Progress toward characterization of the group A Streptococcus metagenome: complete genome sequence of a macrolide-resistant serotype M6 strain.

PubMed

Banks, David J; Porcella, Stephen F; Barbian, Kent D; Beres, Stephen B; Philips, Lauren E; Voyich, Jovanka M; DeLeo, Frank R; Martin, Judith M; Somerville, Greg A; Musser, James M

2004-08-15

We describe the genome sequence of a macrolide-resistant strain (MGAS10394) of serotype M6 group A Streptococcus (GAS). The genome is 1,900,156 bp in length, and 8 prophage-like elements or remnants compose 12.4% of the chromosome. A 8.3-kb prophage remnant encodes the SpeA4 variant of streptococcal pyrogenic exotoxin A. The genome of strain MGAS10394 contains a chimeric genetic element composed of prophage genes and a transposon encoding the mefA gene conferring macrolide resistance. This chimeric element also has a gene encoding a novel surface-exposed protein (designated "R6 protein"), with an LPKTG cell-anchor motif located at the carboxyterminus. Surface expression of this protein was confirmed by flow cytometry. Humans with GAS pharyngitis caused by serotype M6 strains had antibody against the R6 protein present in convalescent, but not acute, serum samples. Our studies add to the theme that GAS prophage-encoded extracellular proteins contribute to host-pathogen interactions in a strain-specific fashion.

Isolation of the Chlamydomonas Regulatory Gene Nit2 by Transposon Tagging

PubMed Central

Schnell, R. A.; Lefebvre, P. A.

1993-01-01

Genetic evidence suggests that the NIT2 gene of Chlamydomonas reinhardtii encodes a positive regulator of the nitrate-assimilation pathway. To learn more about the function of the NIT2 gene product, we isolated the gene using a transposon-tagging strategy. A nit2 mutation caused by the insertion of a transposon was identified by testing spontaneous nit2 mutants for the presence of new copies of Gulliver or TOC1, transposable elements that have been identified in Chlamydomonas. In 2 of the 14 different mutants that were analyzed, a Gulliver element was found to be genetically and phenotypically associated with the nit2 mutation. Using the Gulliver element as a probe, one of the transposon-induced nit2 alleles was isolated, and a sequence adjoining the transposon was used to isolate the corresponding wild-type locus. The NIT2 gene was delimited by mapping DNA rearrangements associated with nit2 mutations and mutant rescue by genetic transformation. The NIT2 gene encodes a 6-kb transcript that was not detected in cells grown in the presence of ammonium. Likewise, NIT2-dependent genes are repressed in ammonium-grown cells. These results suggest that repression of the NIT2 gene may mediate metabolite repression of the nitrate assimilation pathway in Chlamydomonas. PMID:8394263

Comparative genomic and proteomic analyses of two Mycoplasma agalactiae strains: clues to the macro- and micro-events that are shaping mycoplasma diversity.

PubMed

Nouvel, Laurent X; Sirand-Pugnet, Pascal; Marenda, Marc S; Sagné, Eveline; Barbe, Valérie; Mangenot, Sophie; Schenowitz, Chantal; Jacob, Daniel; Barré, Aurélien; Claverol, Stéphane; Blanchard, Alain; Citti, Christine

2010-02-02

While the genomic era is accumulating a tremendous amount of data, the question of how genomics can describe a bacterial species remains to be fully addressed. The recent sequencing of the genome of the Mycoplasma agalactiae type strain has challenged our general view on mycoplasmas by suggesting that these simple bacteria are able to exchange significant amount of genetic material via horizontal gene transfer. Yet, events that are shaping mycoplasma genomes and that are underlining diversity within this species have to be fully evaluated. For this purpose, we compared two strains that are representative of the genetic spectrum encountered in this species: the type strain PG2 which genome is already available and a field strain, 5632, which was fully sequenced and annotated in this study. The two genomes differ by ca. 130 kbp with that of 5632 being the largest (1006 kbp). The make up of this additional genetic material mainly corresponds (i) to mobile genetic elements and (ii) to expanded repertoire of gene families that encode putative surface proteins and display features of highly-variable systems. More specifically, three entire copies of a previously described integrative conjugative element are found in 5632 that accounts for ca. 80 kbp. Other mobile genetic elements, found in 5632 but not in PG2, are the more classical insertion sequences which are related to those found in two other ruminant pathogens, M. bovis and M. mycoides subsp. mycoides SC. In 5632, repertoires of gene families encoding surface proteins are larger due to gene duplication. Comparative proteomic analyses of the two strains indicate that the additional coding capacity of 5632 affects the overall architecture of the surface and suggests the occurrence of new phase variable systems based on single nucleotide polymorphisms. Overall, comparative analyses of two M. agalactiae strains revealed a very dynamic genome which structure has been shaped by gene flow among ruminant mycoplasmas and expansion-reduction of gene repertoires encoding surface proteins, the expression of which is driven by localized genetic micro-events.

