfected into mammalian cells, along with another plasmid that encodes an aminoacyl-tRNA synthetase/tRNA (RS/tRNA) pair that is orthogonal to the host's translational machinery. In the presence of the ncAA, the orthogonal RS/tRNA pair specifically suppresses the Amber codon by incorporating the ncAA into the polypeptide chain of the POI. In the second step, the expressed POI is labeled with a suitably reactive dye derivative that is directly supplied to the growth medium. We provide a detailed protocol for using commercially available ncAAs and dyes for labeling the insulin receptor, and we discuss the optimal surface-labeling conditions and the limitations of labeling living mammalian cells. The protocol involves an initial cloning step that can take 4-7 d, followed by the described transfections and labeling reaction steps, which can take 3-4 d.

Multilocus patterns of polymorphism and selection across the X chromosome of Caenorhabditis remanei.

PubMed

Cutter, Asher D

2008-03-01

Natural selection and neutral processes such as demography, mutation, and gene conversion all contribute to patterns of polymorphism within genomes. Identifying the relative importance of these varied components in evolution provides the principal challenge for population genetics. To address this issue in the nematode Caenorhabditis remanei, I sampled nucleotide polymorphism at 40 loci across the X chromosome. The site-frequency spectrum for these loci provides no evidence for population size change, and one locus presents a candidate for linkage to a target of balancing selection. Selection for codon usage bias leads to the non-neutrality of synonymous sites, and despite its weak magnitude of effect (N(e)s approximately 0.1), is responsible for profound patterns of diversity and divergence in the C. remanei genome. Although gene conversion is evident for many loci, biased gene conversion is not identified as a significant evolutionary process in this sample. No consistent association is observed between synonymous-site diversity and linkage-disequilibrium-based estimators of the population recombination parameter, despite theoretical predictions about background selection or widespread genetic hitchhiking, but genetic map-based estimates of recombination are needed to rigorously test for a diversity-recombination relationship. Coalescent simulations also illustrate how a spurious correlation between diversity and linkage-disequilibrium-based estimators of recombination can occur, due in part to the presence of unbiased gene conversion. These results illustrate the influence that subtle natural selection can exert on polymorphism and divergence, in the form of codon usage bias, and demonstrate the potential of C. remanei for detecting natural selection from genomic scans of polymorphism.

Molecular identification and transcriptional regulation of porcine IFIT2 gene.

PubMed

Yang, Xiuqin; Jing, Xiaoyan; Song, Yanfang; Zhang, Caixia; Liu, Di

2018-04-06

IFN-induced protein with tetratricopeptide repeats 2 (IFIT2) plays important roles in host defense against viral infection as revealed by studies in humans and mice. However, little is known on porcine IFIT2 (pIFIT2). Here, we performed molecular cloning, expression profile, and transcriptional regulation analysis of pIFIT2. pIFIT2 gene, located on chromosome 14, is composed of two exons and have a complete coding sequence of 1407 bp. The encoded polypeptide, 468 aa in length, has three tetratricopeptide repeat motifs. pIFIT2 gene was unevenly distributed in all eleven tissues studied with the most abundance in spleen. Poly(I:C) treatment notably strongly upregulated the mRNA level and promoter activity of pIFIT2 gene. Upstream sequence of 1759 bp from the start codon which was assigned +1 here has promoter activity, and deltaEF1 acts as transcription repressor through binding to sequences at position - 1774 to - 1764. Minimal promoter region exists within nucleotide position - 162 and - 126. Two adjacent interferon-stimulated response elements (ISREs) and two nuclear factor (NF)-κB binding sites were identified within position - 310 and - 126. The ISRE elements act alone and in synergy with the one closer to start codon having more strength, so do the NF-κB binding sites. Synergistic effect was also found between the ISRE and NF-κB binding sites. Additionally, a third ISRE element was identified within position - 1661 to - 1579. These findings will contribute to clarifying the antiviral effect and underlying mechanisms of pIFIT2.

Functional analysis of a proline to serine mutation in codon 453 of the thyroid hormone receptor {beta}1 gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ozata, M.; Suzuki, Satoru; Takeda, Teiji

Mutations in the gene encoding human thyroid hormone receptor {beta}(hTR{beta}) have been associated with generalized resistance to thyroid hormone (GRTH). This disorder is associated with significant behavoral abnormalities. We examined the hTR{beta} gene in a family with members who manifest inappropriately normal TSH, elevated free T{sub 4}, and free and total T{sub 3}. Sequence analysis showed a cytosine to thymine transition at nucleotide 1642 in one allele of the index patient`s genomic DNA. This altered proline to serine at codon 453. The resulting mutant receptor when expressed in vitro bound DNA with high affinity, but the T{sub 3} affinity ofmore » the receptor was impaired. The mutant TR demonstrated a dominant negative effect when cotransfected with two isoforms of wild-type receptor and also in the presence of TR variant {alpha}2 in COS-1 cells. Mutations of codon 453 occur more frequently than at other sites, and four different amino acid substitutions have been reported. Significant differences in phenotype occur among affected individuals, varying from normality to moderately severe GRTH. There is no clear correlation between K{sub a} or in vitro function of the mutant receptor, and phenotype. This study extends the association between GRTH and illness, and indicates that early diagnosis and counseling are needed in families with TR{beta}1 abnormalities. 34 refs., 5 figs., 2 tabs.« less

Purification and characterization of an endoglucanase from Streptomyces lividans 66 and DNA sequence of the gene.

PubMed Central

Théberge, M; Lacaze, P; Shareck, F; Morosoli, R; Kluepfel, D

1992-01-01

The endoglucanase isolated from culture filtrates of Streptomyces lividans IAF74 was shown to have an Mr of 46,000 and a pI of 3.3. The specific enzyme activity of 539 IU/mg, determined by the reducing assay method on carboxymethyl cellulose, is among the highest reported in the literature. The cellulase showed typical endo-type activity when reacting on oligocellodextrins. Optimal enzyme activity was obtained at 50 degrees C and pH 5.5. The kinetic constants for this endoglucanase, determined with carboxymethyl cellulose as the substrate, were a Vmax of 24.9 IU/mg of enzyme and a Km of 4.2 mg/ml. Activity was found against neither methylumbelliferyl- nor p-nitrophenyl-cellobiopyranoside nor with xylan. The DNA sequence contains one possible reading frame validated by the N terminus of the mature purified protein. However, neither ATG nor GTG starting codons were identified near the ribosome-binding site. A putative TTG codon was found as a good candidate for the start codon. Comparison of the primary amino acid sequence of the endoglucanase of S. lividans revealed that the N terminus contains a bacterial cellulose-binding domain. The catalytic domain at the C terminus showed similarity to endoglucanases from a Bacillus sp. Thus, the endoglucanase CelA belongs to family A of cellulases as described before (N. R. Gilkes, B. Henrissat, D. G. Kilburn, R. C. Miller, Jr., and R. A. J. Warren, Microbiol. Rev. 55:303-315, 1991. Images PMID:1575483

Detecting consistent patterns of directional adaptation using differential selection codon models.

PubMed

Parto, Sahar; Lartillot, Nicolas

2017-06-23

Phylogenetic codon models are often used to characterize the selective regimes acting on protein-coding sequences. Recent methodological developments have led to models explicitly accounting for the interplay between mutation and selection, by modeling the amino acid fitness landscape along the sequence. However, thus far, most of these models have assumed that the fitness landscape is constant over time. Fluctuations of the fitness landscape may often be random or depend on complex and unknown factors. However, some organisms may be subject to systematic changes in selective pressure, resulting in reproducible molecular adaptations across independent lineages subject to similar conditions. Here, we introduce a codon-based differential selection model, which aims to detect and quantify the fine-grained consistent patterns of adaptation at the protein-coding level, as a function of external conditions experienced by the organism under investigation. The model parameterizes the global mutational pressure, as well as the site- and condition-specific amino acid selective preferences. This phylogenetic model is implemented in a Bayesian MCMC framework. After validation with simulations, we applied our method to a dataset of HIV sequences from patients with known HLA genetic background. Our differential selection model detects and characterizes differentially selected coding positions specifically associated with two different HLA alleles. Our differential selection model is able to identify consistent molecular adaptations as a function of repeated changes in the environment of the organism. These models can be applied to many other problems, ranging from viral adaptation to evolution of life-history strategies in plants or animals.

Novel mutation in the human immunodeficiency virus type 1 reverse transcriptase gene that encodes cross-resistance to 2',3'-dideoxyinosine and 2',3'-dideoxycytidine.

PubMed Central

Gu, Z; Gao, Q; Li, X; Parniak, M A; Wainberg, M A

1992-01-01

We have used the technique of in vitro selection to generate variants of human immunodeficiency virus type 1 (HIV-1) that are resistant to 2',3'-dideoxyinosine (ddI) and cross-resistant to 2',3'-dideoxycytidine (ddC). The complete reverse transcriptase (RT)-coding regions, plus portions of flanking sequences, of viruses possessing a ddI-resistant phenotype were cloned and sequenced by polymerase chain reaction (PCR)-based methods. We observed that several of these viruses possessed mutations at amino acid sites 184 (Met-->Val; ATG-->GTG) and 294 (Pro-->Ser; CCA-->TCA). These mutations were introduced in the pol gene of infectious, cloned HXB2-D DNA by site-directed mutagenesis. Viral replication assays confirmed the importance of site 184 with regard to resistance to ddI. The recombinant viruses thus generated displayed more than fivefold-greater resistance to ddI than parental HXB2-D did. Moreover, more than fivefold-greater resistance to ddC was also documented; however, the recombinant viruses continued to be inhibited by zidovudine (AZT). No resistance to ddI, ddC, or AZT was introduced by inclusion of mutation site 294 in the pol gene of HXB2-D. PCR analysis performed on viral samples obtained from patients receiving long-term ddI therapy confirmed the presence of mutation site 184 in five of seven cases tested. In three of these five positive cases, the wild-type codon was also detected, indicating that mixtures of viral quasispecies were apparently present. Viruses possessing a ddI resistance phenotype were isolated from both subjects whose viruses contained only the mutated rather than wild-type codon at position 184 as well as from a third individual, whose viruses appeared to be mostly of the mutated variety. Images PMID:1279198

Characterization of the human peroxisome proliferator activated receptor delta gene and its expression.

PubMed

Skogsberg, J; Kannisto, K; Roshani, L; Gagne, E; Hamsten, A; Larsson, C; Ehrenborg, E

2000-07-01

Peroxisome proliferator activated receptors (PPARs) are nuclear receptors regulating the expression of genes involved in lipid and glucose metabolism. Three different PPARs; alpha (PPARA), gamma (PPARG) and delta (PPARD) have been characterized and they are distinguished from each other by tissue distribution and cell activation. In this study, the structure and detailed chromosomal localization of the human PPARD gene was determined. Three genomic clones containing the PPARD gene was isolated from a human P1 library. The gene spans approximately 85 kb of DNA and consists of 9 exons and 8 introns with exons ranging in size from 84 bp to 2.3 kb and introns ranging from 180 bp to 50 kb. All splice acceptor and donor sites conform to the consensus sequences including the AG-GT motif. Although PPARD lacks a TATA box, the gene is transcribed from a unique start site located 380 bp upstream of the ATG initiation codon. The 5' and 3' ends were mapped by rapid amplification of cDNA ends and the mRNA size of PPARD based upon the structure of the gene is 3803 bp. In addition, the chromosomal sublocalization of PPARD was determined by radiation hybrid mapping. The PPARD gene is located at 14 cR from the colipase gene and 15 cR from the serine kinase gene at chromosomal region 6p21.2.

Genome-wide analysis reveals class and gene specific codon usage adaptation in avian paramyxoviruses 1

USDA-ARS?s Scientific Manuscript database

In order to characterize the evolutionary adaptations of avian paramyxovirus 1 (APMV-1) genomes, we have compared codon usage and codon adaptation indexes among groups of Newcastle disease viruses that differ in biological, ecological, and genetic characteristics. We have used available GenBank com...

Enhanced expression of codon optimized Mycobacterium avium subsp. paratuberculosis antigens in Lactobacillus salivarius

USDA-ARS?s Scientific Manuscript database

We have previously identified the mycobacterial high G+C codon usage bias as a limiting factor in heterologous expression of MAP proteins from Lb.salivarius, and demonstrated that codon optimisation of a synthetic coding gene greatly enhances MAP protein production. Here, we effectively demonstrate ...

Complete mitochondrial genome of Palawan peacock-pheasant Polyplectron napoleonis (Galliformes, Phasianidae).

PubMed

Quach, Tommy; Brooks, Daniel M; Miranda, Hector C

2016-01-01

The complete mitochondrial genome of the Palawan peacock-pheasant Polyplectron napoleonis is 16,710 bp and contains 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a control-region. All protein-coding genes use the standard ATG start codon, except for cox1 which has GTG start codon. Seven out of 13 PCGs have TAA stop codons, two have AGG (cox1 and nd6), and three PCGs (nd2, cox2 and nd4) have incomplete stop codon of just T- - nucleotide.

Codon Usage Bias and Determining Forces in Taenia solium Genome.

PubMed

Yang, Xing; Ma, Xusheng; Luo, Xuenong; Ling, Houjun; Zhang, Xichen; Cai, Xuepeng

2015-12-01

The tapeworm Taenia solium is an important human zoonotic parasite that causes great economic loss and also endangers public health. At present, an effective vaccine that will prevent infection and chemotherapy without any side effect remains to be developed. In this study, codon usage patterns in the T. solium genome were examined through 8,484 protein-coding genes. Neutrality analysis showed that T. solium had a narrow GC distribution, and a significant correlation was observed between GC12 and GC3. Examination of an NC (ENC vs GC3s)-plot showed a few genes on or close to the expected curve, but the majority of points with low-ENC (the effective number of codons) values were detected below the expected curve, suggesting that mutational bias plays a major role in shaping codon usage. The Parity Rule 2 plot (PR2) analysis showed that GC and AT were not used proportionally. We also identified 26 optimal codons in the T. solium genome, all of which ended with either a G or C residue. These optimal codons in the T. solium genome are likely consistent with tRNAs that are highly expressed in the cell, suggesting that mutational and translational selection forces are probably driving factors of codon usage bias in the T. solium genome.

Analyzing gene expression from relative codon usage bias in Yeast genome: a statistical significance and biological relevance.

PubMed

Das, Shibsankar; Roymondal, Uttam; Sahoo, Satyabrata

2009-08-15

Based on the hypothesis that highly expressed genes are often characterized by strong compositional bias in terms of codon usage, there are a number of measures currently in use that quantify codon usage bias in genes, and hence provide numerical indices to predict the expression levels of genes. With the recent advent of expression measure from the score of the relative codon usage bias (RCBS), we have explicitly tested the performance of this numerical measure to predict the gene expression level and illustrate this with an analysis of Yeast genomes. In contradiction with previous other studies, we observe a weak correlations between GC content and RCBS, but a selective pressure on the codon preferences in highly expressed genes. The assertion that the expression of a given gene depends on the score of relative codon usage bias (RCBS) is supported by the data. We further observe a strong correlation between RCBS and protein length indicating natural selection in favour of shorter genes to be expressed at higher level. We also attempt a statistical analysis to assess the strength of relative codon bias in genes as a guide to their likely expression level, suggesting a decrease of the informational entropy in the highly expressed genes.

Lost in Translation: Bioinformatic Analysis of Variations Affecting the Translation Initiation Codon in the Human Genome.

PubMed

Abad, Francisco; de la Morena-Barrio, María Eugenia; Fernández-Breis, Jesualdo Tomás; Corral, Javier

2018-06-01

Translation is a key biological process controlled in eukaryotes by the initiation AUG codon. Variations affecting this codon may have pathological consequences by disturbing the correct initiation of translation. Unfortunately, there is no systematic study describing these variations in the human genome. Moreover, we aimed to develop new tools for in silico prediction of the pathogenicity of gene variations affecting AUG codons, because to date, these gene defects have been wrongly classified as missense. Whole-exome analysis revealed the mean of 12 gene variations per person affecting initiation codons, mostly with high (> 0:01) minor allele frequency (MAF). Moreover, analysis of Ensembl data (December 2017) revealed 11,261 genetic variations affecting the initiation AUG codon of 7,205 genes. Most of these variations (99.5%) have low or unknown MAF, probably reflecting deleterious consequences. Only 62 variations had high MAF. Genetic variations with high MAF had closer alternative AUG downstream codons than did those with low MAF. Besides, the high-MAF group better maintained both the signal peptide and reading frame. These differentiating elements could help to determine the pathogenicity of this kind of variation. Data and scripts in Perl and R are freely available at https://github.com/fanavarro/hemodonacion. jfernand@um.es. Supplementary data are available at Bioinformatics online.

Comparative genome analysis of Alkhumra hemorrhagic fever virus with Kyasanur forest disease and tick-borne encephalitis viruses by the in silico approach.

PubMed

Palanisamy, Navaneethan; Akaberi, Dario; Lennerstrand, Johan; Lundkvist, Åke

2018-05-10

Alkhumra hemorrhagic fever virus (AHFV), a relatively new member of the Flaviviruses, was discovered in Saudi Arabia 23 years ago. AHFV is classified in the tick-borne encephalitis virus serocomplex, along with the Kyasanur forest disease virus (KFDV) and tick-borne encephalitis virus (TBEV). Currently, very little is known about the pathologies of AHFV. In this study, using the available genome information of AHFV, KFDV and TBEV, we have predicted and compared the following aspects of these viruses: evolution, nucleotide and protein compositions, recombination, codon frequency, substitution rate, N- and O-glycosylation sites, signal peptide and cleavage site, transmembrane region, secondary structure of 5' and 3' UTRs and RNA-RNA interactions. Additionally, we have modeled the 3D protease and RNA-dependent RNA polymerase structures for AHFV, KFDV and TBEV. Recombination analysis showed no evidence of recombination in the AHFV genome with that of either KFDV or TBEV, although single break point analysis showed that nucleotide position 7399 (in the NS4B) is a breakpoint location. AHFV, KFDV and TBEV are very similar in terms of codon frequency, the number of transmembrane regions, properties of the polyprotein, RNA-RNA interaction sequences, NS3 protease and NS5 polymerase structures and 5' UTR structure. Using genome sequences, we showed the similarities between these closely- related viruses on several different areas.

Transcripts of the NADH-dehydrogenase subunit 3 gene are differentially edited in Oenothera mitochondria.

PubMed Central

Schuster, W; Wissinger, B; Unseld, M; Brennicke, A

1990-01-01

A number of cytosines are altered to be recognized as uridines in transcripts of the nad3 locus in mitochondria of the higher plant Oenothera. Such nucleotide modifications can be found at 16 different sites within the nad3 coding region. Most of these alterations in the mRNA sequence change codon identities to specify amino acids better conserved in evolution. Individual cDNA clones differ in their degree of editing at five nucleotide positions, three of which are silent, while two lead to codon alterations specifying different amino acids. None of the cDNA clones analysed is maximally edited at all possible sites, suggesting slow processing or lowered stringency of editing at these nucleotides. Differentially edited transcripts could be editing intermediates or could code for differing polypeptides. Two edited nucleotides in an open reading frame located upstream of nad3 change two amino acids in the deduced polypeptide. Part of the well-conserved ribosomal protein gene rps12 also encoded downstream of nad3 in other plants, is lost in Oenothera mitochondria by recombination events. The functional rps12 protein must be imported from the cytoplasm since the deleted sequences of this gene are not found in the Oenothera mitochondrial genome. The pseudogene sequence is not edited at any nucleotide position. Images Fig. 3. Fig. 4. Fig. 7. PMID:1688531

Successful COG8 and PDF overlap is mediated by alterations in splicing and polyadenylation signals.

PubMed

Pereira-Castro, Isabel; Quental, Rita; da Costa, Luís T; Amorim, António; Azevedo, Luisa

2012-02-01

Although gene-free areas compose the great majority of eukaryotic genomes, a significant fraction of genes overlaps, i.e., unique nucleotide sequences are part of more than one transcription unit. In this work, the evolutionary history and origin of a same-strand gene overlap is dissected through the analysis of COG8 (component of oligomeric Golgi complex 8) and PDF (peptide deformylase). Comparative genomic surveys reveal that the relative locations of these two genes have been changing over the last 445 million years from distinct chromosomal locations in fish to overlapping in rodents and primates, indicating that the overlap between these genes precedes their divergence. The overlap between the two genes was initiated by the gain of a novel splice donor site between the COG8 stop codon and PDF initiation codon. Splicing is accomplished by the use of the PDF acceptor, leading COG8 to share the 3'end with PDF. In primates, loss of the ancestral polyadenylation signal for COG8 makes the overlap between COG8 and PDF mandatory, while in mouse and rat concurrent overlapping and non-overlapping Cog8 transcripts exist. Altogether, we demonstrate that the origin, evolution and preservation of the COG8/PDF same-strand overlap follow similar mechanistic steps as those documented for antisense overlaps where gain and/or loss of splice sites and polyadenylation signals seems to drive the process.

Pyviko: an automated Python tool to design gene knockouts in complex viruses with overlapping genes.

PubMed

Taylor, Louis J; Strebel, Klaus

2017-01-07

Gene knockouts are a common tool used to study gene function in various organisms. However, designing gene knockouts is complicated in viruses, which frequently contain sequences that code for multiple overlapping genes. Designing mutants that can be traced by the creation of new or elimination of existing restriction sites further compounds the difficulty in experimental design of knockouts of overlapping genes. While software is available to rapidly identify restriction sites in a given nucleotide sequence, no existing software addresses experimental design of mutations involving multiple overlapping amino acid sequences in generating gene knockouts. Pyviko performed well on a test set of over 240,000 gene pairs collected from viral genomes deposited in the National Center for Biotechnology Information Nucleotide database, identifying a point mutation which added a premature stop codon within the first 20 codons of the target gene in 93.2% of all tested gene-overprinted gene pairs. This shows that Pyviko can be used successfully in a wide variety of contexts to facilitate the molecular cloning and study of viral overprinted genes. Pyviko is an extensible and intuitive Python tool for designing knockouts of overlapping genes. Freely available as both a Python package and a web-based interface ( http://louiejtaylor.github.io/pyViKO/ ), Pyviko simplifies the experimental design of gene knockouts in complex viruses with overlapping genes.

Determinants of translation speed are randomly distributed across transcripts resulting in a universal scaling of protein synthesis times

NASA Astrophysics Data System (ADS)

Sharma, Ajeet K.; Ahmed, Nabeel; O'Brien, Edward P.

2018-02-01

Ribosome profiling experiments have found greater than 100-fold variation in ribosome density along mRNA transcripts, indicating that individual codon elongation rates can vary to a similar degree. This wide range of elongation times, coupled with differences in codon usage between transcripts, suggests that the average codon translation-rate per gene can vary widely. Yet, ribosome run-off experiments have found that the average codon translation rate for different groups of transcripts in mouse stem cells is constant at 5.6 AA/s. How these seemingly contradictory results can be reconciled is the focus of this study. Here, we combine knowledge of the molecular factors shown to influence translation speed with genomic information from Escherichia coli, Saccharomyces cerevisiae and Homo sapiens to simulate the synthesis of cytosolic proteins in these organisms. The model recapitulates a near constant average translation rate, which we demonstrate arises because the molecular determinants of translation speed are distributed nearly randomly amongst most of the transcripts. Consequently, codon translation rates are also randomly distributed and fast-translating segments of a transcript are likely to be offset by equally probable slow-translating segments, resulting in similar average elongation rates for most transcripts. We also show that the codon usage bias does not significantly affect the near random distribution of codon translation rates because only about 10 % of the total transcripts in an organism have high codon usage bias while the rest have little to no bias. Analysis of Ribo-Seq data and an in vivo fluorescent assay supports these conclusions.

Insight into pattern of codon biasness and nucleotide base usage in serotonin receptor gene family from different mammalian species.