Comparative genomic and proteomic analyses of two Mycoplasma agalactiae strains: clues to the macro- and micro-events that are shaping mycoplasma diversity

PubMed Central

2010-01-01

Background While the genomic era is accumulating a tremendous amount of data, the question of how genomics can describe a bacterial species remains to be fully addressed. The recent sequencing of the genome of the Mycoplasma agalactiae type strain has challenged our general view on mycoplasmas by suggesting that these simple bacteria are able to exchange significant amount of genetic material via horizontal gene transfer. Yet, events that are shaping mycoplasma genomes and that are underlining diversity within this species have to be fully evaluated. For this purpose, we compared two strains that are representative of the genetic spectrum encountered in this species: the type strain PG2 which genome is already available and a field strain, 5632, which was fully sequenced and annotated in this study. Results The two genomes differ by ca. 130 kbp with that of 5632 being the largest (1006 kbp). The make up of this additional genetic material mainly corresponds (i) to mobile genetic elements and (ii) to expanded repertoire of gene families that encode putative surface proteins and display features of highly-variable systems. More specifically, three entire copies of a previously described integrative conjugative element are found in 5632 that accounts for ca. 80 kbp. Other mobile genetic elements, found in 5632 but not in PG2, are the more classical insertion sequences which are related to those found in two other ruminant pathogens, M. bovis and M. mycoides subsp. mycoides SC. In 5632, repertoires of gene families encoding surface proteins are larger due to gene duplication. Comparative proteomic analyses of the two strains indicate that the additional coding capacity of 5632 affects the overall architecture of the surface and suggests the occurrence of new phase variable systems based on single nucleotide polymorphisms. Conclusion Overall, comparative analyses of two M. agalactiae strains revealed a very dynamic genome which structure has been shaped by gene flow among ruminant mycoplasmas and expansion-reduction of gene repertoires encoding surface proteins, the expression of which is driven by localized genetic micro-events. PMID:20122262

Novel Type V Staphylococcal Cassette Chromosome mec Driven by a Novel Cassette Chromosome Recombinase, ccrC

PubMed Central

Ito, Teruyo; Ma, Xiao Xue; Takeuchi, Fumihiko; Okuma, Keiko; Yuzawa, Harumi; Hiramatsu, Keiichi

2004-01-01

Staphylococcal cassette chromosome mec (SCCmec) is a mobile genetic element composed of the mec gene complex, which encodes methicillin resistance, and the ccr gene complex, which encodes the recombinases responsible for its mobility. The mec gene complex has been classified into four classes, and the ccr gene complex has been classified into three allotypes. Different combinations of mec gene complex classes and ccr gene complex types have so far defined four types of SCCmec elements. Now we introduce the fifth allotype of SCCmec, which was found on the chromosome of a community-acquired methicillin-resistant Staphylococcus aureus strain (strain WIS [WBG8318]) isolated in Australia. The element shared the same chromosomal integration site with the four extant types of SCCmec and the characteristic nucleotide sequences at the chromosome-SCCmec junction regions. The novel SCCmec carried mecA bracketed by IS431 (IS431-mecA-ΔmecR1-IS431), which is designated the class C2 mec gene complex; and instead of ccrA and ccrB genes, it carried a single copy of a gene homologue that encoded cassette chromosome recombinase. Since the open reading frame (ORF) was found to encode an enzyme which catalyzes the precise excision as well as site- and orientation-specific integration of the element, we designated the ORF cassette chromosome recombinase C (ccrC), and we designated the element type V SCCmec. Type V SCCmec is a small SCCmec element (28 kb) and does not carry any antibiotic resistance genes besides mecA. Unlike the extant SCCmec types, it carries a set of foreign genes encoding a restriction-modification system that might play a role in the stabilization of the element on the chromosome. PMID:15215121

Regulation of Bacteriocin Production in Streptococcus mutans by the Quorum-Sensing System Required for Development of Genetic Competence

PubMed Central

van der Ploeg, Jan R.