PubMed

Dass, J Febin Prabhu; Sudandiradoss, C

2012-07-15

5-HT (5-Hydroxy-tryptamine) or serotonin receptors are found both in central and peripheral nervous system as well as in non-neuronal tissues. In the animal and human nervous system, serotonin produces various functional effects through a variety of membrane bound receptors. In this study, we focus on 5-HT receptor family from different mammals and examined the factors that account for codon and nucleotide usage variation. A total of 110 homologous coding sequences from 11 different mammalian species were analyzed using relative synonymous codon usage (RSCU), correspondence analysis (COA) and hierarchical cluster analysis together with nucleotide base usage frequency of chemically similar amino acid codons. The mean effective number of codon (ENc) value of 37.06 for 5-HT(6) shows very high codon bias within the family and may be due to high selective translational efficiency. The COA and Spearman's rank correlation reveals that the nucleotide compositional mutation bias as the major factors influencing the codon usage in serotonin receptor genes. The hierarchical cluster analysis suggests that gene function is another dominant factor that affects the codon usage bias, while species is a minor factor. Nucleotide base usage was reported using Goldman, Engelman, Stietz (GES) scale reveals the presence of high uracil (>45%) content at functionally important hydrophobic regions. Our in silico approach will certainly help for further investigations on critical inference on evolution, structure, function and gene expression aspects of 5-HT receptors family which are potential antipsychotic drug targets. Copyright © 2012 Elsevier B.V. All rights reserved.

Recent evidence for evolution of the genetic code

NASA Technical Reports Server (NTRS)

Osawa, S.; Jukes, T. H.; Watanabe, K.; Muto, A.

1992-01-01

The genetic code, formerly thought to be frozen, is now known to be in a state of evolution. This was first shown in 1979 by Barrell et al. (G. Barrell, A. T. Bankier, and J. Drouin, Nature [London] 282:189-194, 1979), who found that the universal codons AUA (isoleucine) and UGA (stop) coded for methionine and tryptophan, respectively, in human mitochondria. Subsequent studies have shown that UGA codes for tryptophan in Mycoplasma spp. and in all nonplant mitochondria that have been examined. Universal stop codons UAA and UAG code for glutamine in ciliated protozoa (except Euplotes octacarinatus) and in a green alga, Acetabularia. E. octacarinatus uses UAA for stop and UGA for cysteine. Candida species, which are yeasts, use CUG (leucine) for serine. Other departures from the universal code, all in nonplant mitochondria, are CUN (leucine) for threonine (in yeasts), AAA (lysine) for asparagine (in platyhelminths and echinoderms), UAA (stop) for tyrosine (in planaria), and AGR (arginine) for serine (in several animal orders) and for stop (in vertebrates). We propose that the changes are typically preceded by loss of a codon from all coding sequences in an organism or organelle, often as a result of directional mutation pressure, accompanied by loss of the tRNA that translates the codon. The codon reappears later by conversion of another codon and emergence of a tRNA that translates the reappeared codon with a different assignment. Changes in release factors also contribute to these revised assignments. We also discuss the use of UGA (stop) as a selenocysteine codon and the early history of the code.

A condition-specific codon optimization approach for improved heterologous gene expression in Saccharomyces cerevisiae

PubMed Central

2014-01-01

Background Heterologous gene expression is an important tool for synthetic biology that enables metabolic engineering and the production of non-natural biologics in a variety of host organisms. The translational efficiency of heterologous genes can often be improved by optimizing synonymous codon usage to better match the host organism. However, traditional approaches for optimization neglect to take into account many factors known to influence synonymous codon distributions. Results Here we define an alternative approach for codon optimization that utilizes systems level information and codon context for the condition under which heterologous genes are being expressed. Furthermore, we utilize a probabilistic algorithm to generate multiple variants of a given gene. We demonstrate improved translational efficiency using this condition-specific codon optimization approach with two heterologous genes, the fluorescent protein-encoding eGFP and the catechol 1,2-dioxygenase gene CatA, expressed in S. cerevisiae. For the latter case, optimization for stationary phase production resulted in nearly 2.9-fold improvements over commercial gene optimization algorithms. Conclusions Codon optimization is now often a standard tool for protein expression, and while a variety of tools and approaches have been developed, they do not guarantee improved performance for all hosts of applications. Here, we suggest an alternative method for condition-specific codon optimization and demonstrate its utility in Saccharomyces cerevisiae as a proof of concept. However, this technique should be applicable to any organism for which gene expression data can be generated and is thus of potential interest for a variety of applications in metabolic and cellular engineering. PMID:24636000

Codon usage bias reveals genomic adaptations to environmental conditions in an acidophilic consortium.

PubMed

Hart, Andrew; Cortés, María Paz; Latorre, Mauricio; Martinez, Servet

2018-01-01

The analysis of codon usage bias has been widely used to characterize different communities of microorganisms. In this context, the aim of this work was to study the codon usage bias in a natural consortium of five acidophilic bacteria used for biomining. The codon usage bias of the consortium was contrasted with genes from an alternative collection of acidophilic reference strains and metagenome samples. Results indicate that acidophilic bacteria preferentially have low codon usage bias, consistent with both their capacity to live in a wide range of habitats and their slow growth rate, a characteristic probably acquired independently from their phylogenetic relationships. In addition, the analysis showed significant differences in the unique sets of genes from the autotrophic species of the consortium in relation to other acidophilic organisms, principally in genes which code for proteins involved in metal and oxidative stress resistance. The lower values of codon usage bias obtained in this unique set of genes suggest higher transcriptional adaptation to living in extreme conditions, which was probably acquired as a measure for resisting the elevated metal conditions present in the mine.

Identification of four novel HLA-B alleles, B*1590, B*1591, B*2726, and B*4705, from an East African population by high-resolution sequence-based typing.

PubMed

Luo, M; Mao, X; Plummer, F A

2005-02-01

We report here four novel HLA-B alleles, B*1590, B*1591, B*2726, and B*4705, identified from an East African population during sequence-based HLA-B typing. The novel alleles were confirmed by sequencing two separate polymerase chain reaction products, and by molecular cloning and sequencing multiple clones. B*1590 is identical to B*1510 at exon 2 and exon 3, except for a difference (GCCGTC) at codon 158. Sequence differences at codon 152 (GAGGTG) and codon 167 (TGGTCG) differentiate B*1591 from B*1503 at exon 3. B*2726 is identical to B*2708 at exon 2 and exon 3, except for a difference (AAGCAG) at codon 70. B*4705 was identified in three Kenyan women. The allele is identical to B*47010101/02 at exon 2 and exon 3, except for differences at codon 97 (AGGAAT) and codon 99 (TTTTAT). These new alleles have been named by the WHO Nomenclature Committee. Identification of these novel HLA-B alleles reflects the genetic diversity of this East African population.

Energetics of codon-anticodon recognition on the small ribosomal subunit.

PubMed

Almlöf, Martin; Andér, Martin; Aqvist, Johan

2007-01-09

Recent crystal structures of the small ribosomal subunit have made it possible to examine the detailed energetics of codon recognition on the ribosome by computational methods. The binding of cognate and near-cognate anticodon stem loops to the ribosome decoding center, with mRNA containing the Phe UUU and UUC codons, are analyzed here using explicit solvent molecular dynamics simulations together with the linear interaction energy (LIE) method. The calculated binding free energies are in excellent agreement with experimental binding constants and reproduce the relative effects of mismatches in the first and second codon position versus a mismatch at the wobble position. The simulations further predict that the Leu2 anticodon stem loop is about 10 times more stable than the Ser stem loop in complex with the Phe UUU codon. It is also found that the ribosome significantly enhances the intrinsic stability differences of codon-anticodon complexes in aqueous solution. Structural analysis of the simulations confirms the previously suggested importance of the universally conserved nucleotides A1492, A1493, and G530 in the decoding process.

Lack of correlation between p53 codon 72 polymorphism and anal cancer risk

PubMed Central

Contu, Simone S; Agnes, Grasiela; Damin, Andrea P; Contu, Paulo C; Rosito, Mário A; Alexandre, Claudio O; Damin, Daniel C

2009-01-01

AIM: To investigate the potential role of p53 codon 72 polymorphism as a risk factor for development of anal cancer. METHODS: Thirty-two patients with invasive anal carcinoma and 103 healthy blood donors were included in the study. p53 codon 72 polymorphism was analyzed in blood samples through polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing. RESULTS: The relative frequency of each allele was 0.60 for Arg and 0.40 for Pro in patients with anal cancer, and 0.61 for Arg and 0.39 for Pro in normal controls. No significant differences in distribution of the codon 72 genotypes between patients and controls were found. CONCLUSION: These results do not support a role for the p53 codon 72 polymorphism in anal carcinogenesis. PMID:19777616

Random codon re-encoding induces stable reduction of replicative fitness of Chikungunya virus in primate and mosquito cells.

PubMed

Nougairede, Antoine; De Fabritus, Lauriane; Aubry, Fabien; Gould, Ernest A; Holmes, Edward C; de Lamballerie, Xavier

2013-02-01

Large-scale codon re-encoding represents a powerful method of attenuating viruses to generate safe and cost-effective vaccines. In contrast to specific approaches of codon re-encoding which modify genome-scale properties, we evaluated the effects of random codon re-encoding on the re-emerging human pathogen Chikungunya virus (CHIKV), and assessed the stability of the resultant viruses during serial in cellulo passage. Using different combinations of three 1.4 kb randomly re-encoded regions located throughout the CHIKV genome six codon re-encoded viruses were obtained. Introducing a large number of slightly deleterious synonymous mutations reduced the replicative fitness of CHIKV in both primate and arthropod cells, demonstrating the impact of synonymous mutations on fitness. Decrease of replicative fitness correlated with the extent of re-encoding, an observation that may assist in the modulation of viral attenuation. The wild-type and two re-encoded viruses were passaged 50 times either in primate or insect cells, or in each cell line alternately. These viruses were analyzed using detailed fitness assays, complete genome sequences and the analysis of intra-population genetic diversity. The response to codon re-encoding and adaptation to culture conditions occurred simultaneously, resulting in significant replicative fitness increases for both re-encoded and wild type viruses. Importantly, however, the most re-encoded virus failed to recover its replicative fitness. Evolution of these viruses in response to codon re-encoding was largely characterized by the emergence of both synonymous and non-synonymous mutations, sometimes located in genomic regions other than those involving re-encoding, and multiple convergent and compensatory mutations. However, there was a striking absence of codon reversion (<0.4%). Finally, multiple mutations were rapidly fixed in primate cells, whereas mosquito cells acted as a brake on evolution. In conclusion, random codon re-encoding provides important information on the evolution and genetic stability of CHIKV viruses and could be exploited to develop a safe, live attenuated CHIKV vaccine.

A novel start codon mutation of the MERTK gene in a patient with retinitis pigmentosa

PubMed Central

Jinda, Worapoj; Poungvarin, Naravat; Taylor, Todd D.; Suzuki, Yutaka; Thongnoppakhun, Wanna; Limwongse, Chanin; Lertrit, Patcharee; Suriyaphol, Prapat

2016-01-01

Purpose Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous group of inherited retinal degenerations characterized by progressive loss of photoreceptor cells and RPE functions. More than 70 causative genes are known to be responsible for RP. This study aimed to identify the causative gene in a patient from a consanguineous family with childhood-onset severe retinal dystrophy. Methods To identify the defective gene, whole exome sequencing was performed. Candidate causative variants were selected and validated using Sanger sequencing. Segregation analysis of the causative gene was performed in additional family members. To verify that the mutation has an effect on protein synthesis, an expression vector containing the first ten amino acids of the mutant protein fused with the DsRed2 fluorescent protein was constructed and transfected into HEK293T cells. Expression of the fusion protein in the transfected cells was measured using fluorescence microscopy. Results By filtering against public variant databases, a novel homozygous missense mutation (c.3G>A) localized in the start codon of the MERTK gene was detected as a potentially pathogenic mutation for autosomal recessive RP. The c.3G>A mutation cosegregated with the disease phenotype in the family. No expression of the first ten amino acids of the MerTK mutant fused with the DsRed2 fluorescent protein was detected in HEK293T cells, indicating that the mutation affects the translation initiation site of the gene that may lead to loss of function of the MerTK signaling pathway. Conclusions We report a novel missense mutation (c.3G>A, p.0?) in the MERTK gene that causes severe vision impairment in a patient. Taken together with previous reports, our results expand the spectrum of MERTK mutations and extend our understanding of the role of the MerTK protein in the pathogenesis of retinitis pigmentosa. PMID:27122965

Canine parvovirus type 2 (CPV-2) and Feline panleukopenia virus (FPV) codon bias analysis reveals a progressive adaptation to the new niche after the host jump.

PubMed

Franzo, Giovanni; Tucciarone, Claudia Maria; Cecchinato, Mattia; Drigo, Michele

2017-09-01

Based on virus dependence from host cell machinery, their codon usage is expected to show a strong relation with the host one. Even if this association has been stated, especially for bacteria viruses, the linkage is considered to be less consistent for more complex organisms and a codon bias adaptation after host jump has never been proven. Canine parvovirus type 2 (CPV-2) was selected as a model because it represents a well characterized case of host jump, originating from Feline panleukopenia virus (FPV). The current study demonstrates that the adaptation to specific tissue and host codon bias affected CPV-2 evolution. Remarkably, FPV and CPV-2 showed a higher closeness toward the codon bias of the tissues they display the higher tropism for. Moreover, after the host jump, a clear and significant trend was evidenced toward a reduction in the distance between CPV-2 and the dog codon bias over time. This evidence was not confirmed for FPV, suggesting that an equilibrium has been reached during the prolonged virus-host co-evolution. Additionally, the presence of an intermediate pattern displayed by some strains infecting wild species suggests that these could have facilitated the host switch also by acting on codon bias. Copyright © 2017 Elsevier Inc. All rights reserved.

Properties and determinants of codon decoding time distributions

PubMed Central

2014-01-01

Background Codon decoding time is a fundamental property of mRNA translation believed to affect the abundance, function, and properties of proteins. Recently, a novel experimental technology--ribosome profiling--was developed to measure the density, and thus the speed, of ribosomes at codon resolution. Specifically, this method is based on next-generation sequencing, which theoretically can provide footprint counts that correspond to the probability of observing a ribosome in this position for each nucleotide in each transcript. Results In this study, we report for the first time various novel properties of the distribution of codon footprint counts in five organisms, based on large-scale analysis of ribosomal profiling data. We show that codons have distinctive footprint count distributions. These tend to be preserved along the inner part of the ORF, but differ at the 5' and 3' ends of the ORF, suggesting that the translation-elongation stage actually includes three biophysical sub-steps. In addition, we study various basic properties of the codon footprint count distributions and show that some of them correlate with the abundance of the tRNA molecule types recognizing them. Conclusions Our approach emphasizes the advantages of analyzing ribosome profiling and similar types of data via a comparative genomic codon-distribution-centric view. Thus, our methods can be used in future studies related to translation and even transcription elongation. PMID:25572668

Analysis of base and codon usage by rubella virus.

PubMed

Zhou, Yumei; Chen, Xianfeng; Ushijima, Hiroshi; Frey, Teryl K

2012-05-01

Rubella virus (RUBV), a small, plus-strand RNA virus that is an important human pathogen, has the unique feature that the GC content of its genome (70%) is the highest (by 20%) among RNA viruses. To determine the effect of this GC content on genomic evolution, base and codon usage were analyzed across viruses from eight diverse genotypes of RUBV. Despite differences in frequency of codon use, the favored codons in the RUBV genome matched those in the human genome for 18 of the 20 amino acids, indicating adaptation to the host. Although usage patterns were conserved in corresponding genes in the diverse genotypes, within-genome comparison revealed that both base and codon usages varied regionally, particularly in the hypervariable region (HVR) of the P150 replicase gene. While directional mutation pressure was predominant in determining base and codon usage within most of the genome (with the strongest tendency being towards C's at third codon positions), natural selection was predominant in the HVR region. The GC content of this region was the highest in the genome (>80%), and it was not clear if selection at the nucleotide level accompanied selection at the amino acid level. Dinucleotide frequency analysis of the RUBV genome revealed that TpA usage was lower than expected, similar to mammalian genes; however, CpG usage was not suppressed, and TpG usage was not enhanced, as is the case in mammalian genes.

Ribosome rearrangements at the onset of translational bypassing

PubMed Central

Agirrezabala, Xabier; Samatova, Ekaterina; Klimova, Mariia; Zamora, Miguel; Gil-Carton, David; Rodnina, Marina V.; Valle, Mikel

2017-01-01

Bypassing is a recoding event that leads to the translation of two distal open reading frames into a single polypeptide chain. We present the structure of a translating ribosome stalled at the bypassing take-off site of gene 60 of bacteriophage T4. The nascent peptide in the exit tunnel anchors the P-site peptidyl-tRNAGly to the ribosome and locks an inactive conformation of the peptidyl transferase center (PTC). The mRNA forms a short dynamic hairpin in the decoding site. The ribosomal subunits adopt a rolling conformation in which the rotation of the small subunit around its long axis causes the opening of the A-site region. Together, PTC conformation and mRNA structure safeguard against premature termination and read-through of the stop codon and reconfigure the ribosome to a state poised for take-off and sliding along the noncoding mRNA gap. PMID:28630923

Overcoming codon-usage bias in heterologous protein expression in Streptococcus gordonii.

PubMed

Lee, Song F; Li, Yi-Jing; Halperin, Scott A

2009-11-01

One of the limitations facing the development of Streptococcus gordonii into a successful vaccine vector is the inability of this bacterium to express high levels of heterologous proteins. In the present study, we have identified 12 codons deemed as rare codons in S. gordonii and seven other streptococcal species. tRNA genes encoding 10 of the 12 rare codons were cloned into a plasmid. The plasmid was transformed into strains of S. gordonii expressing the fusion protein SpaP/S1, the anti-complement receptor 1 (CR1) single-chain variable fragment (scFv) antibody, or the Toxoplasma gondii cyclophilin C18 protein. These three heterologous proteins contained high percentages of amino acids encoded by rare codons. The results showed that the production of SpaP/S1, anti-CR1 scFv and C18 increased by 2.7-, 120- and 10-fold, respectively, over the control strains. In contrast, the production of the streptococcal SpaP protein without the pertussis toxin S1 fragment was not affected by tRNA gene supplementation, indicating that the increased production of SpaP/S1 protein was due to the ability to overcome the limitation caused by rare codons required for the S1 fragment. The increase in anti-CR1 scFv production was also observed in Streptococcus mutans following tRNA gene supplementation. Collectively, the findings in the present study demonstrate for the first time, to the best of our knowledge, that codon-usage bias exists in Streptococcus spp. and the limitation of heterologous protein expression caused by codon-usage bias can be overcome by tRNA supplementation.

tRNAomics: tRNA gene copy number variation and codon use provide bioinformatic evidence of a new anticodon:codon wobble pair in a eukaryote

PubMed Central

Iben, James R.; Maraia, Richard J.

2012-01-01

tRNA genes are interspersed throughout eukaryotic DNA, contributing to genome architecture and evolution in addition to translation of the transcriptome. Codon use correlates with tRNA gene copy number in noncomplex organisms including yeasts. Synonymous codons impact translation with various outcomes, dependent on relative tRNA abundances. Availability of whole-genome sequences allowed us to examine tRNA gene copy number variation (tgCNV) and codon use in four Schizosaccharomyces species and Saccharomyces cerevisiae. tRNA gene numbers vary from 171 to 322 in the four Schizosaccharomyces despite very high similarity in other features of their genomes. In addition, we performed whole-genome sequencing of several related laboratory strains of Schizosaccharomyces pombe and found tgCNV at a cluster of tRNA genes. We examined for the first time effects of wobble rules on correlation of tRNA gene number and codon use and showed improvement for S. cerevisiae and three of the Schizosaccharomyces species. In contrast, correlation in Schizosaccharomyces japonicus is poor due to markedly divergent tRNA gene content, and much worsened by the wobble rules. In japonicus, some tRNA iso-acceptor genes are absent and others are greatly reduced relative to the other yeasts, while genes for synonymous wobble iso-acceptors are amplified, indicating wobble use not apparent in any other eukaryote. We identified a subset of japonicus-specific wobbles that improves correlation of codon use and tRNA gene content in japonicus. We conclude that tgCNV is high among Schizo species and occurs in related laboratory strains of S. pombe (and expectedly other species), and tRNAome-codon analyses can provide insight into species-specific wobble decoding. PMID:22586155

Physical Model for the Evolution of the Genetic Code

NASA Astrophysics Data System (ADS)

Yamashita, Tatsuro; Narikiyo, Osamu

2011-12-01

Using the shape space of codons and tRNAs we give a physical description of the genetic code evolution on the basis of the codon capture and ambiguous intermediate scenarios in a consistent manner. In the lowest dimensional version of our description, a physical quantity, codon level is introduced. In terms of the codon levels two scenarios are typically classified into two different routes of the evolutional process. In the case of the ambiguous intermediate scenario we perform an evolutional simulation implemented cost selection of amino acids and confirm a rapid transition of the code change. Such rapidness reduces uncomfortableness of the non-unique translation of the code at intermediate state that is the weakness of the scenario. In the case of the codon capture scenario the survival against mutations under the mutational pressure minimizing GC content in genomes is simulated and it is demonstrated that cells which experience only neutral mutations survive.

Reassigning stop codons via translation termination: How a few eukaryotes broke the dogma.

PubMed

Alkalaeva, Elena; Mikhailova, Tatiana

2017-03-01

The genetic code determines how amino acids are encoded within mRNA. It is universal among the vast majority of organisms, although several exceptions are known. Variant genetic codes are found in ciliates, mitochondria, and numerous other organisms. All revealed genetic codes (standard and variant) have at least one codon encoding a translation stop signal. However, recently two new genetic codes with a reassignment of all three stop codons were revealed in studies examining the protozoa transcriptomes. Here, we discuss this finding and the recent studies of variant genetic codes in eukaryotes. We consider the possible molecular mechanisms allowing the use of certain codons as sense and stop signals simultaneously. The results obtained by studying these amazing organisms represent a new and exciting insight into the mechanism of stop codon decoding in eukaryotes. Also see the video abstract here. © 2017 WILEY Periodicals, Inc.

Role of the Integrin-Linked Kinase, ILK, in Mammary Carcinogensis

DTIC Science & Technology

2000-08-01

have been implicated in environmental stress clonei 6-10 responses in yeasts, plants and mammals, as well as regulating abscisic acid signal transduction...phosphatase 2C involved in abscisic acid signal transduction in higher plants. Proc. Natl Acad. Sci. USA, 95, 975-980. Strovel,E.T., Wu,D. and Sussman,D.J...contain a 450bp open reading frame, coding for 149 amino acids and a poly A tail 245bp downstream of the stop codon, although no polyadenylation site

Mitochondrial DNA Mutation Associated with Leber's Hereditary Optic Neuropathy

NASA Astrophysics Data System (ADS)

Wallace, Douglas C.; Singh, Gurparkash; Lott, Marie T.; Hodge, Judy A.; Schurr, Theodore G.; Lezza, Angela M. S.; Elsas, Louis J.; Nikoskelainen, Eeva K.

1988-12-01

Leber's hereditary optic neuropathy is a maternally inherited disease resulting in optic nerve degeneration and cardiac dysrhythmia. A mitochondrial DNA replacement mutation was identified that correlated with this disease in multiple families. This mutation converted a highly conserved arginine to a histidine at codon 340 in the NADH dehydrogenase subunit 4 gene and eliminated an Sfa NI site, thus providing a simple diagnostic test. This finding demonstrated that a nucleotide change in a mitochondrial DNA energy production gene can result in a neurological disease.

Function of the SpoVAEa and SpoVAF Proteins of Bacillus subtilis Spores

DTIC Science & Technology

2014-06-01

outer surface of the spore’s inner membrane, as SpoVAEa was accessible to an external biotinylation agent in spores and SpoVAEa disappeared in parallel...codon was PCR amplified from PS832 chromosomal DNA with primers that inserted BamHI and PstI restriction sites upstream and downstream, respectively... chromosomal structure, and this strain was termed PS4348 (spoVAEa mutant). A B. subtilis strain with a deletion of the spoVF gene was constructed by a two

Burkholderia Mallei tssM Encodes a Secreted Deubiquitinase that is Expressed Inside Infected RAW 264.7 Murine Macrophages

DTIC Science & Technology

2008-10-13

Furthermore, the encoded protein of this gene is only 30 kDa. A potential GTG start codon at position 625 also encodes a protein that is too small...horizontal bar and putative alternate translation initiation sites (ATG, GTG , and TTG) are indicated. The sizes and locations of the proteins encoded... gray line with rounded rectangles showing sequence features and motifs, including the Ala- and Pro-rich N-terminal region and the C-terminal Cys and

PCR-RFLP to Detect Codon 248 Mutation in Exon 7 of "p53" Tumor Suppressor Gene

ERIC Educational Resources Information Center

Ouyang, Liming; Ge, Chongtao; Wu, Haizhen; Li, Suxia; Zhang, Huizhan

2009-01-01

Individual genome DNA was extracted fast from oral swab and followed up with PCR specific for codon 248 of "p53" tumor suppressor gene. "Msp"I restriction mapping showed the G-C mutation in codon 248, which closely relates to cancer susceptibility. Students learn the concepts, detection techniques, and research significance of point mutations or…

Codon influence on protein expression in E. coli correlates with mRNA levels

PubMed Central

Boël, Grégory; Wong, Kam-Ho; Su, Min; Luff, Jon; Valecha, Mayank; Everett, John K.; Acton, Thomas B.; Xiao, Rong; Montelione, Gaetano T.; Aalberts, Daniel P.; Hunt, John F.

2016-01-01

Degeneracy in the genetic code, which enables a single protein to be encoded by a multitude of synonymous gene sequences, has an important role in regulating protein expression, but substantial uncertainty exists concerning the details of this phenomenon. Here we analyze the sequence features influencing protein expression levels in 6,348 experiments using bacteriophage T7 polymerase to synthesize messenger RNA in Escherichia coli. Logistic regression yields a new codon-influence metric that correlates only weakly with genomic codon-usage frequency, but strongly with global physiological protein concentrations and also mRNA concentrations and lifetimes in vivo. Overall, the codon content influences protein expression more strongly than mRNA-folding parameters, although the latter dominate in the initial ~16 codons. Genes redesigned based on our analyses are transcribed with unaltered efficiency but translated with higher efficiency in vitro. The less efficiently translated native sequences show greatly reduced mRNA levels in vivo. Our results suggest that codon content modulates a kinetic competition between protein elongation and mRNA degradation that is a central feature of the physiology and also possibly the regulation of translation in E. coli. PMID:26760206

On the possible origin and evolution of the genetic code

NASA Technical Reports Server (NTRS)

Jukes, T. H.

1974-01-01

The genetic code is examined for indications of possible preceding codes that existed during early evolution. Eight of the 20 amino acids are coded by 'quartets' of codons with fourfold degeneracy, and 16 such quartets can exist, so that an earlier code could have provided for 15 or 16 amino acids, rather than 20. If twofold degeneracy is postulated for the first position of the codon, there could have been ten amino acids in the code. It is speculated that these may have been phenylalanine, valine, proline, alanine, histidine, glutamine, glutanic acid, aspartic acid, cysteine and glycine. There is a notable deficiency of arginine in proteins, despite the fact that it has six codons. Simultaneously, there is more lysine in proteins than would be expected from its two codons, if the four bases in mRNA are equiprobable and are arranged randomly. It is speculated that arginine is an 'intruder' into the genetic code, and that it may have displayed another amino acid such as ornithine, or may even have displayed lysine from some of its previous codon assignments. As a result, natural selection has favored lysine against the fact that it has only two codons.

Demonstration of GTG as an alternative initiation codon for the serpin endopin 2B-2.

PubMed

Hwang, Shin-Rong; Garza, Christina Z; Wegrzyn, Jill L; Hook, Vivian Y H

2005-02-18

This study demonstrates GTG as a novel, alternative initiation codon for translation of bovine endopin 2B-2, a serpin protease inhibitor. Molecular cDNA cloning revealed the endopin 2B-1 and endopin 2B-2 isoforms that are predicted to inhibit papain and elastase. Notably, GTG was demonstrated as the initiation codon for endopin 2B-2, whereas endopin 2B-1 possesses ATG as its initiation codon. GTG mediated in vitro translation of 46kDa endopin 2B-2. GTG also mediated translation of EGFP by in vitro translation and by expression in mammalian cells. Notably, mutagenesis of GTG to GTC resulted in the absence of EGFP expression in cells. GTG produced a lower level of protein expression compared to ATG. The use of GTG as an initiation codon to direct translation of endopin 2B, as well as the heterologous protein EGFP, demonstrates the role of GTG in the regulation of mRNA translation in mammalian cells. Significantly, further analyses of mammalian genomes based on GTG as an alternative initiation codon may predict new candidate gene products expressed by mammalian and human genomes.

Nonneutral GC3 and retroelement codon mimicry in Phytophthora.

PubMed

Jiang, Rays H Y; Govers, Francine

2006-10-01

Phytophthora is a genus entirely comprised of destructive plant pathogens. It belongs to the Stramenopila, a unique branch of eukaryotes, phylogenetically distinct from plants, animals, or fungi. Phytophthora genes show a strong preference for usage of codons ending with G or C (high GC3). The presence of high GC3 in genes can be utilized to differentiate coding regions from noncoding regions in the genome. We found that both selective pressure and mutation bias drive codon bias in Phytophthora. Indicative for selection pressure is the higher GC3 value of highly expressed genes in different Phytophthora species. Lineage specific GC increase of noncoding regions is reminiscent of whole-genome mutation bias, whereas the elevated Phytophthora GC3 is primarily a result of translation efficiency-driven selection. Heterogeneous retrotransposons exist in Phytophthora genomes and many of them vary in their GC content. Interestingly, the most widespread groups of retroelements in Phytophthora show high GC3 and a codon bias that is similar to host genes. Apparently, selection pressure has been exerted on the retroelement's codon usage, and such mimicry of host codon bias might be beneficial for the propagation of retrotransposons.

[Identifying and sequence analysis of HLA-B*2736].

PubMed

Li, Zhen; Zou, Hong-Yan; Shao, Chao-Peng; Tang, Si; Wang, Da-Ming; Cheng, Liang-Hong

2007-11-01

An unknown HLA-B allele which was similar to HLA-B*270401 was detected by FLOW-SSOPCR-SSP and heterozygous sequence-based typing (SBT) in Chinese Han individual. Its anomalous patterns suggested the possible presence of new allele. Amplifying exon 2-5(include intron 2-4) of the HLA-B*27 allele separately by using allele-specific primers and sequencing in both directions. Identifying the difference between the novel B*27 allele and B*270401. The sequence of novel B*27 from exon 2 to partial exon 5 is 1 815 bp. There are 10 nt changes from B*270401 in exon 3-4, at nt634where A-->C(codon130 AGC-->CGC, 130 S-->R); nt670 where A-->T (codon142 ACC-->TCC, 142 T-->S); nt683 where G-->T (codon146 TGG-->TTG, 146 W-->L); nt698 where A-->T (codon151 GAG-->GTG, 151 E-->V); nt774 where G-->C (codon176 GAG-->GAC, 176 E-->D); nt776 where C-->A (codon177 ACG-->AAG, 177 T-->K); nt781 where C-->G (codon179 CAG-->GAG, 179Q-->E); nt789 where G-->T (codon181 GCG-->GCT) resulting no coding change; nt1438 where C-->T (codon206 GGC-->GGT) resulting no coding change; nt1449 where G-->C (codon210 GGG-->GCG, 210G-->A). In IMGT/HLA database, only three alleles (B*270502/2706/2732) have sequences of introns. The same sequence in intron 2 showed homology between the novel HLA-B*27 allele and B*2706, but their homology could not be supported in intron 3-4. Comparing the sequence of the novel B*27 allele in intron 3 and 4 with B*27 group, it showed there are three mutations at nt106 C-->G, nt179 G-->A, nt536 G-->A and one deletion at nt168 in intron 3 and one mutations at nt82 T-->C in intron 4, but the sequence of the novel B*27 allele in intron 3 and 4 was all the same to B*070201. The sequence was submitted to Gen-Bank and the accession number was DQ915176. The allele has been confirmed as an extension of B*2736 by the WHO Nomenclature committee in November 2006.

Zika Virus Attenuation by Codon Pair Deoptimization Induces Sterilizing Immunity in Mouse Models.

PubMed

Li, Penghui; Ke, Xianliang; Wang, Ting; Tan, Zhongyuan; Luo, Dan; Miao, Yuanjiu; Sun, Jianhong; Zhang, Yuan; Liu, Yan; Hu, Qinxue; Xu, Fuqiang; Wang, Hanzhong; Zheng, Zhenhua

2018-06-20

Zika virus (ZIKV) infection during the large epidemics in the Americas is related to congenital abnormities or fetal demise. To date, there is no vaccine, antiviral drug, or other modality available to prevent or treat Zika virus infection. Here we designed novel live attenuated ZIKV vaccine candidates using a codon pair deoptimization strategy. Three codon pair-deoptimized ZIKVs (Min E, Min NS1, and Min E+NS1) were de novo synthesized, and recovered by reverse genetics, containing large amounts of underrepresented codon pairs in E gene and/or NS1 gene. Amino acid sequence was 100% unchanged. The codon pair-deoptimized variants had decreased replication fitness in Vero cells (Min NS1 ≫ Min E > Min E+NS1), replicated more efficiently in insect cells than in mammalian cells, and demonstrated diminished virulence in a mouse model. In particular, Min E+NS1, the most restrictive variant, induced sterilizing immunity with a robust neutralizing antibody titer, and a single immunization achieved complete protection against lethal challenge and vertical ZIKV transmission during pregnancy. More importantly, due to the numerous synonymous substitutions in the codon pair-deoptimized strains, reversion to wild-type virulence through gradual nucleotide sequence mutations is unlikely. Our results collectively demonstrate that ZIKV can be effectively attenuated by codon pair deoptimization, highlighting the potential of Min E+NS1 as a safe vaccine candidate to prevent ZIKV infections. IMPORTANCE Due to unprecedented epidemics of Zika virus (ZIKV) across the Americas and the unexpected clinical symptoms including Guillain-Barré syndrome, microcephaly and other birth defects in human, there is an urgent need for ZIKV vaccine development. Here, we provided the first attenuated versions of ZIKV with two important genes (E and/or NS1) that were subjected to codon pair deoptimization. Compared to parental ZIKV, the codon pair-deoptimized ZIKVs were mammalian-attenuated, and preferred insect to mammalian Cells. Min E+NS1, the most restrictive variant, induced sterilizing immunity with a robust neutralizing antibody titer, and achieved complete protection against lethal challenge and vertical virus transmission during pregnancy. More importantly, the massive synonymous mutational approach made it impossible to revert to wild-type virulence. Our results have proven the feasibility of codon pair deoptimization as a strategy to develop live-attenuated vaccine candidates against flavivirues like ZIKV, Japanese encephalitis virus and West Nile virus. Copyright © 2018 American Society for Microbiology.

Rapid molecular evolution of human bocavirus revealed by Bayesian coalescent inference.

PubMed

Zehender, Gianguglielmo; De Maddalena, Chiara; Canuti, Marta; Zappa, Alessandra; Amendola, Antonella; Lai, Alessia; Galli, Massimo; Tanzi, Elisabetta

2010-03-01

Human bocavirus (HBoV) is a linear single-stranded DNA virus belonging to the Parvoviridae family that has recently been isolated from the upper respiratory tract of children with acute respiratory infection. All of the strains observed so far segregate into two genotypes (1 and 2) with a low level of polymorphism. Given the recent description of the infection and the lack of epidemiological and molecular data, we estimated the virus's rates of molecular evolution and population dynamics. A dataset of forty-nine dated VP2 sequences, including also eight new isolates obtained from pharyngeal swabs of Italian patients with acute respiratory tract infections, was submitted to phylogenetic analysis. The model parameters, evolutionary rates and population dynamics were co-estimated using a Bayesian Markov Chain Monte Carlo approach, and site-specific positive and negative selection was also investigated. Recombination was investigated by seven different methods and one suspected recombinant strain was excluded from further analysis. The estimated mean evolutionary rate of HBoV was 8.6x10(-4)subs/site/year, and that of the 1st+2nd codon positions was more than 15 times less than that of the 3rd codon position. Viral population dynamics analysis revealed that the two known genotypes diverged recently (mean tMRCA: 24 years), and that the epidemic due to HBoV genotype 2 grew exponentially at a rate of 1.01year(-1). Selection analysis of the partial VP2 showed that 8.5% of sites were under significant negative pressure and the absence of positive selection. Our results show that, like other parvoviruses, HBoV is characterised by a rapid evolution. The low level of polymorphism is probably due to a relatively recent divergence between the circulating genotypes and strong purifying selection acting on viral antigens.

Genome-wide A-to-I RNA editing in fungi independent of ADAR enzymes

PubMed Central

Liu, Huiquan; Wang, Qinhu; He, Yi; Chen, Lingfeng; Hao, Chaofeng; Jiang, Cong; Li, Yang; Dai, Yafeng; Kang, Zhensheng; Xu, Jin-Rong

2016-01-01

Yeasts and filamentous fungi do not have adenosine deaminase acting on RNA (ADAR) orthologs and are believed to lack A-to-I RNA editing, which is the most prevalent editing of mRNA in animals. However, during this study with the PUK1 (FGRRES_01058) pseudokinase gene important for sexual reproduction in Fusarium graminearum, we found that two tandem stop codons, UA1831GUA1834G, in its kinase domain were changed to UG1831GUG1834G by RNA editing in perithecia. To confirm A-to-I editing of PUK1 transcripts, strand-specific RNA-seq data were generated with RNA isolated from conidia, hyphae, and perithecia. PUK1 was almost specifically expressed in perithecia, and 90% of transcripts were edited to UG1831GUG1834G. Genome-wide analysis identified 26,056 perithecium-specific A-to-I editing sites. Unlike those in animals, 70.5% of A-to-I editing sites in F. graminearum occur in coding regions, and more than two-thirds of them result in amino acid changes, including editing of 69 PUK1-like pseudogenes with stop codons in ORFs. PUK1 orthologs and other pseudogenes also displayed stage-specific expression and editing in Neurospora crassa and F. verticillioides. Furthermore, F. graminearum differs from animals in the sequence preference and structure selectivity of A-to-I editing sites. Whereas A's embedded in RNA stems are targeted by ADARs, RNA editing in F. graminearum preferentially targets A's in hairpin loops, which is similar to the anticodon loop of tRNA targeted by adenosine deaminases acting on tRNA (ADATs). Overall, our results showed that A-to-I RNA editing occurs specifically during sexual reproduction and mainly in the coding regions in filamentous ascomycetes, involving adenosine deamination mechanisms distinct from metazoan ADARs. PMID:26934920

Evolutionary analysis of Old World arenaviruses reveals a major adaptive contribution of the viral polymerase.

PubMed

Pontremoli, Chiara; Forni, Diego; Cagliani, Rachele; Pozzoli, Uberto; Riva, Stefania; Bravo, Ignacio G; Clerici, Mario; Sironi, Manuela

2017-10-01

The Old World (OW) arenavirus complex includes several species of rodent-borne viruses, some of which (i.e., Lassa virus, LASV and Lymphocytic choriomeningitis virus, LCMV) cause human diseases. Most LCMV and LASV infections are caused by rodent-to-human transmissions. Thus, viral evolution is largely determined by events that occur in the wildlife reservoirs. We used a set of human- and rodent-derived viral sequences to investigate the evolutionary history underlying OW arenavirus speciation, as well as the more recent selective events that accompanied LASV spread in West Africa. We show that the viral RNA polymerase (L protein) was a major positive selection target in OW arenaviruses and during LASV out-of-Nigeria migration. No evidence of selection was observed for the glycoprotein, whereas positive selection acted on the nucleoprotein (NP) during LCMV speciation. Positively selected sites in L and NP are surrounded by highly conserved residues, and the bulk of the viral genome evolves under purifying selection. Several positively selected sites are likely to modulate viral replication/transcription. In both L and NP, structural features (solvent exposed surface area) are important determinants of site-wise evolutionary rate variation. By incorporating several rodent-derived sequences, we also performed an analysis of OW arenavirus codon adaptation to the human host. Results do not support a previously hypothesized role of codon adaptation in disease severity for non-Nigerian strains. In conclusion, L and NP represent the major selection targets and possible determinants of disease presentation; these results suggest that field surveys and experimental studies should primarily focus on these proteins. © 2017 John Wiley & Sons Ltd.

Monitoring mis-acylated tRNA suppression efficiency in mammalian cells via EGFP fluorescence recovery

PubMed Central

Ilegems, Erwin; Pick, Horst M.; Vogel, Horst

2002-01-01

A reporter assay was developed to detect and quantify nonsense codon suppression by chemically aminoacylated tRNAs in mammalian cells. It is based on the cellular expression of the enhanced green fluorescent protein (EGFP) as a reporter for the site-specific amino acid incorporation in its sequence using an orthogonal suppressor tRNA derived from Escherichia coli. Suppression of an engineered amber codon at position 64 in the EGFP run-off transcript could be achieved by the incorporation of a leucine via an in vitro aminoacylated suppressor tRNA. Microinjection of defined amounts of mutagenized EGFP mRNA and suppressor tRNA into individual cells allowed us to accurately determine suppression efficiencies by measuring the EGFP fluorescence intensity in individual cells using laser-scanning confocal microscopy. Control experiments showed the absence of natural suppression or aminoacylation of the synthetic tRNA by endogenous aminoacyl-tRNA synthetases. This reporter assay opens the way for the optimization of essential experimental parameters for expanding the scope of the suppressor tRNA technology to different cell types. PMID:12466560

The complete genome sequence of freesia mosaic virus and its relationship to other potyviruses.

PubMed

Choi, H I; Lim, H R; Song, Y S; Kim, M J; Choi, S H; Song, Y S; Bae, S C; Ryu, K H

2010-07-01

We have completed the genomic sequence of a potyvirus, freesia mosaic virus (FreMV), and compared it to those of other known potyviruses. The full-length genome sequence of FreMV consists of 9,489 nucleotides. The large protein contains 3,077 amino acids, with an AUG start codon and UAA stop codon, containing one open reading frame typical of a potyvirus polyprotein. The polyprotein of FreMV-Kr gives rise to eleven proteins (P1, HC-pro, P3, PIPO, 6K1, CI, 6K2, VPg, NIa, NIb and CP), and putative cleavage sites of each protein were identified by sequence comparison to those of other known potyviruses. Phylogenetic analysis of the polyprotein revealed that FreMV-Kr was most closely related to PeMoV and was related to BtMV, BaRMV and PeLMV, which belong to the BCMV subgroup. This is the first information on the complete genome structure of FreMV, and the sequence information clearly supports the status of FreMV as a member of a distinct species in the genus Potyvirus.

Conformational Differences between Open and Closed States of the Eukaryotic Translation Initiation Complex

PubMed Central

Llácer, Jose L.; Hussain, Tanweer; Marler, Laura; Aitken, Colin Echeverría; Thakur, Anil; Lorsch, Jon R.; Hinnebusch, Alan G.; Ramakrishnan, V.

2015-01-01

Summary Translation initiation in eukaryotes begins with the formation of a pre-initiation complex (PIC) containing the 40S ribosomal subunit, eIF1, eIF1A, eIF3, ternary complex (eIF2-GTP-Met-tRNAi), and eIF5. The PIC, in an open conformation, attaches to the 5′ end of the mRNA and scans to locate the start codon, whereupon it closes to arrest scanning. We present single particle cryo-electron microscopy (cryo-EM) reconstructions of 48S PICs from yeast in these open and closed states, at 6.0 Å and 4.9 Å, respectively. These reconstructions show eIF2β as well as a configuration of eIF3 that appears to encircle the 40S, occupying part of the subunit interface. Comparison of the complexes reveals a large conformational change in the 40S head from an open mRNA latch conformation to a closed one that constricts the mRNA entry channel and narrows the P site to enclose tRNAi, thus elucidating key events in start codon recognition. PMID:26212456

Identification of genomic islands in six plant pathogens.

PubMed

Chen, Ling-Ling

2006-06-07

Genomic islands (GIs) play important roles in microbial evolution, which are acquired by horizontal gene transfer. In this paper, the GIs of six completely sequenced plant pathogens are identified using a windowless method based on Z curve representation of DNA sequences. Consequently, four, eight, four, one, two and four GIs are recognized with the length greater than 20-Kb in plant pathogens Agrobacterium tumefaciens str. C58, Rolstonia solanacearum GMI1000, Xanthomonas axonopodis pv. citri str. 306 (Xac), Xanthomonas campestris pv. campestris str. ATCC33913 (Xcc), Xylella fastidiosa 9a5c and Pseudomonas syringae pv. tomato str. DC3000, respectively. Most of these regions share a set of conserved features of GIs, including an abrupt change in GC content compared with that of the rest of the genome, the existence of integrase genes at the junction, the use of tRNA as the integration sites, the presence of genetic mobility genes, the difference of codon usage, codon preference and amino acid usage, etc. The identification of these GIs will benefit the research for the six important phytopathogens.

Substitution rate and natural selection in parvovirus B19

PubMed Central

Stamenković, Gorana G.; Ćirković, Valentina S.; Šiljić, Marina M.; Blagojević, Jelena V.; Knežević, Aleksandra M.; Joksić, Ivana D.; Stanojević, Maja P.

2016-01-01

The aim of this study was to estimate substitution rate and imprints of natural selection on parvovirus B19 genotype 1. Studied datasets included 137 near complete coding B19 genomes (positions 665 to 4851) for phylogenetic and substitution rate analysis and 146 and 214 partial genomes for selection analyses in open reading frames ORF1 and ORF2, respectively, collected 1973–2012 and including 9 newly sequenced isolates from Serbia. Phylogenetic clustering assigned majority of studied isolates to G1A. Nucleotide substitution rate for total coding DNA was 1.03 (0.6–1.27) x 10−4 substitutions/site/year, with higher values for analyzed genome partitions. In spite of the highest evolutionary rate, VP2 codons were found to be under purifying selection with rare episodic positive selection, whereas codons under diversifying selection were found in the unique part of VP1, known to contain B19 immune epitopes important in persistent infection. Analyses of overlapping gene regions identified nucleotide positions under opposite selective pressure in different ORFs, suggesting complex evolutionary mechanisms of nucleotide changes in B19 viral genomes. PMID:27775080

Ensemble cryo-EM elucidates the mechanism of translation fidelity

PubMed Central

Loveland, Anna B.; Demo, Gabriel; Grigorieff, Nikolaus; Korostelev, Andrei A.

2017-01-01

SUMMARY Faithful gene translation depends on accurate decoding, whose structural mechanism remains a matter of debate. Ribosomes decode mRNA codons by selecting cognate aminoacyl-tRNAs delivered by EF-Tu. We present high-resolution structural ensembles of ribosomes with cognate or near-cognate aminoacyl-tRNAs delivered by EF-Tu. Both cognate and near-cognate tRNA anticodons explore the A site of an open 30S subunit, while inactive EF-Tu is separated from the 50S subunit. A transient conformation of decoding-center nucleotide G530 stabilizes the cognate codon-anticodon helix, initiating step-wise “latching” of the decoding center. The resulting 30S domain closure docks EF-Tu at the sarcin-ricin loop of the 50S subunit, activating EF-Tu for GTP hydrolysis and ensuing aminoacyl-tRNA accommodation. By contrast, near-cognate complexes fail to induce the G530 latch, thus favoring open 30S pre-accommodation intermediates with inactive EF-Tu. This work unveils long-sought structural differences between the pre-accommodation of cognate and near-cognate tRNA that elucidate the mechanism of accurate decoding. PMID:28538735

Decoding Mechanisms by which Silent Codon Changes Influence Protein Biogenesis and Function

PubMed Central

Bali, Vedrana; Bebok, Zsuzsanna

2015-01-01

Scope Synonymous codon usage has been a focus of investigation since the discovery of the genetic code and its redundancy. The occurrences of synonymous codons vary between species and within genes of the same genome, known as codon usage bias. Today, bioinformatics and experimental data allow us to compose a global view of the mechanisms by which the redundancy of the genetic code contributes to the complexity of biological systems from affecting survival in prokaryotes, to fine tuning the structure and function of proteins in higher eukaryotes. Studies analyzing the consequences of synonymous codon changes in different organisms have revealed that they impact nucleic acid stability, protein levels, structure and function without altering amino acid sequence. As such, synonymous mutations inevitably contribute to the pathogenesis of complex human diseases. Yet, fundamental questions remain unresolved regarding the impact of silent mutations in human disorders. In the present review we describe developments in this area concentrating on mechanisms by which synonymous mutations may affect protein function and human health. Purpose This synopsis illustrates the significance of synonymous mutations in disease pathogenesis. We review the different steps of gene expression affected by silent mutations, and assess the benefits and possible harmful effects of codon optimization applied in the development of therapeutic biologics. Physiological and medical relevance Understanding mechanisms by which synonymous mutations contribute to complex diseases such as cancer, neurodegeneration and genetic disorders, including the limitations of codon-optimized biologics, provides insight concerning interpretation of silent variants and future molecular therapies. PMID:25817479

Codon Usage Patterns of Tyrosinase Genes in Clonorchis sinensis.

PubMed

Bae, Young-An

2017-04-01

Codon usage bias (CUB) is a unique property of genomes and has contributed to the better understanding of the molecular features and the evolution processes of particular gene. In this study, genetic indices associated with CUB, including relative synonymous codon usage and effective numbers of codons, as well as the nucleotide composition, were investigated in the Clonorchis sinensis tyrosinase genes and their platyhelminth orthologs, which play an important role in the eggshell formation. The relative synonymous codon usage patterns substantially differed among tyrosinase genes examined. In a neutrality analysis, the correlation between GC 12 and GC 3 was statistically significant, and the regression line had a relatively gradual slope (0.218). NC-plot, i.e., GC 3 vs effective number of codons (ENC), showed that most of the tyrosinase genes were below the expected curve. The codon adaptation index (CAI) values of the platyhelminth tyrosinases had a narrow distribution between 0.685/0.714 and 0.797/0.837, and were negatively correlated with their ENC. Taken together, these results suggested that CUB in the tyrosinase genes seemed to be basically governed by selection pressures rather than mutational bias, although the latter factor provided an additional force in shaping CUB of the C. sinensis and Opisthorchis viverrini genes. It was also apparent that the equilibrium point between selection pressure and mutational bias is much more inclined to selection pressure in highly expressed C. sinensis genes, than in poorly expressed genes.

The proto-oncogene KRAS and BRAF profiles and some clinical characteristics in colorectal cancer in the Turkish population.

PubMed

Ozen, Filiz; Ozdemir, Semra; Zemheri, Ebru; Hacimuto, Gizem; Silan, Fatma; Ozdemir, Ozturk

2013-02-01

The aim of the current study was to investigate the prevalence and predictive significance of the KRAS and BRAF mutations in Turkish patients with colorectal cancer (CRC). Totally, 53 fresh tumoral tissue specimens were investigated in patients with CRC. All specimens were obtained during routine surgery of patients who were histopathologically diagnosed and genotyped for common KRAS and BRAF point mutations. After DNA extraction, the target mutations were analyzed using the AutoGenomics INFINITI(®) assay, and some samples were confirmed by quantitative real-time polymerase chain reaction fluorescence melting curve analyses. KRAS mutations were found in 26 (49.05%) CRC samples. Twenty-seven samples (50.95%) had wild-type profiles for KRAS codon 12, 13, and 61 in the current cohort. In 17 (65.38%) samples, codon 12; in 7 (26.93%) samples, codon 13; and in 2 (7.69%) samples, codon 61 were found to be mutated, particularly in grade 2 of tumoral tissues. No point mutation was detected in BRAF codon Val600Glu for the studied CRC patients. Our study, based on a representative collection of human CRC tumors, indicates that KRAS gene mutations were detected in 49.05% of the samples, and the most frequent mutation was in the G12D codon. Results also showed that codons 12 and 13 of KRAS are relatively frequently without BRAF mutation in a CRC cohort from the Turkish population.

Ribosome stalling and peptidyl-tRNA drop-off during translational delay at AGA codons

PubMed Central

Cruz-Vera, Luis Rogelio; Magos-Castro, Marco Antonio; Zamora-Romo, Efraín; Guarneros, Gabriel

2004-01-01

Minigenes encoding the peptide Met–Arg–Arg have been used to study the mechanism of toxicity of AGA codons proximal to the start codon or prior to the termination codon in bacteria. The codon sequences of the ‘mini-ORFs’ employed were initiator, combinations of AGA and CGA, and terminator. Both, AGA and CGA are low-usage Arg codons in ORFs of Escherichia coli but, whilst AGA is translated by the scarce tRNAArg4, CGA is recognized by the abundant tRNAArg2. Overexpression of minigenes harbouring AGA in the third position, next to a termination codon, was deleterious to the cell and led to the accumulation of peptidyl-tRNAArg4 and of the peptidyl-tRNA cognate to the preceding CGA or AGA Arg triplet. The minigenes carrying CGA in the third position were not toxic. Minigene-mediated toxicity and peptidyl-tRNA accumulation were suppressed by overproduction of tRNAArg4 but not by overproduction of peptidyl-tRNA hydrolase, an enzyme that is only active on substrates that have been released from the ribosome. Consistent with these findings, peptidyl-tRNAArg4 was identified to be mainly associated with ribosomes in a stand-by complex. These and previous results support the hypothesis that the primary mechanism of inhibition of protein synthesis by AGA triplets in pth+ cells involves sequestration of tRNAs as peptidyl-tRNA on the stalled ribosome. PMID:15317870

Codon optimisation to improve expression of a Mycobacterium avium ssp. paratuberculosis-specific membrane-associated antigen by Lactobacillus salivarius.

PubMed

Johnston, Christopher; Douarre, Pierre E; Soulimane, Tewfik; Pletzer, Daniel; Weingart, Helge; MacSharry, John; Coffey, Aidan; Sleator, Roy D; O'Mahony, Jim

2013-06-01

Subunit and DNA-based vaccines against Mycobacterium avium ssp. paratuberculosis (MAP) attempt to overcome inherent issues associated with whole-cell formulations. However, these vaccines can be hampered by poor expression of recombinant antigens from a number of disparate hosts. The high G+C content of MAP invariably leads to a codon bias throughout gene expression. To investigate if the codon bias affects recombinant MAP antigen expression, the open reading frame of a MAP-specific antigen MptD (MAP3733c) was codon optimised for expression against a Lactobacillus salivarius host. Of the total 209 codons which constitute MAP3733c, 172 were modified resulting in a reduced G+C content from 61% for the native gene to 32.7% for the modified form. Both genes were placed under the transcriptional control of the PnisA promoter; allowing controlled heterologous expression in L. salivarius. Expression was monitored using fluorescence microscopy and microplate fluorometry via GFP tags translationally fused to the C-termini of the two MptD genes. A > 37-fold increase in expression was observed for the codon-optimised MAP3733synth variant over the native gene. Due to the low cost and improved expression achieved, codon optimisation significantly improves the potential of L. salivarius as an oral vaccine stratagem against Johne's disease. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Severe Hemophilia A in a Male Old English Sheep Dog with a C→T Transition that Created a Premature Stop Codon in Factor VIII

PubMed Central

Lozier, Jay N; Kloos, Mark T; Merricks, Elizabeth P; Lemoine, Nathaly; Whitford, Margaret H; Raymer, Robin A; Bellinger, Dwight A; Nichols, Timothy C

2016-01-01

Animals with hemophilia are models for gene therapy, factor replacement, and inhibitor development in humans. We have actively sought dogs with severe hemophilia A that have novel factor VIII mutations unlike the previously described factor VIII intron 22 inversion. A male Old English Sheepdog with recurrent soft-tissue hemorrhage and hemarthrosis was diagnosed with severe hemophilia A (factor VIII activity less than 1% of normal). We purified genomic DNA from this dog and ruled out the common intron 22 inversion; we then sequenced all 26 exons. Comparing the results with the normal canine factor VIII sequence revealed a C→T transition in exon 12 of the factor VIII gene that created a premature stop codon at amino acid 577 in the A2 domain of the protein. In addition, 2 previously described polymorphisms that do not cause hemophilia were present at amino acids 909 and 1184. The hemophilia mutation creates a new TaqI site that facilitates rapid genotyping of affected offspring by PCR and restriction endonuclease analyses. This mutation is analogous to the previously described human factor VIII mutation at Arg583, which likewise is a CpG dinucleotide transition causing a premature stop codon in exon 12. Thus far, despite extensive treatment with factor VIII, this dog has not developed neutralizing antibodies (‘inhibitors’) to the protein. This novel mutation in a dog gives rise to severe hemophilia A analogous to a mutation seen in humans. This model will be useful for studies of the treatment of hemophilia. PMID:27780008

Identification of a splicing enhancer in MLH1 using COMPARE a new assay for determination of relative RNA splicing efficiencies

PubMed Central

Xu, Dong-Qing; Mattox, William

2006-01-01

Exonic splicing enhancers (ESEs) are sequences that facilitate recognition of splice sites and prevent exon-skipping. Because ESEs are often embedded within proteincoding sequences, alterations in them can also often be interpreted as nonsense, missense or silent mutations. To correctly interpret exonic mutations and their roles in disease, it is important to develop strategies that identify ESE mutations. Potential ESEs can be found computationally in many exons but it has proven difficult to predict if a given mutation will have effects on splicing based on sequence alone. Here we describe a flexible in vitro method that can be used to functionally compare the effects of multiple sequence variants on ESE activity in a single in vitro splicing reaction. We have applied this method in parallel with conventional splicing assays to test for a splicing enhancer in exon 17 of the human MLH1 gene. Point mutations associated with hereditary nonpolyposis colorectal cancer (HNPCC) have previously been found to correlate with exon-skipping in both lymphocytes and tumors from patients. We show that sequences from this exon can replace an ESE from the mouse IgM gene to support RNA splicing in HeLa nuclear extracts. ESE activity was reduced by HNPCC point mutations in codon 659 indicating that their primary effect is on splicing. Surprisingly the strongest enhancer function mapped to a different region of the exon upstream of this codon. Together our results indicate that HNPCC point mutations in codon 659 affect an auxillary element that augments the enhancer function to ensure exon inclusion. PMID:16357104

Kinetic modeling predicts a stimulatory role for ribosome collisions at elongation stall sites in bacteria

PubMed Central

Ferrin, Michael A; Subramaniam, Arvind R

2017-01-01

Ribosome stalling on mRNAs can decrease protein expression. To decipher ribosome kinetics at stall sites, we induced ribosome stalling at specific codons by starving the bacterium Escherichia coli for the cognate amino acid. We measured protein synthesis rates from a reporter library of over 100 variants that encoded systematic perturbations of translation initiation rate, the number of stall sites, and the distance between stall sites. Our measurements are quantitatively inconsistent with two widely-used kinetic models for stalled ribosomes: ribosome traffic jams that block initiation, and abortive (premature) termination of stalled ribosomes. Rather, our measurements support a model in which collision with a trailing ribosome causes abortive termination of the stalled ribosome. In our computational analysis, ribosome collisions selectively stimulate abortive termination without fine-tuning of kinetic rate parameters at ribosome stall sites. We propose that ribosome collisions serve as a robust timer for translational quality control pathways to recognize stalled ribosomes. DOI: http://dx.doi.org/10.7554/eLife.23629.001 PMID:28498106

Hepatitis D virus RNA editing is inhibited by a GFP fusion protein containing a C-terminally deleted delta antigen.

PubMed

Shih, Ko-Nien; Chuang, Ya-Ting; Liu, Hsuan; Lo, Szecheng J

2004-04-01

During its life cycle, hepatitis D virus (HDV) produces two forms of delta antigen (HDAg), small delta antigen (SDAg) and large delta antigen (LDAg), which differ in their C-terminal 19 amino acids. Host enzymes termed ADARs (adenosine deaminases that act on double-stranded RNA) are required for LDAg production. These enzymes change the stop codon (UAG) of SDAg to a tryptophan codon (UGG). However, the temporal and spatial regulation of HDV RNA editing is largely unknown. In this study, we constructed three GFP fusion proteins containing different lengths of SDAg and characterized their cellular localization and effects on HDV replication. One of these fusion proteins, designated D(1-88)-GFP, inhibited LDAg but not SDAg production, suggesting that D(1-88)-GFP inhibits HDV RNA editing. Two experiments further supported this supposition: (i). RT-PCR analysis combined with NcoI restriction enzyme digestion revealed that HDV RNA editing was reduced by 42% in HeLa-D(1-88)-GFP when compared with HeLa cells; and (ii). the ratio of SDAg/LDAg production from the reporter RNAs was reduced in cells co-transfected with ADAR-expressing and reporter plasmids in the presence of D(1-88)-GFP. Double fluorescence microscopy found that D(1-88)-GFP was either associated with SC-35 or was adjacent to PML (premyelocytic leukaemia antigen) at nuclear speckles, but D(1-88)-GFP was not co-localized with ADAR, which was mainly located in the nucleolus. In situ hybridization showing co-localization of HDV RNA with D(1-88)-GFP at nuclear speckles suggested that HDV RNA editing might occur in the nuclear speckles and require other nuclear factor(s), in addition to ADAR.

Elevation of the Yields of Very Long Chain Polyunsaturated Fatty Acids via Minimal Codon Optimization of Two Key Biosynthetic Enzymes

PubMed Central

Zheng, Desong; Sun, Quanxi; Liu, Jiang; Li, Yaxiao; Hua, Jinping

2016-01-01

Eicosapentaenoic acid (EPA, 20:5Δ5,8,11,14,17) and Docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,16,19) are nutritionally beneficial to human health. Transgenic production of EPA and DHA in oilseed crops by transferring genes originating from lower eukaryotes, such as microalgae and fungi, has been attempted in recent years. However, the low yield of EPA and DHA produced in these transgenic crops is a major hurdle for the commercialization of these transgenics. Many factors can negatively affect transgene expression, leading to a low level of converted fatty acid products. Among these the codon bias between the transgene donor and the host crop is one of the major contributing factors. Therefore, we carried out codon optimization of a fatty acid delta-6 desaturase gene PinD6 from the fungus Phytophthora infestans, and a delta-9 elongase gene, IgASE1 from the microalga Isochrysis galbana for expression in Saccharomyces cerevisiae and Arabidopsis respectively. These are the two key genes encoding enzymes for driving the first catalytic steps in the Δ6 desaturation/Δ6 elongation and the Δ9 elongation/Δ8 desaturation pathways for EPA/DHA biosynthesis. Hence expression levels of these two genes are important in determining the final yield of EPA/DHA. Via PCR-based mutagenesis we optimized the least preferred codons within the first 16 codons at their N-termini, as well as the most biased CGC codons (coding for arginine) within the entire sequences of both genes. An expression study showed that transgenic Arabidopsis plants harbouring the codon-optimized IgASE1 contained 64% more elongated fatty acid products than plants expressing the native IgASE1 sequence, whilst Saccharomyces cerevisiae expressing the codon optimized PinD6 yielded 20 times more desaturated products than yeast expressing wild-type (WT) PinD6. Thus the codon optimization strategy we developed here offers a simple, effective and low-cost alternative to whole gene synthesis for high expression of foreign genes in yeast and Arabidopsis. PMID:27433934

Improved Prefusion Stability, Optimized Codon Usage, and Augmented Virion Packaging Enhance the Immunogenicity of Respiratory Syncytial Virus Fusion Protein in a Vectored-Vaccine Candidate

PubMed Central

Liang, Bo; Ngwuta, Joan O.; Surman, Sonja; Kabatova, Barbora; Liu, Xiang; Lingemann, Matthias; Liu, Xueqiao; Yang, Lijuan; Herbert, Richard; Swerczek, Joanna; Chen, Man; Moin, Syed M.; Kumar, Azad; McLellan, Jason S.; Kwong, Peter D.; Graham, Barney S.; Collins, Peter L.

2017-01-01

ABSTRACT Respiratory syncytial virus (RSV) is the most important viral agent of severe pediatric respiratory tract disease worldwide, but it lacks a licensed vaccine or suitable antiviral drug. A live attenuated chimeric bovine/human parainfluenza virus type 3 (rB/HPIV3) was developed previously as a vector expressing RSV fusion (F) protein to confer bivalent protection against RSV and HPIV3. In a previous clinical trial in virus-naive children, rB/HPIV3 was well tolerated but the immunogenicity of wild-type RSV F was unsatisfactory. We previously modified RSV F with a designed disulfide bond (DS) to increase stability in the prefusion (pre-F) conformation and to be efficiently packaged in the vector virion. Here, we further stabilized pre-F by adding both disulfide and cavity-filling mutations (DS-Cav1), and we also modified RSV F codon usage to have a lower CpG content and a higher level of expression. This RSV F open reading frame was evaluated in rB/HPIV3 in three forms: (i) pre-F without vector-packaging signal, (ii) pre-F with vector-packaging signal, and (iii) secreted pre-F ectodomain trimer. Despite being efficiently expressed, the secreted pre-F was poorly immunogenic. DS-Cav1 stabilized pre-F, with or without packaging, induced higher titers of pre-F specific antibodies in hamsters, and improved the quality of RSV-neutralizing serum antibodies. Codon-optimized RSV F containing fewer CpG dinucleotides had higher F expression, replicated more efficiently in vivo, and was more immunogenic. The combination of DS-Cav1 pre-F stabilization, optimized codon usage, reduced CpG content, and vector packaging significantly improved vector immunogenicity and protective efficacy against RSV. This provides an improved vectored RSV vaccine candidate suitable for pediatric clinical evaluation. IMPORTANCE RSV and HPIV3 are the first and second leading viral causes of severe pediatric respiratory disease worldwide. Licensed vaccines or suitable antiviral drugs are not available. We are developing a chimeric rB/HPIV3 vector expressing RSV F as a bivalent RSV/HPIV3 vaccine and have been evaluating means to increase RSV F immunogenicity. In this study, we evaluated the effects of improved stabilization of F in the pre-F conformation and of codon optimization resulting in reduced CpG content and greater pre-F expression. Reduced CpG content dampened the interferon response to infection, promoting higher replication and increased F expression. We demonstrate that improved pre-F stabilization and strategic manipulation of codon usage, together with efficient pre-F packaging into vector virions, significantly increased F immunogenicity in the bivalent RSV/HPIV3 vaccine. The improved immunogenicity included induction of increased titers of high-quality complement-independent antibodies with greater pre-F site Ø binding and greater protection against RSV challenge. PMID:28539444

Pervasive positive selection on duplicated and nonduplicated vertebrate protein coding genes.

PubMed

Studer, Romain A; Penel, Simon; Duret, Laurent; Robinson-Rechavi, Marc

2008-09-01

A stringent branch-site codon model was used to detect positive selection in vertebrate evolution. We show that the test is robust to the large evolutionary distances involved. Positive selection was detected in 77% of 884 genes studied. Most positive selection concerns a few sites on a single branch of the phylogenetic tree: Between 0.9% and 4.7% of sites are affected by positive selection depending on the branches. No functional category was overrepresented among genes under positive selection. Surprisingly, whole genome duplication had no effect on the prevalence of positive selection, whether the fish-specific genome duplication or the two rounds at the origin of vertebrates. Thus positive selection has not been limited to a few gene classes, or to specific evolutionary events such as duplication, but has been pervasive during vertebrate evolution.

An MSC2 Promoter-lacZ Fusion Gene Reveals Zinc-Responsive Changes in Sites of Transcription Initiation That Occur across the Yeast Genome

PubMed Central

Wu, Yi-Hsuan; Taggart, Janet; Song, Pamela Xiyao; MacDiarmid, Colin; Eide, David J.

2016-01-01

The Msc2 and Zrg17 proteins of Saccharomyces cerevisiae form a complex to transport zinc into the endoplasmic reticulum. ZRG17 is transcriptionally induced in zinc-limited cells by the Zap1 transcription factor. In this report, we show that MSC2 mRNA also increases (~1.5 fold) in zinc-limited cells. The MSC2 gene has two in-frame ATG codons at its 5’ end, ATG1 and ATG2; ATG2 is the predicted initiation codon. When the MSC2 promoter was fused at ATG2 to the lacZ gene, we found that unlike the chromosomal gene this reporter showed a 4-fold decrease in lacZ mRNA in zinc-limited cells. Surprisingly, β-galactosidase activity generated by this fusion gene increased ~7 fold during zinc deficiency suggesting the influence of post-transcriptional factors. Transcription of MSC2ATG2-lacZ was found to start upstream of ATG1 in zinc-replete cells. In zinc-limited cells, transcription initiation shifted to sites just upstream of ATG2. From the results of mutational and polysome profile analyses, we propose the following explanation for these effects. In zinc-replete cells, MSC2ATG2-lacZ mRNA with long 5’ UTRs fold into secondary structures that inhibit translation. In zinc-limited cells, transcripts with shorter unstructured 5’ UTRs are generated that are more efficiently translated. Surprisingly, chromosomal MSC2 did not show start site shifts in response to zinc status and only shorter 5’ UTRs were observed. However, the shifts that occur in the MSC2ATG2-lacZ construct led us to identify significant transcription start site changes affecting the expression of ~3% of all genes. Therefore, zinc status can profoundly alter transcription initiation across the yeast genome. PMID:27657924

Disruption of the Opal Stop Codon Attenuates Chikungunya Virus-Induced Arthritis and Pathology.

PubMed

Jones, Jennifer E; Long, Kristin M; Whitmore, Alan C; Sanders, Wes; Thurlow, Lance R; Brown, Julia A; Morrison, Clayton R; Vincent, Heather; Peck, Kayla M; Browning, Christian; Moorman, Nathaniel; Lim, Jean K; Heise, Mark T

2017-11-14

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus responsible for several significant outbreaks of debilitating acute and chronic arthritis and arthralgia over the past decade. These include a recent outbreak in the Caribbean islands and the Americas that caused more than 1 million cases of viral arthralgia. Despite the major impact of CHIKV on global health, viral determinants that promote CHIKV-induced disease are incompletely understood. Most CHIKV strains contain a conserved opal stop codon at the end of the viral nsP3 gene. However, CHIKV strains that encode an arginine codon in place of the opal stop codon have been described, and deep-sequencing analysis of a CHIKV isolate from the Caribbean identified both arginine and opal variants within this strain. Therefore, we hypothesized that the introduction of the arginine mutation in place of the opal termination codon may influence CHIKV virulence. We tested this by introducing the arginine mutation into a well-characterized infectious clone of a CHIKV strain from Sri Lanka and designated this virus Opal524R. This mutation did not impair viral replication kinetics in vitro or in vivo Despite this, the Opal524R virus induced significantly less swelling, inflammation, and damage within the feet and ankles of infected mice. Further, we observed delayed induction of proinflammatory cytokines and chemokines, as well as reduced CD4 + T cell and NK cell recruitment compared to those in the parental strain. Therefore, the opal termination codon plays an important role in CHIKV pathogenesis, independently of effects on viral replication. IMPORTANCE Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes significant outbreaks of viral arthralgia. Studies with CHIKV and other alphaviruses demonstrated that the opal termination codon within nsP3 is highly conserved. However, some strains of CHIKV and other alphaviruses contain mutations in the opal termination codon. These mutations alter the virulence of related alphaviruses in mammalian and mosquito hosts. Here, we report that a clinical isolate of a CHIKV strain from the recent outbreak in the Caribbean islands contains a mixture of viruses encoding either the opal termination codon or an arginine mutation. Mutating the opal stop codon to an arginine residue attenuates CHIKV-induced disease in a mouse model. Compared to infection with the opal-containing parental virus, infection with the arginine mutant causes limited swelling and inflammation, as well as dampened recruitment of immune mediators of pathology, including CD4 + T cells and NK cells. We propose that the opal termination codon plays an essential role in the induction of severe CHIKV disease. Copyright © 2017 Jones et al.

Nuclear localization of Merkel cell polyomavirus large T antigen in Merkel cell carcinoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakamura, Tomoyuki; Sato, Yuko; Watanabe, Daisuke

2010-03-15

To clarify whether mutations in the large T gene encoded by Merkel cell polyomavirus affect the expression and function of large T antigen in Merkel cell carcinoma cases, we investigated the expression of large T antigen in vitro and in vivo. Immunohistochemistry using a rabbit polyclonal antibody revealed that large T antigen was expressed in the nuclei of Merkel cell carcinoma cells with Merkel cell polyomavirus infection. Deletion mutant analyses identified an Arg-Lys-Arg-Lys sequence (amino acids 277-280) as a nuclear localization signal in large T antigen. Sequence analyses revealed that there were no mutations in the nuclear localization signal inmore » any of the eleven Merkel cell polyomavirus strains examined. Furthermore, stop codons were not observed in the upstream of the nuclear localization signal in any of the Merkel cell carcinoma cases examined. These data suggest that the nuclear localization signal is highly conserved and functional in Merkel cell carcinoma cases.« less

Diverse Dengue Type 2 Virus Populations Contain Recombinant and Both Parental Viruses in a Single Mosquito Host

PubMed Central

Craig, Scott; Thu, Hlaing Myat; Lowry, Kym; Wang, Xiao-fang; Holmes, Edward C.; Aaskov, John

2003-01-01

Envelope (E) protein genes sampled from populations of dengue 2 (DEN-2) virus in individual Aedes aegypti mosquitoes and in serum from dengue patients were copied to cDNA, cloned, and sequenced. The nucleotide sequences of the E genes in more than 70% of the clones differed from the consensus sequence for the corresponding virus population at up to 11 sites, and 24 of the 94 clones contained at least one stop codon. Virus populations recovered up to 2 years apart yielded clones with similar polymorphisms in the E gene. For one mosquito, the clones obtained fell into two genotypes. One group of sequences was closely related to those of viruses recovered from dengue patients in the same locality (Yangon, Myanmar) since 1995 and were classified as Asian 1 genotype. The second group were Cosmopolitan genotype viruses which were also circulating in Yangon in 2000 and which were related to DEN-2 viruses sampled from southern China in 1999. Finally, one clone was identified as a recombinant genome composed of portions of these two “parental” genotypes. This is the first report of recombinant and parental dengue viruses in a single host. PMID:12634407

Identification of four novel XPC mutations in two xeroderma pigmentosum complementation group C patients and functional study of XPC Q320X mutant.

PubMed

Gu, Yajuan; Chang, Xiaodan; Dai, Shan; Song, Qinghua; Zhao, Hongshan; Lei, Pengcheng

2017-09-10

Xeroderma pigmentosum (XP) is a rare, recessive hereditary disease characterized by sunlight hypersensitivity and high incidence of skin cancer with clinical and genetic heterogeneity. We collected two unrelated Chinese patients showing typical symptoms of XPC without neurologic symptoms. Direct sequencing of XPC gene revealed that patient 1 carried IVS1+1G>A and c.958 C>T mutations, and patient 2 carried c.545_546delTA and c.2257_2258insC mutations. All these four mutations introduced premature terminal codons (PTCs) in XPC gene. The nonsense mutation c.958 C>T yielded truncated mutant Q320X, and we studied its function for global genome repair kinetics. Overexpressed Q320X mutant can localize to site of DNA damage, but it is defective in CPD and 6-4PP repair. Readthrough of PTCs is a new approach to treatment of genetic diseases. We found that aminoglycosides could significantly increase the full length protein expression of Q320X mutant, but NER defects were not rescued in vitro. Copyright © 2017 Elsevier B.V. All rights reserved.

Transformation of NIH3T3 Cells with Synthetic c‐Ha‐ras Genes

PubMed Central

Kamiya, Hiroyuki; Miura, Kazunobu; Ohtomo, Noriko; Koda, Toshiaki; Kakinuma, Mitsuaki; Nishimura, Susumu

1989-01-01

Synthetic human c‐Ha‐ras genes in which amino acid codons were altered to those which are frequently used in highly expressed Escherichia coli genes were ligated to the 3′‐end of Rous sarcoma virus long terminal repeat. When NIH3T3 cells were transfected with the plasmids having those genes with valine at codon 12, leucine at codon 61 or arginine at codon 61, transformants were efficiently produced. These results indicated that the synthetic c‐Ha‐ras genes are expressed in a mammalian system even though their codon usage is altered to correspond with that of E. colt. This expression vector system should he useful for studies on the structure‐function relationships of c‐Ha‐ras, since the synthetic gene can be easily modified to have multiple base alterations, and can also be used simultaneously for the production of large amounts of p21 in E. coli for biochemical and biophysical studies. PMID:2542206

RNA Editing in Plant Mitochondria

NASA Astrophysics Data System (ADS)

Hiesel, Rudolf; Wissinger, Bernd; Schuster, Wolfgang; Brennicke, Axel

1989-12-01

Comparative sequence analysis of genomic and complementary DNA clones from several mitochondrial genes in the higher plant Oenothera revealed nucleotide sequence divergences between the genomic and the messenger RNA-derived sequences. These sequence alterations could be most easily explained by specific post-transcriptional nucleotide modifications. Most of the nucleotide exchanges in coding regions lead to altered codons in the mRNA that specify amino acids better conserved in evolution than those encoded by the genomic DNA. Several instances show that the genomic arginine codon CGG is edited in the mRNA to the tryptophan codon TGG in amino acid positions that are highly conserved as tryptophan in the homologous proteins of other species. This editing suggests that the standard genetic code is used in plant mitochondria and resolves the frequent coincidence of CGG codons and tryptophan in different plant species. The apparently frequent and non-species-specific equivalency of CGG and TGG codons in particular suggests that RNA editing is a common feature of all higher plant mitochondria.

An analysis of the metabolic theory of the origin of the genetic code

NASA Technical Reports Server (NTRS)

Amirnovin, R.; Bada, J. L. (Principal Investigator)

1997-01-01

A computer program was used to test Wong's coevolution theory of the genetic code. The codon correlations between the codons of biosynthetically related amino acids in the universal genetic code and in randomly generated genetic codes were compared. It was determined that many codon correlations are also present within random genetic codes and that among the random codes there are always several which have many more correlations than that found in the universal code. Although the number of correlations depends on the choice of biosynthetically related amino acids, the probability of choosing a random genetic code with the same or greater number of codon correlations as the universal genetic code was found to vary from 0.1% to 34% (with respect to a fairly complete listing of related amino acids). Thus, Wong's theory that the genetic code arose by coevolution with the biosynthetic pathways of amino acids, based on codon correlations between biosynthetically related amino acids, is statistical in nature.

Complete mitochondrial genome of the Yellownose skate: Zearaja chilensis (Rajiformes, Rajidae).

PubMed

Jeong, Dageum; Lee, Youn-Ho

2016-01-01

The complete sequence of mitochondrial DNA of a Yellownose skate, Zearaja chilensis was determined for the first time. It is 16,909 bp in length covering 2 rRNA, 22 tRNA and 13 protein coding genes with the identical gene order and structure as those of other Rajidae species. The nucleotide of L-strand is composed of low G (14.3%), and slightly high A + T (58.9%) nucleotides. The strong codon usage bias against the use of G (6.0%) is found at the third codon positions. Twelve of the 13 protein coding genes use ATG as the start codon while COX1 starts with GTG. As for the stop codon, only ND4 shows an incomplete stop codon TA. This is the first report of the mitogenome for a species in the genus Zearaja, providing a valuable source of genetic information on the evolution of the family Rajidae and the genus Zearaja as well as for establishment of a sustainble fishery management plan of the species.

Bayesian estimation of post-Messinian divergence times in Balearic Island lizards.

PubMed

Brown, R P; Terrasa, B; Pérez-Mellado, V; Castro, J A; Hoskisson, P A; Picornell, A; Ramon, M M

2008-07-01

Phylogenetic relationships and timings of major cladogenesis events are investigated in the Balearic Island lizards Podarcislilfordi and P.pityusensis using 2675bp of mitochondrial and nuclear DNA sequences. Partitioned Bayesian and Maximum Parsimony analyses provided a well-resolved phylogeny with high node-support values. Bayesian MCMC estimation of node dates was investigated by comparing means of posterior distributions from different subsets of the sequence against the most robust analysis which used multiple partitions and allowed for rate heterogeneity among branches under a rate-drift model. Evolutionary rates were systematically underestimated and thus divergence times overestimated when sequences containing lower numbers of variable sites were used (based on ingroup node constraints). The following analyses allowed the best recovery of node times under the constant-rate (i.e., perfect clock) model: (i) all cytochrome b sequence (partitioned by codon position), (ii) cytochrome b (codon position 3 alone), (iii) NADH dehydrogenase (subunits 1 and 2; partitioned by codon position), (iv) cytochrome b and NADH dehydrogenase sequence together (six gene-codon partitions), (v) all unpartitioned sequence, (vi) a full multipartition analysis (nine partitions). Of these, only (iv) and (vi) performed well under the rate-drift model. These findings have significant implications for dating of recent divergence times in other taxa. The earliest P.lilfordi cladogenesis event (divergence of Menorcan populations), occurred before the end of the Pliocene, some 2.6Ma. Subsequent events led to a West Mallorcan lineage (2.0Ma ago), followed 1.2Ma ago by divergence of populations from the southern part of the Cabrera archipelago from a widely-distributed group from north Cabrera, northern and southern Mallorcan islets. Divergence within P.pityusensis is more recent with the main Ibiza and Formentera clades sharing a common ancestor at about 1.0Ma ago. Climatic and sea level changes are likely to have initiated cladogenesis, with lineages making secondary contact during periodic landbridge formation. This oscillating cross-archipelago pattern in which ancient divergence is followed by repeated contact resembles that seen between East-West refugia populations from mainland Europe.

Contribution of single amino acid and codon substitutions to the production and secretion of a lipase by Bacillus subtilis.

PubMed

Skoczinski, Pia; Volkenborn, Kristina; Fulton, Alexander; Bhadauriya, Anuseema; Nutschel, Christina; Gohlke, Holger; Knapp, Andreas; Jaeger, Karl-Erich

2017-09-25

Bacillus subtilis produces and secretes proteins in amounts of up to 20 g/l under optimal conditions. However, protein production can be challenging if transcription and cotranslational secretion are negatively affected, or the target protein is degraded by extracellular proteases. This study aims at elucidating the influence of a target protein on its own production by a systematic mutational analysis of the homologous B. subtilis model protein lipase A (LipA). We have covered the full natural diversity of single amino acid substitutions at 155 positions of LipA by site saturation mutagenesis excluding only highly conserved residues and qualitatively and quantitatively screened about 30,000 clones for extracellular LipA production. Identified variants with beneficial effects on production were sequenced and analyzed regarding B. subtilis growth behavior, extracellular lipase activity and amount as well as changes in lipase transcript levels. In total, 26 LipA variants were identified showing an up to twofold increase in either amount or activity of extracellular lipase. These variants harbor single amino acid or codon substitutions that did not substantially affect B. subtilis growth. Subsequent exemplary combination of beneficial single amino acid substitutions revealed an additive effect solely at the level of extracellular lipase amount; however, lipase amount and activity could not be increased simultaneously. Single amino acid and codon substitutions can affect LipA secretion and production by B. subtilis. Several codon-related effects were observed that either enhance lipA transcription or promote a more efficient folding of LipA. Single amino acid substitutions could improve LipA production by increasing its secretion or stability in the culture supernatant. Our findings indicate that optimization of the expression system is not sufficient for efficient protein production in B. subtilis. The sequence of the target protein should also be considered as an optimization target for successful protein production. Our results further suggest that variants with improved properties might be identified much faster and easier if mutagenesis is prioritized towards elements that contribute to enzymatic activity or structural integrity.

Cell lines that support replication of a novel herpes simplex virus 1 U{sub L}31 deletion mutant can properly target U{sub L}34 protein to the nuclear rim in the absence of U{sub L}31

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liang Li; Tanaka, Michiko; Kawaguchi, Yasushi

2004-11-10

Previous results indicated that the herpes simplex virus 1 (HSV-1) U{sub L}31 gene is necessary and sufficient for localization of the U{sub L}34 protein exclusively to the nuclear membrane of infected Hep2 cells. In the current studies, a bacterial artificial chromosome containing the entire HSV-1 strain F genome was used to construct a recombinant viral genome in which a gene encoding kanamycin resistance was inserted in place of 262 codons of the 306 codon U{sub L}31 open reading frame. The deletion virus produced virus titers approximately 10- to 50-fold lower in rabbit skin cells, more than 2000-fold lower in Veromore » cells, and more than 1500-fold lower in CV1 cells, compared to a virus bearing a restored U{sub L}31 gene. The replication of the U{sub L}31 deletion virus was restored on U{sub L}31-complementing cell lines derived either from rabbit skin cells or CV1 cells. Confocal microscopy indicated that the majority of U{sub L}34 protein localized aberrantly in the cytoplasm and nucleoplasm of Vero cells and CV1 cells, whereas U{sub L}34 protein localized at the nuclear membrane in rabbit skin cells, and U{sub L}31 complementing CV1 cells infected with the U{sub L}31 deletion virus. We conclude that rabbit skin cells encode a function that allows proper localization of U{sub L}34 protein to the nuclear membrane. We speculate that this function partially complements that of U{sub L}31 and may explain why U{sub L}31 is less critical for replication in rabbit skin cells as opposed to Vero and CV1 cells.« less

Comprehensive analysis of the codon usage patterns in the envelope glycoprotein E2 gene of the classical swine fever virus

PubMed Central

Chi, Xiaojuan; Wang, Song; Ma, Yanmei; Chen, Jilong

2017-01-01

The classical swine fever virus (CSFV), circulating worldwide, is a highly contagious virus. Since the emergence of CSFV, it has caused great economic loss in swine industry. The envelope glycoprotein E2 gene of the CSFV is an immunoprotective antigen that induces the immune system to produce neutralizing antibodies. Therefore, it is essential to study the codon usage of the E2 gene of the CSFV. In this study, 140 coding sequences of the E2 gene were analyzed. The value of effective number of codons (ENC) showed low codon usage bias in the E2 gene. Our study showed that codon usage could be described mainly by mutation pressure ENC plot analysis combined with principal component analysis (PCA) and translational selection-correlation analysis between the general average hydropathicity (Gravy) and aromaticity (Aroma), and nucleotides at the third position of codons (A3s, T3s, G3s, C3s and GC3s). Furthermore, the neutrality analysis, which explained the relationship between GC12s and GC3s, revealed that natural selection had a key role compared with mutational bias during the evolution of the E2 gene. These results lay a foundation for further research on the molecular evolution of CSFV. PMID:28880881

Comprehensive analysis of the codon usage patterns in the envelope glycoprotein E2 gene of the classical swine fever virus.

PubMed

Chen, Ye; Li, Xinxin; Chi, Xiaojuan; Wang, Song; Ma, Yanmei; Chen, Jilong

2017-01-01

The classical swine fever virus (CSFV), circulating worldwide, is a highly contagious virus. Since the emergence of CSFV, it has caused great economic loss in swine industry. The envelope glycoprotein E2 gene of the CSFV is an immunoprotective antigen that induces the immune system to produce neutralizing antibodies. Therefore, it is essential to study the codon usage of the E2 gene of the CSFV. In this study, 140 coding sequences of the E2 gene were analyzed. The value of effective number of codons (ENC) showed low codon usage bias in the E2 gene. Our study showed that codon usage could be described mainly by mutation pressure ENC plot analysis combined with principal component analysis (PCA) and translational selection-correlation analysis between the general average hydropathicity (Gravy) and aromaticity (Aroma), and nucleotides at the third position of codons (A3s, T3s, G3s, C3s and GC3s). Furthermore, the neutrality analysis, which explained the relationship between GC12s and GC3s, revealed that natural selection had a key role compared with mutational bias during the evolution of the E2 gene. These results lay a foundation for further research on the molecular evolution of CSFV.

Differential Reprogramming of Isogenic Colorectal Cancer Cells by Distinct Activating KRAS Mutations

PubMed Central

2015-01-01

Oncogenic mutations of Ras at codons 12, 13, or 61, that render the protein constitutively active, are found in ∼16% of all cancer cases. Among the three major Ras isoforms, KRAS is the most frequently mutated isoform in cancer. Each Ras isoform and tumor type displays a distinct pattern of codon-specific mutations. In colon cancer, KRAS is typically mutated at codon 12, but a significant fraction of patients have mutations at codon 13. Clinical data suggest different outcomes and responsiveness to treatment between these two groups. To investigate the differential effects upon cell status associated with KRAS mutations we performed a quantitative analysis of the proteome and phosphoproteome of isogenic SW48 colon cancer cell lines in which one allele of the endogenous gene has been edited to harbor specific KRAS mutations (G12V, G12D, or G13D). Each mutation generates a distinct signature, with the most variability seen between G13D and the codon 12 KRAS mutants. One notable example of specific up-regulation in KRAS codon 12 mutant SW48 cells is provided by the short form of the colon cancer stem cell marker doublecortin-like Kinase 1 (DCLK1) that can be reversed by suppression of KRAS. PMID:25599653

Modification of orthogonal tRNAs: unexpected consequences for sense codon reassignment.

PubMed

Biddle, Wil; Schmitt, Margaret A; Fisk, John D

2016-12-01

Breaking the degeneracy of the genetic code via sense codon reassignment has emerged as a way to incorporate multiple copies of multiple non-canonical amino acids into a protein of interest. Here, we report the modification of a normally orthogonal tRNA by a host enzyme and show that this adventitious modification has a direct impact on the activity of the orthogonal tRNA in translation. We observed nearly equal decoding of both histidine codons, CAU and CAC, by an engineered orthogonal M. jannaschii tRNA with an AUG anticodon: tRNA Opt We suspected a modification of the tRNA Opt AUG anticodon was responsible for the anomalous lack of codon discrimination and demonstrate that adenosine 34 of tRNA Opt AUG is converted to inosine. We identified tRNA Opt AUG anticodon loop variants that increase reassignment of the histidine CAU codon, decrease incorporation in response to the histidine CAC codon, and improve cell health and growth profiles. Recognizing tRNA modification as both a potential pitfall and avenue of directed alteration will be important as the field of genetic code engineering continues to infiltrate the genetic codes of diverse organisms. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Cloning and expression of codon-optimized recombinant darbepoetin alfa in Leishmania tarentolae T7-TR.

PubMed

Kianmehr, Anvarsadat; Golavar, Raziyeh; Rouintan, Mandana; Mahrooz, Abdolkarim; Fard-Esfahani, Pezhman; Oladnabi, Morteza; Khajeniazi, Safoura; Mostafavi, Seyede Samaneh; Omidinia, Eskandar

2016-02-01

Darbepoetin alfa is an engineered and hyperglycosylated analog of recombinant human erythropoietin (EPO) which is used as a drug in treating anemia in patients with chronic kidney failure and cancer. This study desribes the secretory expression of a codon-optimized recombinant form of darbepoetin alfa in Leishmania tarentolae T7-TR. Synthetic codon-optimized gene was amplified by PCR and cloned into the pLEXSY-I-blecherry3 vector. The resultant expression vector, pLEXSYDarbo, was purified, digested, and electroporated into the L. tarentolae. Expression of recombinant darbepoetin alfa was evaluated by ELISA, reverse-transcription PCR (RT-PCR), Western blotting, and biological activity. After codon optimization, codon adaptation index (CAI) of the gene raised from 0.50 to 0.99 and its GC% content changed from 56% to 58%. Expression analysis confirmed the presence of a protein band at 40 kDa. Furthermore, reticulocyte experiment results revealed that the activity of expressed darbepoetin alfa was similar to that of its equivalent expressed in Chinese hamster ovary (CHO) cells. These data suggested that the codon optimization and expression in L. tarentolae host provided an efficient approach for high level expression of darbepoetin alfa. Copyright © 2015 Elsevier Inc. All rights reserved.

Effect of KRAS codon13 mutations in patients with advanced colorectal cancer (advanced CRC) under oxaliplatin containing chemotherapy. Results from a translational study of the AIO colorectal study group

PubMed Central

2012-01-01

Background To evaluate the value of KRAS codon 13 mutations in patients with advanced colorectal cancer (advanced CRC) treated with oxaliplatin and fluoropyrimidines. Methods Tumor specimens from 201 patients with advanced CRC from a randomized, phase III trial comparing oxaliplatin/5-FU vs. oxaliplatin/capecitabine were retrospectively analyzed for KRAS mutations. Mutation data were correlated to response data (Overall response rate, ORR), progression-free survival (PFS) and overall survival (OS). Results 201 patients were analysed for KRAS mutation (61.2% males; mean age 64.2 ± 8.6 years). KRAS mutations were identified in 36.3% of tumors (28.8% in codon 12, 7.4% in codon 13). The ORR in codon 13 patients compared to codon 12 and wild type patients was significantly lower (p = 0.008). There was a tendency for a better overall survival in KRAS wild type patients compared to mutants (p = 0.085). PFS in all patients was not different in the three KRAS genetic groups (p = 0.72). However, we found a marked difference in PFS between patients with codon 12 and 13 mutant tumors treated with infusional 5-FU versus capecitabine based regimens. Conclusions Our data suggest that the type of KRAS mutation may be of clinical relevance under oxaliplatin combination chemotherapies without the addition of monoclonal antibodies in particular when overall response rates are important. Trial registration number 2002-04-017 PMID:22876876

Mitochondrial genetic codes evolve to match amino acid requirements of proteins.

PubMed

Swire, Jonathan; Judson, Olivia P; Burt, Austin

2005-01-01

Mitochondria often use genetic codes different from the standard genetic code. Now that many mitochondrial genomes have been sequenced, these variant codes provide the first opportunity to examine empirically the processes that produce new genetic codes. The key question is: Are codon reassignments the sole result of mutation and genetic drift? Or are they the result of natural selection? Here we present an analysis of 24 phylogenetically independent codon reassignments in mitochondria. Although the mutation-drift hypothesis can explain reassignments from stop to an amino acid, we found that it cannot explain reassignments from one amino acid to another. In particular--and contrary to the predictions of the mutation-drift hypothesis--the codon involved in such a reassignment was not rare in the ancestral genome. Instead, such reassignments appear to take place while the codon is in use at an appreciable frequency. Moreover, the comparison of inferred amino acid usage in the ancestral genome with the neutral expectation shows that the amino acid gaining the codon was selectively favored over the amino acid losing the codon. These results are consistent with a simple model of weak selection on the amino acid composition of proteins in which codon reassignments are selected because they compensate for multiple slightly deleterious mutations throughout the mitochondrial genome. We propose that the selection pressure is for reduced protein synthesis cost: most reassignments give amino acids that are less expensive to synthesize. Taken together, our results strongly suggest that mitochondrial genetic codes evolve to match the amino acid requirements of proteins.

Modifications modulate anticodon loop dynamics and codon recognition of E. coli tRNA(Arg1,2).

PubMed

Cantara, William A; Bilbille, Yann; Kim, Jia; Kaiser, Rob; Leszczyńska, Grażyna; Malkiewicz, Andrzej; Agris, Paul F

2012-03-02

Three of six arginine codons are read by two tRNA(Arg) isoacceptors in Escherichia coli. The anticodon stem and loop of these isoacceptors (ASL(Arg1,2)) differs only in that the position 32 cytidine of tRNA(Arg1) is posttranscriptionally modified to 2-thiocytidine (s(2)C(32)). The tRNA(Arg1,2) are also modified at positions 34 (inosine, I(34)) and 37 (2-methyladenosine, m(2)A(37)). To investigate the roles of modifications in the structure and function, we analyzed six ASL(Arg1,2) constructs differing in their array of modifications by spectroscopy and codon binding assays. Thermal denaturation and circular dichroism spectroscopy indicated that modifications contribute thermodynamic and base stacking properties, resulting in more order but less stability. NMR-derived structures of the ASL(Arg1,2) showed that the solution structures of the ASLs were nearly identical. Surprisingly, none possessed the U-turn conformation required for effective codon binding on the ribosome. Yet, all ASL(Arg1,2) constructs efficiently bound the cognate CGU codon. Three ASLs with I(34) were able to decode CGC, whereas only the singly modified ASL(Arg1,2)(ICG) with I(34) was able to decode CGA. The dissociation constants for all codon bindings were physiologically relevant (0.4-1.4 μM). However, with the introduction of s(2)C(32) or m(2)A(37) to ASL(Arg1,2)(ICG), the maximum amount of ASL bound to CGU and CGC was significantly reduced. These results suggest that, by allowing loop flexibility, the modifications modulate the conformation of the ASL(Arg1,2), which takes one structure free in solution and two others when bound to the cognate arginyl-tRNA synthetase or to codons on the ribosome where modifications reduce or restrict binding to specific codons. Copyright © 2011 Elsevier Ltd. All rights reserved.

Genetic and codon usage bias analyses of polymerase genes of equine influenza virus and its relation to evolution.

PubMed

Bera, Bidhan Ch; Virmani, Nitin; Kumar, Naveen; Anand, Taruna; Pavulraj, S; Rash, Adam; Elton, Debra; Rash, Nicola; Bhatia, Sandeep; Sood, Richa; Singh, Raj Kumar; Tripathi, Bhupendra Nath

2017-08-23

Equine influenza is a major health problem of equines worldwide. The polymerase genes of influenza virus have key roles in virus replication, transcription, transmission between hosts and pathogenesis. Hence, the comprehensive genetic and codon usage bias of polymerase genes of equine influenza virus (EIV) were analyzed to elucidate the genetic and evolutionary relationships in a novel perspective. The group - specific consensus amino acid substitutions were identified in all polymerase genes of EIVs that led to divergence of EIVs into various clades. The consistent amino acid changes were also detected in the Florida clade 2 EIVs circulating in Europe and Asia since 2007. To study the codon usage patterns, a total of 281,324 codons of polymerase genes of EIV H3N8 isolates from 1963 to 2015 were systemically analyzed. The polymerase genes of EIVs exhibit a weak codon usage bias. The ENc-GC3s and Neutrality plots indicated that natural selection is the major influencing factor of codon usage bias, and that the impact of mutation pressure is comparatively minor. The methods for estimating host imposed translation pressure suggested that the polymerase acidic (PA) gene seems to be under less translational pressure compared to polymerase basic 1 (PB1) and polymerase basic 2 (PB2) genes. The multivariate statistical analysis of polymerase genes divided EIVs into four evolutionary diverged clusters - Pre-divergent, Eurasian, Florida sub-lineage 1 and 2. Various lineage specific amino acid substitutions observed in all polymerase genes of EIVs and especially, clade 2 EIVs underwent major variations which led to the emergence of a phylogenetically distinct group of EIVs originating from Richmond/1/07. The codon usage bias was low in all the polymerase genes of EIVs that was influenced by the multiple factors such as the nucleotide compositions, mutation pressure, aromaticity and hydropathicity. However, natural selection was the major influencing factor in defining the codon usage patterns and evolution of polymerase genes of EIVs.

Evidence for Context-Dependent Complementarity of Non-Shine-Dalgarno Ribosome Binding Sites to Escherichia coli rRNA

PubMed Central

Barendt, Pamela A.; Shah, Najaf A.; Barendt, Gregory A.; Kothari, Parth A.; Sarkar, Casim A.

2013-01-01

While the ribosome has evolved to function in complex intracellular environments, these contexts do not easily allow for the study of its inherent capabilities. We have used a synthetic, well-defined, Escherichia coli (E. coli)-based translation system in conjunction with ribosome display, a powerful in vitro selection method, to identify ribosome binding sites (RBSs) that can promote the efficient translation of messenger RNAs (mRNAs) with a leader length representative of natural E. coli mRNAs. In previous work, we used a longer leader sequence and unexpectedly recovered highly efficient cytosine-rich sequences with complementarity to the 16S ribosomal RNA (rRNA) and similarity to eukaryotic RBSs. In the current study, Shine-Dalgarno (SD) sequences were prevalent but non-SD sequences were also heavily enriched and were dominated by novel guanine- and uracil-rich motifs which showed statistically significant complementarity to the 16S rRNA. Additionally, only SD motifs exhibited position-dependent decreases in sequence entropy, indicating that non-SD motifs likely operate by increasing the local concentration of ribosomes in the vicinity of the start codon, rather than by a position-dependent mechanism. These results further support the putative generality of mRNA-rRNA complementarity in facilitating mRNA translation, but also suggest that context (e.g., leader length and composition) dictates the specific subset of possible RBSs that are used for efficient translation of a given transcript. PMID:23427812

Simultaneous identification of 36 mutations in KRAS codons 61and 146, BRAF, NRAS, and PIK3CA in a single reaction by multiplex assay kit

PubMed Central

2013-01-01

Background Retrospective analyses in the West suggest that mutations in KRAS codons 61 and 146, BRAF, NRAS, and PIK3CA are negative predictive factors for cetuximab treatment in colorectal cancer patients. We developed a novel multiplex kit detecting 36 mutations in KRAS codons 61 and 146, BRAF, NRAS, and PIK3CA using Luminex (xMAP) assay in a single reaction. Methods Tumor samples and clinical data from Asian colorectal cancer patients treated with cetuximab were collected. We investigated KRAS, BRAF, NRAS, and PIK3CA mutations using both the multiplex kit and direct sequencing methods, and evaluated the concordance between the 2 methods. Objective response, progression-free survival (PFS), and overall survival (OS) were also evaluated according to mutational status. Results In total, 82 of 83 samples (78 surgically resected specimens and 5 biopsy specimens) were analyzed using both methods. All multiplex assays were performed using 50 ng of template DNA. The concordance rate between the methods was 100%. Overall, 49 (59.8%) patients had all wild-type tumors, 21 (25.6%) had tumors harboring KRAS codon 12 or 13 mutations, and 12 (14.6%) had tumors harboring KRAS codon 61, KRAS codon 146, BRAF, NRAS, or PIK3CA mutations. The response rates in these patient groups were 38.8%, 4.8%, and 0%, respectively. Median PFS in these groups was 6.1 months (95% confidence interval (CI): 3.1–9.2), 2.7 months (1.2–4.2), and 1.6 months (1.5–1.7); median OS was 13.8 months (9.2–18.4), 8.2 months (5.7–10.7), and 6.3 months (1.3–11.3), respectively. Statistically significant differences in both PFS and OS were found between patients with all wild-type tumors and those with KRAS codon 61, KRAS codon 146, BRAF, NRAS, or PIK3CA mutations (PFS: 95% CI, 0.11–0.44; P < 0.0001; OS: 95% CI, 0.15–0.61; P < 0.0001). Conclusions Our newly developed multiplex kit is practical and feasible for investigation of a range of sample types. Moreover, mutations in KRAS codon 61, KRAS codon 146, BRAF, NRAS, or PIK3CA detected in Asian patients were not predictive of clinical benefits from cetuximab treatment, similar to the result obtained in European studies. PMID:24006859

Red Fluorescent Proteins for Gene Expression and Protein Localization Studies in Streptococcus pneumoniae and Efficient Transformation with DNA Assembled via the Gibson Assembly Method.

PubMed

Beilharz, Katrin; van Raaphorst, Renske; Kjos, Morten; Veening, Jan-Willem

2015-10-01

During the last decades, a wide range of fluorescent proteins (FPs) have been developed and improved. This has had a great impact on the possibilities in biological imaging and the investigation of cellular processes at the single-cell level. Recently, we have benchmarked a set of green fluorescent proteins (GFPs) and generated a codon-optimized superfolder GFP for efficient use in the important human pathogen Streptococcus pneumoniae and other low-GC Gram-positive bacteria. In the present work, we constructed and compared four red fluorescent proteins (RFPs) in S. pneumoniae. Two orange-red variants, mOrange2 and TagRFP, and two far-red FPs, mKate2 and mCherry, were codon optimized and examined by fluorescence microscopy and plate reader assays. Notably, protein fusions of the RFPs to FtsZ were constructed by direct transformation of linear Gibson assembly (isothermal assembly) products, a method that speeds up the strain construction process significantly. Our data show that mCherry is the fastest-maturing RFP in S. pneumoniae and is best suited for studying gene expression, while mKate2 and TagRFP are more stable and are the preferred choices for protein localization studies. The RFPs described here will be useful for cell biology studies that require multicolor labeling in S. pneumoniae and related organisms. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Oligo kernels for datamining on biological sequences: a case study on prokaryotic translation initiation sites

PubMed Central

Meinicke, Peter; Tech, Maike; Morgenstern, Burkhard; Merkl, Rainer

2004-01-01

Background Kernel-based learning algorithms are among the most advanced machine learning methods and have been successfully applied to a variety of sequence classification tasks within the field of bioinformatics. Conventional kernels utilized so far do not provide an easy interpretation of the learnt representations in terms of positional and compositional variability of the underlying biological signals. Results We propose a kernel-based approach to datamining on biological sequences. With our method it is possible to model and analyze positional variability of oligomers of any length in a natural way. On one hand this is achieved by mapping the sequences to an intuitive but high-dimensional feature space, well-suited for interpretation of the learnt models. On the other hand, by means of the kernel trick we can provide a general learning algorithm for that high-dimensional representation because all required statistics can be computed without performing an explicit feature space mapping of the sequences. By introducing a kernel parameter that controls the degree of position-dependency, our feature space representation can be tailored to the characteristics of the biological problem at hand. A regularized learning scheme enables application even to biological problems for which only small sets of example sequences are available. Our approach includes a visualization method for transparent representation of characteristic sequence features. Thereby importance of features can be measured in terms of discriminative strength with respect to classification of the underlying sequences. To demonstrate and validate our concept on a biochemically well-defined case, we analyze E. coli translation initiation sites in order to show that we can find biologically relevant signals. For that case, our results clearly show that the Shine-Dalgarno sequence is the most important signal upstream a start codon. The variability in position and composition we found for that signal is in accordance with previous biological knowledge. We also find evidence for signals downstream of the start codon, previously introduced as transcriptional enhancers. These signals are mainly characterized by occurrences of adenine in a region of about 4 nucleotides next to the start codon. Conclusions We showed that the oligo kernel can provide a valuable tool for the analysis of relevant signals in biological sequences. In the case of translation initiation sites we could clearly deduce the most discriminative motifs and their positional variation from example sequences. Attractive features of our approach are its flexibility with respect to oligomer length and position conservation. By means of these two parameters oligo kernels can easily be adapted to different biological problems. PMID:15511290

Strong Purifying Selection at Synonymous Sites in D. melanogaster

PubMed Central

Lawrie, David S.; Messer, Philipp W.; Hershberg, Ruth; Petrov, Dmitri A.

2013-01-01

Synonymous sites are generally assumed to be subject to weak selective constraint. For this reason, they are often neglected as a possible source of important functional variation. We use site frequency spectra from deep population sequencing data to show that, contrary to this expectation, 22% of four-fold synonymous (4D) sites in Drosophila melanogaster evolve under very strong selective constraint while few, if any, appear to be under weak constraint. Linking polymorphism with divergence data, we further find that the fraction of synonymous sites exposed to strong purifying selection is higher for those positions that show slower evolution on the Drosophila phylogeny. The function underlying the inferred strong constraint appears to be separate from splicing enhancers, nucleosome positioning, and the translational optimization generating canonical codon bias. The fraction of synonymous sites under strong constraint within a gene correlates well with gene expression, particularly in the mid-late embryo, pupae, and adult developmental stages. Genes enriched in strongly constrained synonymous sites tend to be particularly functionally important and are often involved in key developmental pathways. Given that the observed widespread constraint acting on synonymous sites is likely not limited to Drosophila, the role of synonymous sites in genetic disease and adaptation should be reevaluated. PMID:23737754

Analysis of amino acid and codon usage in Paramecium bursaria.

PubMed

Dohra, Hideo; Fujishima, Masahiro; Suzuki, Haruo

2015-10-07

The ciliate Paramecium bursaria harbors the green-alga Chlorella symbionts. We reassembled the P. bursaria transcriptome to minimize falsely fused transcripts, and investigated amino acid and codon usage using the transcriptome data. Surface proteins preferentially use smaller amino acid residues like cysteine. Unusual synonymous codon and amino acid usage in highly expressed genes can reflect a balance between translational selection and other factors. A correlation of gene expression level with synonymous codon or amino acid usage is emphasized in genes down-regulated in symbiont-bearing cells compared to symbiont-free cells. Our results imply that the selection is associated with P. bursaria-Chlorella symbiosis. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Role of a Novel I1781T Mutation and Other Mechanisms in Conferring Resistance to Acetyl-CoA Carboxylase Inhibiting Herbicides in a Black-Grass Population

PubMed Central

Kaundun, Shiv Shankhar; Hutchings, Sarah-Jane; Dale, Richard P.; McIndoe, Eddie

2013-01-01

Background Knowledge of the mechanisms of herbicide resistance is important for designing long term sustainable weed management strategies. Here, we have used an integrated biology and molecular approach to investigate the mechanisms of resistance to acetyl-CoA carboxylase inhibiting herbicides in a UK black-grass population (BG2). Methodology/Principal Findings Comparison between BG2 phenotypes using single discriminant rates of herbicides and genotypes based on ACCase gene sequencing showed that the I1781L, a novel I1781T, but not the W2027C mutations, were associated with resistance to cycloxydim. All plants were killed with clethodim and a few individuals containing the I1781L mutation were partially resistant to tepraloxydim. Whole plant dose response assays demonstrated that a single copy of the mutant T1781 allele conferred fourfold resistance levels to cycloxydim and clodinafop-propargyl. In contrast, the impact of the I1781T mutation was low (Rf = 1.6) and non-significant on pinoxaden. BG2 was also characterised by high levels of resistance, very likely non-target site based, to the two cereal selective herbicides clodinafop-propargyl and pinoxaden and not to the poorly metabolisable cyclohexanedione herbicides. Analysis of 480 plants from 40 cycloxydim resistant black grass populations from the UK using two very effective and high throughput dCAPS assays established for detecting any amino acid changes at the 1781 ACCase codon and for positively identifying the threonine residue, showed that the occurrence of the T1781 is extremely rare compared to the L1781 allele. Conclusion/Significance This study revealed a novel mutation at ACCase codon position 1781 and adequately assessed target site and non-target site mechanisms in conferring resistance to several ACCase herbicides in a black-grass population. It highlights that over time the level of suspected non-target site resistance to some cereal selective ACCase herbicides have in some instances surpassed that of target site resistance, including the one endowed by the most commonly encountered I1781L mutation. PMID:23936046

Adaptive molecular evolution of the two-pore channel 1 gene TPC1 in the karst-adapted genus Primulina (Gesneriaceae)

PubMed Central

Tao, Junjie; Feng, Chao; Ai, Bin; Kang, Ming

2016-01-01

Background and Aims Limestone karst areas possess high floral diversity and endemism. The genus Primulina, which contributes to the unique calcicole flora, has high species richness and exhibit specific soil-based habitat associations that are mainly distributed on calcareous karst soils. The adaptive molecular evolutionary mechanism of the genus to karst calcium-rich environments is still not well understood. The Ca2+-permeable channel TPC1 was used in this study to test whether its gene is involved in the local adaptation of Primulina to karst high-calcium soil environments. Methods Specific amplification and sequencing primers were designed and used to amplify the full-length coding sequences of TPC1 from cDNA of 76 Primulina species. The sequence alignment without recombination and the corresponding reconstructed phylogeny tree were used in molecular evolutionary analyses at the nucleic acid level and amino acid level, respectively. Finally, the identified sites under positive selection were labelled on the predicted secondary structure of TPC1. Key Results Seventy-six full-length coding sequences of Primulina TPC1 were obtained. The length of the sequences varied between 2220 and 2286 bp and the insertion/deletion was located at the 5′ end of the sequences. No signal of substitution saturation was detected in the sequences, while significant recombination breakpoints were detected. The molecular evolutionary analyses showed that TPC1 was dominated by purifying selection and the selective pressures were not significantly different among species lineages. However, significant signals of positive selection were detected at both TPC1 codon level and amino acid level, and five sites under positive selective pressure were identified by at least three different methods. Conclusions The Ca2+-permeable channel TPC1 may be involved in the local adaptation of Primulina to karst Ca2+-rich environments. Different species lineages suffered similar selective pressure associated with calcium in karst environments, and episodic diversifying selection at a few sites may play a major role in the molecular evolution of Primulina TPC1. PMID:27582362

Polymorphism in xeroderma pigmentosum complementation group C codon 939 and aflatoxin B1-related hepatocellular carcinoma in the Guangxi population.

PubMed

Long, Xi-Dai; Ma, Yun; Zhou, Yuan-Feng; Ma, Ai-Min; Fu, Guo-Hui

2010-10-01

Genetic polymorphisms in DNA repair genes may influence individual variations in DNA repair capacity, and this may be associated with the risk and outcome of hepatocellular carcinoma (HCC) related to aflatoxin B1 (AFB1) exposure. In this study, we focused on the polymorphism of xeroderma pigmentosum complementation group C (XPC) codon 939 (rs#2228001), which is involved in nucleotide excision repair. We conducted a case-control study including 1156 HCC cases and 1402 controls without any evidence of hepatic disease to evaluate the associations between this polymorphism and HCC risk and prognosis in the Guangxi population. AFB1 DNA adduct levels, XPC genotypes, and XPC protein levels were tested with a comparative enzyme-linked immunosorbent assay, TaqMan polymerase chain reaction for XPC genotypes, and immunohistochemistry, respectively. Higher AFB1 exposure was observed among HCC patients versus the control group [odds ratio (OR) = 9.88 for AFB1 exposure years and OR = 6.58 for AFB1 exposure levels]. The XPC codon 939 Gln alleles significantly increased HCC risk [OR = 1.25 (95% confidence interval = 1.03-1.52) for heterozygotes of the XPC codon 939 Lys and Gln alleles (XPC-LG) and OR = 1.81 (95% confidence interval = 1.36-2.40) for homozygotes of the XPC codon 939 Gln alleles (XPC-GG)]. Significant interactive effects between genotypes and AFB1 exposure status were also observed in the joint-effects analysis. This polymorphism, moreover, was correlated with XPC expression levels in cancerous tissues (r = -0.369, P < 0.001) and with the overall survival of HCC patients (the median survival times were 30, 25, and 19 months for patients with homozygotes of the XPC codon 939 Lys alleles, XPC-LG, and XPC-GG, respectively), especially under high AFB1 exposure conditions. Like AFB1 exposure, the XPC codon 939 polymorphism was an independent prognostic factor influencing the survival of HCC. Additionally, this polymorphism multiplicatively interacted with the xeroderma pigmentosum complementation group D codon 751 polymorphism with respect to HCC risk (OR(interaction) = 1.71). These results suggest that the XPC codon 939 polymorphism may be associated with the risk and outcome of AFB1-related HCC in the Guangxi population and may interact with AFB1 exposure in the process of HCC induction by AFB1.

Molecular Characterization of β-Thalassemia Mutations in Central Vietnam.

PubMed

Doro, Maria G; Casu, Giuseppina; Frogheri, Laura; Persico, Ivana; Triet, Le Phan Minh; Hoa, Phan Thi Thuy; Hoang, Nguyen Huy; Pirastru, Monica; Mereu, Paolo; Cucca, Francesco; Masala, Bruno

2017-03-01

The molecular basis of β-thalassemia (β-thal) mutations in North and in South Vietnam have been described during the past 15 years, whereas limited data were available concerning the central area of the country. In this study, we describe the molecular characterization and frequency of β-globin gene mutations in the Thua Thien Hue Province of Central Vietnam as the result of a first survey conducted in 22 transfusion-dependent patients, and four unrelated heterozygotes. Nine different known mutations were identified (seven of the β 0 and two of the β + type) in a total of 48 chromosomes. The most common was codon 26 (G>A) or Hb E (HBB: c.79 G>A) accounting for 29.2% of the total studied chromosomes, followed by codon 17 (A>T) (HBB: c.52 A>T) (25.0%), and codons 41/42 (-TTCT) (HBB: c.126_129delCTTT) (18.8%). Other mutations with appreciable frequencies (6.3-8.3%) were IVS-I-1 (G>T) (HBB: c.92+1 G>T), codon 26 (G>T) (HBB: c.79 G>T) and codons 71/72 (+A) (HBB: c.216_217insA). Relatively rarer (2.0%) were the promoter -28 (A>G) (HBB: c.78 A>G) mutation, the codon 95 (+A) (HBB: c.287_288insA), which is reported only in the Vietnamese, and the codons 14/15 (+G) (HBB: c.45_46insG) mutation, thus far observed only in Thailand. Results are relevant for implementing appropriate measures for β-thal prevention and control in the region as well as in the whole country.

Hand gesture recognition by analysis of codons

NASA Astrophysics Data System (ADS)

Ramachandra, Poornima; Shrikhande, Neelima

2007-09-01

The problem of recognizing gestures from images using computers can be approached by closely understanding how the human brain tackles it. A full fledged gesture recognition system will substitute mouse and keyboards completely. Humans can recognize most gestures by looking at the characteristic external shape or the silhouette of the fingers. Many previous techniques to recognize gestures dealt with motion and geometric features of hands. In this thesis gestures are recognized by the Codon-list pattern extracted from the object contour. All edges of an image are described in terms of sequence of Codons. The Codons are defined in terms of the relationship between maxima, minima and zeros of curvature encountered as one traverses the boundary of the object. We have concentrated on a catalog of 24 gesture images from the American Sign Language alphabet (Letter J and Z are ignored as they are represented using motion) [2]. The query image given as an input to the system is analyzed and tested against the Codon-lists, which are shape descriptors for external parts of a hand gesture. We have used the Weighted Frequency Indexing Transform (WFIT) approach which is used in DNA sequence matching for matching the Codon-lists. The matching algorithm consists of two steps: 1) the query sequences are converted to short sequences and are assigned weights and, 2) all the sequences of query gestures are pruned into match and mismatch subsequences by the frequency indexing tree based on the weights of the subsequences. The Codon sequences with the most weight are used to determine the most precise match. Once a match is found, the identified gesture and corresponding interpretation are shown as output.

The impact of KRAS mutations on VEGF-A production and tumour vascular network

PubMed Central

2013-01-01

Background The malignant potential of tumour cells may be influenced by the molecular nature of KRAS mutations being codon 13 mutations less aggressive than codon 12 ones. Their metabolic profile is also different, with an increased anaerobic glycolytic metabolism in cells harbouring codon 12 KRAS mutations compared with cells containing codon 13 mutations. We hypothesized that this distinct metabolic behaviour could be associated with different HIF-1α expression and a distinct angiogenic profile. Methods Codon13 KRAS mutation (ASP13) or codon12 KRAS mutation (CYS12) NIH3T3 transfectants were analyzed in vitro and in vivo. Expression of HIF-1α, and VEGF-A was studied at RNA and protein levels. Regulation of VEGF-A promoter activity was assessed by means of luciferase assays using different plasmid constructs. Vascular network was assessed in tumors growing after subcutaneous inoculation. Non parametric statistics were used for analysis of results. Results Our results show that in normoxic conditions ASP13 transfectants exhibited less HIF-1α protein levels and activity than CYS12. In contrast, codon 13 transfectants exhibited higher VEGF-A mRNA and protein levels and enhanced VEGF-A promoter activity. These differences were due to a differential activation of Sp1/AP2 transcription elements of the VEGF-A promoter associated with increased ERKs signalling in ASP13 transfectants. Subcutaneous CYS12 tumours expressed less VEGF-A and showed a higher microvessel density (MVD) than ASP13 tumours. In contrast, prominent vessels were only observed in the latter. Conclusion Subtle changes in the molecular nature of KRAS oncogene activating mutations occurring in tumour cells have a major impact on the vascular strategy devised providing with new insights on the role of KRAS mutations on angiogenesis. PMID:23506169

Analysis of phylogeny and codon usage bias and relationship of GC content, amino acid composition with expression of the structural nif genes.

PubMed

Mondal, Sunil Kanti; Kundu, Sudip; Das, Rabindranath; Roy, Sujit

2016-08-01

Bacteria and archaea have evolved with the ability to fix atmospheric dinitrogen in the form of ammonia, catalyzed by the nitrogenase enzyme complex which comprises three structural genes nifK, nifD and nifH. The nifK and nifD encodes for the beta and alpha subunits, respectively, of component 1, while nifH encodes for component 2 of nitrogenase. Phylogeny based on nifDHK have indicated that Cyanobacteria is closer to Proteobacteria alpha and gamma but not supported by the tree based on 16SrRNA. The evolutionary ancestor for the different trees was also different. The GC1 and GC2% analysis showed more consistency than GC3% which appeared to below for Firmicutes, Cyanobacteria and Euarchaeota while highest in Proteobacteria beta and clearly showed the proportional effect on the codon usage with a few exceptions. Few genes from Firmicutes, Euryarchaeota, Proteobacteria alpha and delta were found under mutational pressure. These nif genes with low and high GC3% from different classes of organisms showed similar expected number of codons. Distribution of the genes and codons, based on codon usage demonstrated opposite pattern for different orientation of mirror plane when compared with each other. Overall our results provide a comprehensive analysis on the evolutionary relationship of the three structural nif genes, nifK, nifD and nifH, respectively, in the context of codon usage bias, GC content relationship and amino acid composition of the encoded proteins and exploration of crucial statistical method for the analysis of positive data with non-constant variance to identify the shape factors of codon adaptation index.

Negative and Translation Termination-Dependent Positive Control of FLI-1 Protein Synthesis by Conserved Overlapping 5′ Upstream Open Reading Frames in Fli-1 mRNA

PubMed Central

Sarrazin, Sandrine; Starck, Joëlle; Gonnet, Colette; Doubeikovski, Alexandre; Melet, Fabrice; Morle, François

2000-01-01

The proto-oncogene Fli-1 encodes a transcription factor of the ets family whose overexpression is associated with multiple virally induced leukemias in mouse, inhibits murine and avian erythroid cell differentiation, and induces drastic perturbations of early development in Xenopus. This study demonstrates the surprisingly sophisticated regulation of Fli-1 mRNA translation. We establish that two FLI-1 protein isoforms (of 51 and 48 kDa) detected by Western blotting in vivo are synthesized by alternative translation initiation through the use of two highly conserved in-frame initiation codons, AUG +1 and AUG +100. Furthermore, we show that the synthesis of these two FLI-1 isoforms is regulated by two short overlapping 5′ upstream open reading frames (uORF) beginning at two highly conserved upstream initiation codons, AUG −41 and GUG −37, and terminating at two highly conserved stop codons, UGA +35 and UAA +15. The mutational analysis of these two 5′ uORF revealed that each of them negatively regulates FLI-1 protein synthesis by precluding cap-dependent scanning to the 48- and 51-kDa AUG codons. Simultaneously, the translation termination of the two 5′ uORF appears to enhance 48-kDa protein synthesis, by allowing downstream reinitiation at the 48-kDa AUG codon, and 51-kDa protein synthesis, by allowing scanning ribosomes to pile up and consequently allowing upstream initiation at the 51-kDa AUG codon. To our knowledge, this is the first example of a cellular mRNA displaying overlapping 5′ uORF whose translation termination appears to be involved in the positive control of translation initiation at both downstream and upstream initiation codons. PMID:10757781

Increasing the fidelity of noncanonical amino acid incorporation in cell-free protein synthesis.

PubMed

Gan, Qinglei; Fan, Chenguang

2017-11-01

Cell-free protein synthesis provides a robust platform for co-translational incorporation of noncanonical amino acid (ncAA) into proteins to facilitate biological studies and biotechnological applications. Recently, eliminating the activity of release factor 1 has been shown to increase ncAA incorporation in response to amber codons. However, this approach could promote mis-incorporation of canonical amino acids by near cognate suppression. We performed a facile protocol to remove near cognate tRNA isoacceptors of the amber codon from total tRNAs, and used the phosphoserine (Sep) incorporation system as validation. By manipulating codon usage of target genes and tRNA species introduced into the cell-free protein synthesis system, we increased the fidelity of Sep incorporation at a specific position. By removing three near cognate tRNA isoacceptors of the amber stop codon [tRNA Lys , tRNA Tyr , and tRNA Gln (CUG)] from the total tRNA, the near cognate suppression decreased by 5-fold without impairing normal protein synthesis in the cell-free protein synthesis system. Mass spectrometry analyses indicated that the fidelity of ncAA incorporation was improved. Removal of near cognate tRNA isoacceptors of the amber codon could increase ncAA incorporation fidelity towards the amber stop codon in release factor deficiency systems. We provide a general strategy to improve fidelity of ncAA incorporation towards stop, quadruplet and sense codons in cell-free protein synthesis systems. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2016 Elsevier B.V. All rights reserved.

Genotyping of beta thalassemia trait by high-resolution DNA melting analysis.

PubMed

Saetung, Rattika; Ongchai, Siriwan; Charoenkwan, Pimlak; Sanguansermsri, Torpong

2013-11-01

Beta thalassemia is a common hereditary hemalogogical disease in Thailand, with a prevalence of 5-8%. In this study, we evaluated the high resolution DNA melting (HRM) assay to identify beta thalassemia mutation in samples from 143 carriers of the beta thalassemia traits in at risk couples. The DNA was isolated from venous blood samples and tested for mutation under a series of 5 PCR-HRM (A, B, C, D and E primers) protocols. The A primers were for detection of beta thalassemia mutations in the HBB promoter region, the B primers for mutations in exon I, the C primers for exon II, the D primers for exon III and the E primers for the 3.4 kb deletion mutation. The mutations were diagnosed by comparing the complete melting curve profiles of a wild type control with those for each mutant sample. With the PCR-HRM technique, fourteen types of beta thalassemia mutations were detected. Each mutation had a unique and specific melting profile. The mutations included 36.4% (52 cases) codon 41/42-CTTT, 26.6% (38 cases) codon 17 A-T, 11.2% (16 cases) IVS1-1 G-T, 8.4% (12 cases) codon 71/72 +A, 8.4% (12 cases) of the 3.4 kb deletion and 3.5% (5 cases) -28 A-G. The remainder included one instance each of -87 C-A, -31 A-C, codon 27/28 +C, codon 30 G-A, IVS1-5 G-C, codon 35 C-A, codon 41-C and IVSII -654 C-T. Of the total cases, 85.8% of the mutations could be detected by primers B and C. The PCR-HRM method provides a rapid, simple and highly feasible strategy for mutation screening of beta thalassemia traits.

A Rapid Method to Test for Chloroplast DNA Involvement in Atrazine Resistance

PubMed Central

McNally, Sheila; Bettini, Priscilla; Sevignac, Mireille; Darmency, Henry; Gasquez, Jacques; Dron, Michel

1987-01-01

A point mutation in the chloroplast psbA gene at codon 264 resulting in an animo acid substitution (ser-gly) manifests itself as atrazine resistance in all recognized weed species studied to date. The single base substitution overlaps a highly conserved Mae1 restriction site which is present in susceptible but not in resistant plants. This restriction enzyme, recently commercialized, has been used to show that it is now possible to discriminate rapidly between the two biotypes without the need for DNA sequencing. Images Fig. 1 PMID:16665229

An expanded genetic code in mammalian cells with a functional quadruplet codon.

PubMed

Niu, Wei; Schultz, Peter G; Guo, Jiantao

2013-07-19

We have utilized in vitro evolution to identify tRNA variants with significantly enhanced activity for the incorporation of unnatural amino acids into proteins in response to a quadruplet codon in both bacterial and mammalian cells. This approach will facilitate the creation of an optimized and standardized system for the genetic incorporation of unnatural amino acids using quadruplet codons, which will allow the biosynthesis of biopolymers that contain multiple unnatural building blocks.

Regions of extreme synonymous codon selection in mammalian genes

PubMed Central

Schattner, Peter; Diekhans, Mark

2006-01-01

Recently there has been increasing evidence that purifying selection occurs among synonymous codons in mammalian genes. This selection appears to be a consequence of either cis-regulatory motifs, such as exonic splicing enhancers (ESEs), or mRNA secondary structures, being superimposed on the coding sequence of the gene. We have developed a program to identify regions likely to be enriched for such motifs by searching for extended regions of extreme codon conservation between homologous genes of related species. Here we present the results of applying this approach to five mammalian species (human, chimpanzee, mouse, rat and dog). Even with very conservative selection criteria, we find over 200 regions of extreme codon conservation, ranging in length from 60 to 178 codons. The regions are often found within genes involved in DNA-binding, RNA-binding or zinc-ion-binding. They are highly depleted for synonymous single nucleotide polymorphisms (SNPs) but not for non-synonymous SNPs, further indicating that the observed codon conservation is being driven by negative selection. Forty-three percent of the regions overlap conserved alternative transcript isoforms and are enriched for known ESEs. Other regions are enriched for TpA dinucleotides and may contain conserved motifs/structures relating to mRNA stability and/or degradation. We anticipate that this tool will be useful for detecting regions enriched in other classes of coding-sequence motifs and structures as well. PMID:16556911

Effects of tRNA modification on translational accuracy depend on intrinsic codon-anticodon strength.

PubMed

Manickam, Nandini; Joshi, Kartikeya; Bhatt, Monika J; Farabaugh, Philip J

2016-02-29

Cellular health and growth requires protein synthesis to be both efficient to ensure sufficient production, and accurate to avoid producing defective or unstable proteins. The background of misreading error frequency by individual tRNAs is as low as 2 × 10(-6) per codon but is codon-specific with some error frequencies above 10(-3) per codon. Here we test the effect on error frequency of blocking post-transcriptional modifications of the anticodon loops of four tRNAs in Escherichia coli. We find two types of responses to removing modification. Blocking modification of tRNA(UUC)(Glu) and tRNA(QUC)(Asp) increases errors, suggesting that the modifications act at least in part to maintain accuracy. Blocking even identical modifications of tRNA(UUU)(Lys) and tRNA(QUA)(Tyr) has the opposite effect of decreasing errors. One explanation could be that the modifications play opposite roles in modulating misreading by the two classes of tRNAs. Given available evidence that modifications help preorder the anticodon to allow it to recognize the codons, however, the simpler explanation is that unmodified 'weak' tRNAs decode too inefficiently to compete against cognate tRNAs that normally decode target codons, which would reduce the frequency of misreading. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

The effect of tRNA levels on decoding times of mRNA codons.

PubMed

Dana, Alexandra; Tuller, Tamir

2014-08-01

The possible effect of transfer ribonucleic acid (tRNA) concentrations on codons decoding time is a fundamental biomedical research question; however, due to a large number of variables affecting this process and the non-direct relation between them, a conclusive answer to this question has eluded so far researchers in the field. In this study, we perform a novel analysis of the ribosome profiling data of four organisms which enables ranking the decoding times of different codons while filtering translational phenomena such as experimental biases, extreme ribosomal pauses and ribosome traffic jams. Based on this filtering, we show for the first time that there is a significant correlation between tRNA concentrations and the codons estimated decoding time both in prokaryotes and in eukaryotes in natural conditions (-0.38 to -0.66, all P values <0.006); in addition, we show that when considering tRNA concentrations, codons decoding times are not correlated with aminoacyl-tRNA levels. The reported results support the conjecture that translation efficiency is directly influenced by the tRNA levels in the cell. Thus, they should help to understand the evolution of synonymous aspects of coding sequences via the adaptation of their codons to the tRNA pool. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Genotype-specific signal generation based on digestion of 3-way DNA junctions: application to KRAS variation detection.

PubMed

Amicarelli, Giulia; Adlerstein, Daniel; Shehi, Erlet; Wang, Fengfei; Makrigiorgos, G Mike

2006-10-01

Genotyping methods that reveal single-nucleotide differences are useful for a wide range of applications. We used digestion of 3-way DNA junctions in a novel technology, OneCutEventAmplificatioN (OCEAN) that allows sequence-specific signal generation and amplification. We combined OCEAN with peptide-nucleic-acid (PNA)-based variant enrichment to detect and simultaneously genotype v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) codon 12 sequence variants in human tissue specimens. We analyzed KRAS codon 12 sequence variants in 106 lung cancer surgical specimens. We conducted a PNA-PCR reaction that suppresses wild-type KRAS amplification and genotyped the product with a set of OCEAN reactions carried out in fluorescence microplate format. The isothermal OCEAN assay enabled a 3-way DNA junction to form between the specific target nucleic acid, a fluorescently labeled "amplifier", and an "anchor". The amplifier-anchor contact contains the recognition site for a restriction enzyme. Digestion produces a cleaved amplifier and generation of a fluorescent signal. The cleaved amplifier dissociates from the 3-way DNA junction, allowing a new amplifier to bind and propagate the reaction. The system detected and genotyped KRAS sequence variants down to approximately 0.3% variant-to-wild-type alleles. PNA-PCR/OCEAN had a concordance rate with PNA-PCR/sequencing of 93% to 98%, depending on the exact implementation. Concordance rate with restriction endonuclease-mediated selective-PCR/sequencing was 89%. OCEAN is a practical and low-cost novel technology for sequence-specific signal generation. Reliable analysis of KRAS sequence alterations in human specimens circumvents the requirement for sequencing. Application is expected in genotyping KRAS codon 12 sequence variants in surgical specimens or in bodily fluids, as well as single-base variations and sequence alterations in other genes.

Molecular Characterization of the Complete Genome of Three Basal-BR Isolates of Turnip mosaic virus Infecting Raphanus sativus in China.

PubMed

Zhu, Fuxiang; Sun, Ying; Wang, Yan; Pan, Hongyu; Wang, Fengting; Zhang, Xianghui; Zhang, Yanhua; Liu, Jinliang

2016-06-04

Turnip mosaic virus (TuMV) infects crops of plant species in the family Brassicaceae worldwide. TuMV isolates were clustered to five lineages corresponding to basal-B, basal-BR, Asian-BR, world-B and OMs. Here, we determined the complete genome sequences of three TuMV basal-BR isolates infecting radish from Shandong and Jilin Provinces in China. Their genomes were all composed of 9833 nucleotides, excluding the 3'-terminal poly(A) tail. They contained two open reading frames (ORFs), with the large one encoding a polyprotein of 3164 amino acids and the small overlapping ORF encoding a PIPO protein of 61 amino acids, which contained the typically conserved motifs found in members of the genus Potyvirus. In pairwise comparison with 30 other TuMV genome sequences, these three isolates shared their highest identities with isolates from Eurasian countries (Germany, Italy, Turkey and China). Recombination analysis showed that the three isolates in this study had no "clear" recombination. The analyses of conserved amino acids changed between groups showed that the codons in the TuMV out group (OGp) and OMs group were the same at three codon sites (852, 1006, 1548), and the other TuMV groups (basal-B, basal-BR, Asian-BR, world-B) were different. This pattern suggests that the codon in the OMs progenitor did not change but that in the other TuMV groups the progenitor sequence did change at divergence. Genetic diversity analyses indicate that the PIPO gene was under the highest selection pressure and the selection pressure on P3N-PIPO and P3 was almost the same. It suggests that most of the selection pressure on P3 was probably imposed through P3N-PIPO.

An initiator codon mutation in SDE2 causes recessive embryonic lethality in Holstein cattle.

PubMed

Fritz, Sébastien; Hoze, Chris; Rebours, Emmanuelle; Barbat, Anne; Bizard, Méline; Chamberlain, Amanda; Escouflaire, Clémentine; Vander Jagt, Christy; Boussaha, Mekki; Grohs, Cécile; Allais-Bonnet, Aurélie; Philippe, Maëlle; Vallée, Amélie; Amigues, Yves; Hayes, Benjamin J; Boichard, Didier; Capitan, Aurélien

2018-04-18

Researching depletions in homozygous genotypes for specific haplotypes among the large cohorts of animals genotyped for genomic selection is a very efficient strategy to map recessive lethal mutations. In this study, by analyzing real or imputed Illumina BovineSNP50 (Illumina Inc., San Diego, CA) genotypes from more than 250,000 Holstein animals, we identified a new locus called HH6 showing significant negative effects on conception rate and nonreturn rate at 56 d in at-risk versus control mating. We fine-mapped this locus in a 1.1-Mb interval and analyzed genome sequence data from 12 carrier and 284 noncarrier Holstein bulls. We report the identification of a strong candidate mutation in the gene encoding SDE2 telomere maintenance homolog (SDE2), a protein essential for genomic stability in eukaryotes. This A-to-G transition changes the initiator ATG (methionine) codon to ACG because the gene is transcribed on the reverse strand. Using RNA sequencing and quantitative reverse-transcription PCR, we demonstrated that this mutation does not significantly affect SDE2 splicing and expression level in heterozygous carriers compared with control animals. Initiation of translation at the closest in-frame methionine codon would truncate the SDE2 precursor by 83 amino acids, including the cleavage site necessary for its activation. Finally, no homozygote for the G allele was observed in a large population of nearly 29,000 individuals genotyped for the mutation. The low frequency (1.3%) of the derived allele in the French population and the availability of a diagnostic test on the Illumina EuroG10K SNP chip routinely used for genomic evaluation will enable rapid and efficient selection against this deleterious mutation. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Alignment-based and alignment-free methods converge with experimental data on amino acids coded by stop codons at split between nuclear and mitochondrial genetic codes.

PubMed

Seligmann, Hervé

2018-05-01

Genetic codes mainly evolve by reassigning punctuation codons, starts and stops. Previous analyses assuming that undefined amino acids translate stops showed greater divergence between nuclear and mitochondrial genetic codes. Here, three independent methods converge on which amino acids translated stops at split between nuclear and mitochondrial genetic codes: (a) alignment-free genetic code comparisons inserting different amino acids at stops; (b) alignment-based blast analyses of hypothetical peptides translated from non-coding mitochondrial sequences, inserting different amino acids at stops; (c) biases in amino acid insertions at stops in proteomic data. Hence short-term protein evolution models reconstruct long-term genetic code evolution. Mitochondria reassign stops to amino acids otherwise inserted at stops by codon-anticodon mismatches (near-cognate tRNAs). Hence dual function (translation termination and translation by codon-anticodon mismatch) precedes mitochondrial reassignments of stops to amino acids. Stop ambiguity increases coded information, compensates endocellular mitogenome reduction. Mitochondrial codon reassignments might prevent viral infections. Copyright © 2018 Elsevier B.V. All rights reserved.

Complete mitochondrial genome of Chocolate Pansy, Junonia iphita (Lepidoptera: Nymphalidae: Nymphalinae).

PubMed

Vanlalruati, Catherine; Mandal, Surajit De; Gurusubramanian, Guruswami; Senthil Kumar, Nachimuthu

2016-07-01

The complete mitochondrial genome of Junonia iphita was determined to be 15,433 bp in length, including 37 typical mitochondrial genes and an AT-rich region. All the protein coding genes (PCGs) are initiated by typical ATN codons, except cox1 gene that is by CGA codon. Eight genes use complete termination codon (TAA), whereas the cox1, cox2 and nad5 genes end with single T; nad4 and nad1 ends with stop codon TA. All the tRNA show secondary cloverleaf structures except trnS1 (AGN). The A + T rich region is 546 bp in length containing ATAGA motif followed by a 18 bp poly-T stretch, two microsatellite-like (TA)9 elements and 8 bp poly-A stretch immediately upstream of trnM gene.

Entomologic and Virologic Investigation of Chikungunya, Singapore

PubMed Central

Tan, Li-Kiang; Tan, Cheong-Huat; Tan, Sharon S.Y.; Hapuarachchi, Hapuarachchige C.; Pok, Kwoon-Yong; Lai, Yee-Ling; Lam-Phua, Sai-Gek; Bucht, Göran; Lin, Raymond T.P.; Leo, Yee-Sin; Tan, Boon-Hian; Han, Hwi-Kwang; Ooi, Peng-Lim S; James, Lyn; Khoo, Seow-Poh

2009-01-01

Local transmission of chikungunya, a debilitating mosquito-borne viral disease, was first reported in Singapore in January 2008. After 3 months of absence, locally acquired Chikungunya cases resurfaced in May 2008, causing an outbreak that resulted in a total of 231 cases by September 2008. The circulating viruses were related to East, Central, and South African genotypes that emerged in the Indian Ocean region in 2005. The first local outbreak was due to a wild-type virus (alanine at codon 226 of the envelope 1 gene) and occurred in an area where Aedes aegypti mosquitoes were the primary vector. Strains isolated during subsequent outbreaks showed alanine to valine substitution (A226V) and largely spread in areas predominated by Ae. albopictus mosquitoes. These findings led to a revision of the current vector control strategy in Singapore. This report highlights the use of entomologic and virologic data to assist in the control of chikungunya in disease-endemic areas. PMID:19751586

Entomologic and virologic investigation of Chikungunya, Singapore.

PubMed

Ng, Lee-Ching; Tan, Li-Kiang; Tan, Cheong-Huat; Tan, Sharon S Y; Hapuarachchi, Hapuarachchige C; Pok, Kwoon-Yong; Lai, Yee-Ling; Lam-Phua, Sai-Gek; Bucht, Göran; Lin, Raymond T P; Leo, Yee-Sin; Tan, Boon-Hian; Han, Hwi-Kwang; Ooi, Peng-Lim S; James, Lyn; Khoo, Seow-Poh

2009-08-01

Local transmission of chikungunya, a debilitating mosquito-borne viral disease, was first reported in Singapore in January 2008. After 3 months of absence, locally acquired Chikungunya cases resurfaced in May 2008, causing an outbreak that resulted in a total of 231 cases by September 2008. The circulating viruses were related to East, Central, and South African genotypes that emerged in the Indian Ocean region in 2005. The first local outbreak was due to a wild-type virus (alanine at codon 226 of the envelope 1 gene) and occurred in an area where Aedes aegypti mosquitoes were the primary vector. Strains isolated during subsequent outbreaks showed alanine to valine substitution (A226V) and largely spread in areas predominated by Ae. albopictus mosquitoes. These findings led to a revision of the current vector control strategy in Singapore. This report highlights the use of entomologic and virologic data to assist in the control of chikungunya in disease-endemic areas.

Genomic adaptation of the ISA virus to Salmo salar codon usage

PubMed Central

2013-01-01

Background The ISA virus (ISAV) is an Orthomyxovirus whose genome encodes for at least 10 proteins. Low protein identity and lack of genetic tools have hampered the study of the molecular mechanism behind its virulence. It has been shown that viral codon usage controls several processes such as translational efficiency, folding, tuning of protein expression, antigenicity and virulence. Despite this, the possible role that adaptation to host codon usage plays in virulence and viral evolution has not been studied in ISAV. Methods Intergenomic adaptation between viral and host genomes was calculated using the codon adaptation index score with EMBOSS software and the Kazusa database. Classification of host genes according to GeneOnthology was performed using Blast2go. A non parametric test was applied to determine the presence of significant correlations among CAI, mortality and time. Results Using the codon adaptation index (CAI) score, we found that the encoding genes for nucleoprotein, matrix protein M1 and antagonist of Interferon I signaling (NS1) are the ISAV genes that are more adapted to host codon usage, in agreement with their requirement for production of viral particles and inactivation of antiviral responses. Comparison to host genes showed that ISAV shares CAI values with less than 0.45% of Salmo salar genes. GeneOntology classification of host genes showed that ISAV genes share CAI values with genes from less than 3% of the host biological process, far from the 14% shown by Influenza A viruses and closer to the 5% shown by Influenza B and C. As well, we identified a positive correlation (p<0.05) between CAI values of a virus and the duration of the outbreak disease in given salmon farms, as well as a weak relationship between codon adaptation values of PB1 and the mortality rates of a set of ISA viruses. Conclusions Our analysis shows that ISAV is the least adapted viral Salmo salar pathogen and Orthomyxovirus family member less adapted to host codon usage, avoiding the general behavior of host genes. This is probably due to its recent emergence among farmed Salmon populations. PMID:23829271

Genomic adaptation of the ISA virus to Salmo salar codon usage.

PubMed

Tello, Mario; Vergara, Francisco; Spencer, Eugenio

2013-07-05

The ISA virus (ISAV) is an Orthomyxovirus whose genome encodes for at least 10 proteins. Low protein identity and lack of genetic tools have hampered the study of the molecular mechanism behind its virulence. It has been shown that viral codon usage controls several processes such as translational efficiency, folding, tuning of protein expression, antigenicity and virulence. Despite this, the possible role that adaptation to host codon usage plays in virulence and viral evolution has not been studied in ISAV. Intergenomic adaptation between viral and host genomes was calculated using the codon adaptation index score with EMBOSS software and the Kazusa database. Classification of host genes according to GeneOnthology was performed using Blast2go. A non parametric test was applied to determine the presence of significant correlations among CAI, mortality and time. Using the codon adaptation index (CAI) score, we found that the encoding genes for nucleoprotein, matrix protein M1 and antagonist of Interferon I signaling (NS1) are the ISAV genes that are more adapted to host codon usage, in agreement with their requirement for production of viral particles and inactivation of antiviral responses. Comparison to host genes showed that ISAV shares CAI values with less than 0.45% of Salmo salar genes. GeneOntology classification of host genes showed that ISAV genes share CAI values with genes from less than 3% of the host biological process, far from the 14% shown by Influenza A viruses and closer to the 5% shown by Influenza B and C. As well, we identified a positive correlation (p<0.05) between CAI values of a virus and the duration of the outbreak disease in given salmon farms, as well as a weak relationship between codon adaptation values of PB1 and the mortality rates of a set of ISA viruses. Our analysis shows that ISAV is the least adapted viral Salmo salar pathogen and Orthomyxovirus family member less adapted to host codon usage, avoiding the general behavior of host genes. This is probably due to its recent emergence among farmed Salmon populations.

On the Origin of Sequence

PubMed Central

van der Gulik, Peter T. S.

2015-01-01

Three aspects which make planet Earth special, and which must be taken in consideration with respect to the emergence of peptides, are the mineralogical composition, the Moon which is in the same size class, and the triple environment consisting of ocean, atmosphere, and continent. GlyGly is a remarkable peptide because it stimulates peptide bond formation in the Salt-Induced Peptide Formation reaction. The role glycine and aspartic acid play in the active site of RNA polymerase is remarkable too. GlyGly might have been the original product of coded peptide synthesis because of its importance in stimulating the production of oligopeptides with a high aspartic acid content, which protected small RNA molecules by binding Mg2+ ions. The feedback loop, which is closed by having RNA molecules producing GlyGly, is proposed as the essential element fundamental to life. Having this system running, longer sequences could evolve, gradually solving the problem of error catastrophe. The basic structure of the standard genetic code (8 fourfold degenerate codon boxes and 8 split codon boxes) is an example of the way information concerning the emergence of life is frozen in the biological constitution of organisms: the structure of the code contains historical information. PMID:26580656

New Insights into the Fructosyltransferase Activity of Schwanniomyces occidentalis β-Fructofuranosidase, Emerging from Nonconventional Codon Usage and Directed Mutation▿

PubMed Central

Álvaro-Benito, Miguel; de Abreu, Miguel; Portillo, Francisco; Sanz-Aparicio, Julia; Fernández-Lobato, María

2010-01-01

Schwanniomyces occidentalis β-fructofuranosidase (Ffase) releases β-fructose from the nonreducing ends of β-fructans and synthesizes 6-kestose and 1-kestose, both considered prebiotic fructooligosaccharides. Analyzing the amino acid sequence of this protein revealed that it includes a serine instead of a leucine at position 196, caused by a nonuniversal decoding of the unique mRNA leucine codon CUG. Substitution of leucine for Ser196 dramatically lowers the apparent catalytic efficiency (kcat/Km) of the enzyme (approximately 1,000-fold), but surprisingly, its transferase activity is enhanced by almost 3-fold, as is the enzymes' specificity for 6-kestose synthesis. The influence of 6 Ffase residues on enzyme activity was analyzed on both the Leu196/Ser196 backgrounds (Trp47, Asn49, Asn52, Ser111, Lys181, and Pro232). Only N52S and P232V mutations improved the transferase activity of the wild-type enzyme (about 1.6-fold). Modeling the transfructosylation products into the active site, in combination with an analysis of the kinetics and transfructosylation reactions, defined a new region responsible for the transferase specificity of the enzyme. PMID:20851958

Homology between DNA polymerases of poxviruses, herpesviruses, and adenoviruses: nucleotide sequence of the vaccinia virus DNA polymerase gene.

PubMed Central

Earl, P L; Jones, E V; Moss, B

1986-01-01

A 5400-base-pair segment of the vaccinia virus genome was sequenced and an open reading frame of 938 codons was found precisely where the DNA polymerase had been mapped by transfer of a phosphonoacetate-resistance marker. A single nucleotide substitution changing glycine at position 347 to aspartic acid accounts for the drug resistance of the mutant vaccinia virus. The 5' end of the DNA polymerase mRNA was located 80 base pairs before the methionine codon initiating the open reading frame. Correspondence between the predicted Mr 108,577 polypeptide and the 110,000 purified enzyme indicates that little or no proteolytic processing occurs. Extensive homology, extending over 435 amino acids, was found upon comparing the DNA polymerase of vaccinia virus and DNA polymerase of Epstein-Barr virus. A highly conserved sequence of 14 amino acids in the carboxyl-terminal regions of the above DNA polymerases is also present at a similar location in adenovirus DNA polymerase. This structure, which is predicted to form a turn flanked by beta-pleated sheets, may form part of an essential binding or catalytic site that accounts for its presence in DNA polymerases of poxviruses, herpesviruses, and adenoviruses. Images PMID:3012524

Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry.

PubMed

Siverino, Claudia; Tabisz, Barbara; Lühmann, Tessa; Meinel, Lorenz; Müller, Thomas; Walles, Heike; Nickel, Joachim

2018-03-29

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present several limitations that have led to the use of biomaterials in combination with osteogenic growth factors, such as bone morphogenetic proteins (BMPs). Commonly used absorption or encapsulation methods require supra-physiological amounts of BMP2, typically resulting in a so-called initial burst release effect that provokes several severe adverse side effects. A possible strategy to overcome these problems would be to covalently couple the protein to the scaffold. Moreover, coupling should be performed in a site-specific manner in order to guarantee a reproducible product outcome. Therefore, we created a BMP2 variant, in which an artificial amino acid (propargyl-L-lysine) was introduced into the mature part of the BMP2 protein by codon usage expansion (BMP2-K3Plk). BMP2-K3Plk was coupled to functionalized beads through copper catalyzed azide-alkyne cycloaddition (CuAAC). The biological activity of the coupled BMP2-K3Plk was proven in vitro and the osteogenic activity of the BMP2-K3Plk-functionalized beads was proven in cell based assays. The functionalized beads in contact with C2C12 cells were able to induce alkaline phosphatase (ALP) expression in locally restricted proximity of the bead. Thus, by this technique, functionalized scaffolds can be produced that can trigger cell differentiation towards an osteogenic lineage. Additionally, lower BMP2 doses are sufficient due to the controlled orientation of site-directed coupled BMP2. With this method, BMPs are always exposed to their receptors on the cell surface in the appropriate orientation, which is not the case if the factors are coupled via non-site-directed coupling techniques. The product outcome is highly controllable and, thus, results in materials with homogeneous properties, improving their applicability for the repair of critical size bone defects.

A single U/C nucleotide substitution changing alanine to valine in the beet necrotic yellow vein virus P25 protein promotes increased virus accumulation in roots of mechanically inoculated, partially resistant sugar beet seedlings.

PubMed

Koenig, R; Loss, S; Specht, J; Varrelmann, M; Lüddecke, P; Deml, G

2009-03-01

Beet necrotic yellow vein virus (BNYVV) A type isolates E12 and S8, originating from areas where resistance-breaking had or had not been observed, respectively, served as starting material for studying the influence of sequence variations in BNYVV RNA 3 on virus accumulation in partially resistant sugar beet varieties. Sub-isolates containing only RNAs 1 and 2 were obtained by serial local lesion passages; biologically active cDNA clones were prepared for RNAs 3 which differed in their coding sequences for P25 aa 67, 68 and 129. Sugar beet seedlings were mechanically inoculated with RNA 1+2/RNA 3 pseudorecombinants. The origin of RNAs 1+2 had little influence on virus accumulation in rootlets. E12 RNA 3 coding for V(67)C(68)Y(129) P25, however, enabled a much higher virus accumulation than S8 RNA 3 coding for A(67)H(68)H(129) P25. Mutants revealed that this was due only to the V(67) 'GUU' codon as opposed to the A(67) 'GCU' codon.

Evolutionary Consequences of DNA Methylation in a Basal Metazoan

PubMed Central

Dixon, Groves B.; Bay, Line K.; Matz, Mikhail V.

2016-01-01

Gene body methylation (gbM) is an ancestral and widespread feature in Eukarya, yet its adaptive value and evolutionary implications remain unresolved. The occurrence of gbM within protein-coding sequences is particularly puzzling, because methylation causes cytosine hypermutability and hence is likely to produce deleterious amino acid substitutions. We investigate this enigma using an evolutionarily basal group of Metazoa, the stony corals (order Scleractinia, class Anthozoa, phylum Cnidaria). We show that patterns of coral gbM are similar to other invertebrate species, predicting wide and active transcription and slower sequence evolution. We also find a strong correlation between gbM and codon bias, resulting from systematic replacement of CpG bearing codons. We conclude that gbM has strong effects on codon evolution and speculate that this may influence establishment of optimal codons. PMID:27189563

Molecular investigations of β-thalassemic children in Erbil governorate

NASA Astrophysics Data System (ADS)

Hasan, Ahmad N.; Al-Attar, Mustafa S.

2017-09-01

The present work studies the molecular investigation of 40 thalassemic carriers using polymerase chain reaction. Forty thalassemic carriers who were registered and treated at Erbil thalassemic center and twenty apparently healthy children have been included in the present study. Ages of both groups ranged between 1-18 years. Four primers used to detect four different beta thalassemia mutations they were codon 8/9, codon 8, codon 41/42 and IVS-1-5. The two most common mutations detected among thalassemia group were Cd8/9 with 8 cases (20%) and Cd-8 with 6 cases (15%) followed by codon 41/42 with 4 cases (10%) which investigated and detected for the first time in Erbil governorate through the present study and finally IVS-1-5 with 3 cases (7.5%), while no any cases detected among control group.

Experience with the use of the Codonics Safe Label System(™) to improve labelling compliance of anaesthesia drugs.

PubMed

Ang, S B L; Hing, W C; Tung, S Y; Park, T

2014-07-01

The Codonics Safe Labeling System(™) (http://www.codonics.com/Products/SLS/flash/) is a piece of equipment that is able to barcode scan medications, read aloud the medication and the concentration and print a label of the appropriate concentration in the appropriate colour code. We decided to test this system in our facility to identify risks, benefits and usability. Our project comprised a baseline survey (25 anaesthesia cases during which 212 syringes were prepared from 223 drugs), an observational study (47 cases with 330 syringes prepared) and a user acceptability survey. The baseline compliance with all labelling requirements was 58%. In the observational study the compliance using the Codonics system was 98.6% versus 63.8% with conventional labelling. In the user acceptability survey the majority agreed the Codonics machine was easy to use, more legible and adhered with better security than the conventional preprinted label. However, most were neutral when asked about the likelihood of flexibility and customisation and were dissatisfied with the increased workload. Our findings suggest that the Codonics labelling machine is user-friendly and it improved syringe labelling compliance in our study. However, staff need to be willing to follow proper labelling workflow rather than batch label during preparation. Future syringe labelling equipment developers need to concentrate on user interface issues to reduce human factor and workflow problems. Support logistics are also an important consideration prior to implementation of any new labelling system.

Codon usage in Chlamydia trachomatis is the result of strand-specific mutational biases and a complex pattern of selective forces

PubMed Central

Romero, Héctor; Zavala, Alejandro; Musto, Héctor

2000-01-01

The patterns of synonymous codon choices of the completely sequenced genome of the bacterium Chlamydia trachomatis were analysed. We found that the most important source of variation among the genes results from whether the sequence is located on the leading or lagging strand of replication, resulting in an over representation of G or C, respectively. This can be explained by different mutational biases associated to the different enzymes that replicate each strand. Next we found that most highly expressed sequences are located on the leading strand of replication. From this result, replicational-transcriptional selection can be invoked. Then, when the genes located on the leading strand are studied separately, the correspondence analysis detects a principal trend which discriminates between lowly and highly expressed sequences, the latter displaying a different codon usage pattern than the former, suggesting selection for translation, which is reinforced by the fact that Ks values between orthologous sequences from C.trachomatis and Chlamydia pneumoniae are much smaller in highly expressed genes. Finally, synonymous codon choices appear to be influenced by the hydropathy of each encoded protein and by the degree of amino acid conservation. Therefore, synonymous codon usage in C.trachomatis seems to be the result of a very complex balance among different factors, which rises the problem of whether the forces driving codon usage patterns among microorganisms are rather more complex than generally accepted. PMID:10773076

Codon usage in Chlamydia trachomatis is the result of strand-specific mutational biases and a complex pattern of selective forces.

PubMed

Romero, H; Zavala, A; Musto, H

2000-05-15

The patterns of synonymous codon choices of the completely sequenced genome of the bacterium Chlamydia trachomatis were analysed. We found that the most important source of variation among the genes results from whether the sequence is located on the leading or lagging strand of replication, resulting in an over representation of G or C, respectively. This can be explained by different mutational biases associated to the different enzymes that replicate each strand. Next we found that most highly expressed sequences are located on the leading strand of replication. From this result, replicational-transcriptional selection can be invoked. Then, when the genes located on the leading strand are studied separately, the correspondence analysis detects a principal trend which discriminates between lowly and highly expressed sequences, the latter displaying a different codon usage pattern than the former, suggesting selection for translation, which is reinforced by the fact that Ks values between orthologous sequences from C. trachomatis and Chlamydia pneumoniae are much smaller in highly expressed genes. Finally, synonymous codon choices appear to be influenced by the hydropathy of each encoded protein and by the degree of amino acid conservation. Therefore, synonymous codon usage in C.trachomatis seems to be the result of a very complex balance among different factors, which rises the problem of whether the forces driving codon usage patterns among microorganisms are rather more complex than generally accepted.

Molecular Mechanism of Scanning and Start Codon Selection in Eukaryotes

PubMed Central

Hinnebusch, Alan G.

2011-01-01

Summary: The correct translation of mRNA depends critically on the ability to initiate at the right AUG codon. For most mRNAs in eukaryotic cells, this is accomplished by the scanning mechanism, wherein the small (40S) ribosomal subunit attaches to the 5′ end of the mRNA and then inspects the leader base by base for an AUG in a suitable context, using complementarity with the anticodon of methionyl initiator tRNA (Met-tRNAiMet) as the key means of identifying AUG. Over the past decade, a combination of yeast genetics, biochemical analysis in reconstituted systems, and structural biology has enabled great progress in deciphering the mechanism of ribosomal scanning. A robust molecular model now exists, describing the roles of initiation factors, notably eukaryotic initiation factor 1 (eIF1) and eIF1A, in stabilizing an “open” conformation of the 40S subunit with Met-tRNAiMet bound in a low-affinity state conducive to scanning and in triggering rearrangement into a “closed” conformation incompatible with scanning, which features Met-tRNAiMet more tightly bound to the “P” site and base paired with AUG. It has also emerged that multiple DEAD-box RNA helicases participate in producing a single-stranded “landing pad” for the 40S subunit and in removing the secondary structure to enable the mRNA to traverse the 40S mRNA-binding channel in the single-stranded form for base-by-base inspection in the P site. PMID:21885680

Major histocompatibility complex alleles associated with parasite susceptibility in wild giant pandas.

PubMed

Zhang, L; Wu, Q; Hu, Y; Wu, H; Wei, F

2015-01-01

Major histocompatibility complex (MHC) polymorphism is thought to be driven by antagonistic coevolution between pathogens and hosts, mediated through either overdominance or frequency-dependent selection. However, investigations under natural conditions are still rare for endangered mammals which often exhibit depleted variation, and the mechanism of selection underlying the maintenance of characteristics remains a considerable debate. In this study, 87 wild giant pandas were used to investigate MHC variation associated with parasite load. With the knowledge of the MHC profile provided by the genomic data of the giant panda, seven DRB1, seven DQA1 and eight DQA2 alleles were identified at each single locus. Positive selection evidenced by a significantly higher number of non-synonymous substitutions per non-synonymous codon site relative to synonymous substitutions per synonymous codon site could only be detected at the DRB1 locus, which leads to the speculation that DRB1 may have a more important role in dealing with parasite infection for pandas. Coprological analyses revealed that 55.17% of individuals exhibited infection with 1-2 helminthes and 95.3% of infected pandas carried Baylisascaris shroederi. Using a generalized linear model, we found that Aime-DRB1*10 was significantly associated with parasite infection, but no resistant alleles could be detected. MHC heterozygosity of the pandas was found to be uncorrelated with the infection status or the infection intensity. These results suggested that the possible selection mechanisms in extant wild pandas may be frequency dependent rather than being determined by overdominance selection. Our findings could guide the candidate selection for the ongoing reintroduction or translocation of pandas.

Major histocompatibility complex alleles associated with parasite susceptibility in wild giant pandas

PubMed Central

Zhang, L; Wu, Q; Hu, Y; Wu, H; Wei, F

2015-01-01

Major histocompatibility complex (MHC) polymorphism is thought to be driven by antagonistic coevolution between pathogens and hosts, mediated through either overdominance or frequency-dependent selection. However, investigations under natural conditions are still rare for endangered mammals which often exhibit depleted variation, and the mechanism of selection underlying the maintenance of characteristics remains a considerable debate. In this study, 87 wild giant pandas were used to investigate MHC variation associated with parasite load. With the knowledge of the MHC profile provided by the genomic data of the giant panda, seven DRB1, seven DQA1 and eight DQA2 alleles were identified at each single locus. Positive selection evidenced by a significantly higher number of non-synonymous substitutions per non-synonymous codon site relative to synonymous substitutions per synonymous codon site could only be detected at the DRB1 locus, which leads to the speculation that DRB1 may have a more important role in dealing with parasite infection for pandas. Coprological analyses revealed that 55.17% of individuals exhibited infection with 1–2 helminthes and 95.3% of infected pandas carried Baylisascaris shroederi. Using a generalized linear model, we found that Aime-DRB1*10 was significantly associated with parasite infection, but no resistant alleles could be detected. MHC heterozygosity of the pandas was found to be uncorrelated with the infection status or the infection intensity. These results suggested that the possible selection mechanisms in extant wild pandas may be frequency dependent rather than being determined by overdominance selection. Our findings could guide the candidate selection for the ongoing reintroduction or translocation of pandas. PMID:25248466

Structure-function analysis of Sua5 protein reveals novel functional motifs required for the biosynthesis of the universal t6A tRNA modification.

PubMed

Pichard-Kostuch, Adeline; Zhang, Wenhua; Liger, Dominique; Daugeron, Marie-Claire; Letoquart, Juliette; Li de la Sierra-Gallay, Ines; Forterre, Patrick; Collinet, Bruno; van Tilbeurgh, Herman; Basta, Tamara

2018-04-12

N6-threonyl-carbamoyl adenosine (t6A) is a universal tRNA modification found at position 37, next to the anticodon, in almost all tRNAs decoding ANN codons (where N = A, U, G or C). t6A stabilizes the codon-anticodon interaction and hence promotes translation fidelity. The first step of the biosynthesis of t6A, the production of threonyl-carbamoyl adenylate (TC-AMP), is catalyzed by the Sua5/TsaC family of enzymes. While TsaC is a single domain protein, Sua5 enzymes are composed of the TsaC-like domain, a linker and an extra domain called SUA5 of unknown function. In the present study, we report structure-function analysis of Pyrococcus abyssi Sua5 (Pa-Sua5). Crystallographic data revealed binding sites for bicarbonate substrate and pyrophosphate product. The linker of Pa-Sua5 forms a loop structure that folds into the active site gorge and closes it. Using structure-guided mutational analysis we established that the conserved sequence motifs in the linker and the domain-domain interface are essential for the function of Pa-Sua5. We propose that the linker participates actively in the biosynthesis of TC-AMP by binding to ATP/PPi and by stabilizing the N-carboxy-L-threonine intermediate. Hence, TsaC orthologs which lack such a linker and SUA5 domain use different mechanism for TC-AMP synthesis. Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Abolition of Peroxiredoxin-5 Mitochondrial Targeting during Canid Evolution

PubMed Central

Van der Eecken, Valérie; Clippe, André; Dekoninck, Sophie; Goemaere, Julie; Walbrecq, Geoffroy; Van Veldhoven, Paul P.; Knoops, Bernard

2013-01-01

In human, the subcellular targeting of peroxiredoxin-5 (PRDX5), a thioredoxin peroxidase, is dependent on the use of multiple alternative transcription start sites and two alternative in-frame translation initiation sites, which determine whether or not the region encoding a mitochondrial targeting sequence (MTS) is translated. In the present study, the abolition of PRDX5 mitochondrial targeting in dog is highlighted and the molecular mechanism underlying the loss of mitochondrial PRDX5 during evolution is examined. Here, we show that the absence of mitochondrial PRDX5 is generalized among the extant canids and that the first events leading to PRDX5 MTS abolition in canids involve a mutation in the more 5′ translation initiation codon as well as the appearance of a STOP codon. Furthermore, we found that PRDX5 MTS functionality is maintained in giant panda and northern elephant seal, which are phylogenetically closely related to canids. Also, the functional consequences of the restoration of mitochondrial PRDX5 in dog Madin-Darby canine kidney (MDCK) cells were investigated. The restoration of PRDX5 mitochondrial targeting in MDCK cells, instead of protecting, provokes deleterious effects following peroxide exposure independently of its peroxidase activity, indicating that mitochondrial PRDX5 gains cytotoxic properties under acute oxidative stress in MDCK cells. Altogether our results show that, although mitochondrial PRDX5 cytoprotective function against oxidative stress has been clearly demonstrated in human and rodents, PRDX5 targeting to mitochondria has been evolutionary lost in canids. Moreover, restoration of mitochondrial PRDX5 in dog MDCK cells, instead of conferring protection against peroxide exposure, makes them more vulnerable. PMID:24023783

Replacement of arginine 773 by cysteine or histidine in the human androgen receptor causes complete androgen insensitivity with different receptor phenotypes

PubMed Central

Prior, Lynn; Bordet, Sylvie; Trifiro, Mark A.; Mhatre, Anand; Kaufman, Morris; Pinsky, Leonard; Wrogeman, Klaus; Belsham, Denise D.; Pereira, Fred; Greenberg, Cheryl; Trapman, Jan; Brinkman, Albert O.; Chang, Chawnshang; Liao, Shutsung

1992-01-01

We have discovered two different point mutations in a single codon of the X-linked androgen-receptor (AR) gene in two pairs of unrelated families who have complete androgen insensitivity (resistance) associated with different AR phenotypes in their genital skin fibroblasts. One mutation is a C-to-T transition at a CpG sequence near the 5' terminus of exon 6; it changes the sense of codon 773 from arginine to cysteine, ablates specific androgen-binding activity at 37°C, and eliminates a unique KpnI site at the intron-exon boundary. The other mutation is a G-to-A transition that changes amino acid 773 to histidine and eliminates an SphI site. This mutant AR has a normal androgen-binding capacity at 37°C but has a reduced affinity for androgens and is thermolabile in their presence. Transient transfection of COS cells with cDNA expression vectors yielded little androgen-binding activity at 37°C from Arg773Cys and abundant activity with abnormal properties from Arg773His, thereby proving the pathogenicity of both sequence alterations. This conclusion coincides with the following facts about evolutionary preservation of the position homologous to Arg773 in the AR: it is occupied by Arg or lysine in the progesterone, glucocorticoid, and mineralocorticoid receptors, and it is within a 14-amino-acid region of their steroid-binding domains that share ∼85% amino acid identity. ImagesFigure 7Figure 2Figure 3Figure 5Figure 6Figure 8 PMID:1609793