2005-01-01

In Streptococcus mutans, competence for genetic transformation and biofilm formation are dependent on the two-component signal transduction system ComDE together with the inducer peptide pheromone competence-stimulating peptide (CSP) (encoded by comC). Here, it is shown that the same system is also required for expression of the nlmAB genes, which encode a two-peptide nonlantibiotic bacteriocin. Expression from a transcriptional nlmAB′-lacZ fusion was highest at high cell density and was increased up to 60-fold following addition of CSP, but it was abolished when the comDE genes were interrupted. Two more genes, encoding another putative bacteriocin and a putative bacteriocin immunity protein, were also regulated by this system. The regions upstream of these genes and of two further putative bacteriocin-encoding genes and a gene encoding a putative bacteriocin immunity protein contained a conserved 9-bp repeat element just upstream of the transcription start, which suggests that expression of these genes is also dependent on the ComCDE regulatory system. Mutations in the repeat element of the nlmAB promoter region led to a decrease in CSP-dependent expression of nlmAB′-lacZ. In agreement with these results, a comDE mutant and mutants unable to synthesize or export CSP did not produce bacteriocins. It is speculated that, at high cell density, bacteriocin production is induced to liberate DNA from competing streptococci. PMID:15937160

The endogenous transposable element Tgm9 is suitable for functional analyses of soybean genes and generating novel mutants for genetic improvement of soybean

USDA-ARS?s Scientific Manuscript database

In soybean, variegated flowers can be caused by somatic excision of the CACTA-type transposable element Tgm9 from intron 2 of the DFR2 gene encoding dihydroflavonol-4-reductase in the anthocyanin pigment biosynthetic pathway. DFR2 has been mapped to the W4 locus where the allele containing the elem...

ICE Afe 1, an actively excising genetic element from the biomining bacterium Acidithiobacillus ferrooxidans.

PubMed

Bustamante, Paula; Covarrubias, Paulo C; Levicán, Gloria; Katz, Assaf; Tapia, Pablo; Holmes, David; Quatrini, Raquel; Orellana, Omar

2012-01-01

Integrative conjugative elements (ICEs) are self-transferred mobile genetic elements that contribute to horizontal gene transfer. An ICE (ICEAfe1) was identified in the genome of Acidithiobacillus ferrooxidans ATCC 23270. Excision of the element and expression of relevant genes under normal and DNA-damaging growth conditions was analyzed. Bioinformatic tools and DNA amplification methods were used to identify and to assess the excision and expression of genes related to the mobility of the element. Both basal and mitomycin C-inducible excision as well as expression and induction of the genes for integration/excision are demonstrated, suggesting that ICEAfe1 is an actively excising SOS-regulated mobile genetic element. The presence of a complete set of genes encoding self-transfer functions that are induced in response to DNA damage caused by mitomycin C additionally suggests that this element is capable of conjugative transfer to suitable recipient strains. Transfer of ICEAfe1 may provide selective advantages to other acidophiles in this ecological niche through dissemination of gene clusters expressing transfer RNAs, CRISPRs, and exopolysaccharide biosynthesis enzymes, probably by modification of translation efficiency, resistance to bacteriophage infection and biofilm formation, respectively. These data open novel avenues of research on conjugative transformation of biotechnologically relevant microorganisms recalcitrant to genetic manipulation. Copyright © 2013 S. Karger AG, Basel.

Disease-associated variants in different categories of disease located in distinct regulatory elements.

PubMed

Ma, Meng; Ru, Ying; Chuang, Ling-Shiang; Hsu, Nai-Yun; Shi, Li-Song; Hakenberg, Jörg; Cheng, Wei-Yi; Uzilov, Andrew; Ding, Wei; Glicksberg, Benjamin S; Chen, Rong

2015-01-01

The invention of high throughput sequencing technologies has led to the discoveries of hundreds of thousands of genetic variants associated with thousands of human diseases. Many of these genetic variants are located outside the protein coding regions, and as such, it is challenging to interpret the function of these genetic variants by traditional genetic approaches. Recent genome-wide functional genomics studies, such as FANTOM5 and ENCODE have uncovered a large number of regulatory elements across hundreds of different tissues or cell lines in the human genome. These findings provide an opportunity to study the interaction between regulatory elements and disease-associated genetic variants. Identifying these diseased-related regulatory elements will shed light on understanding the mechanisms of how these variants regulate gene expression and ultimately result in disease formation and progression. In this study, we curated and categorized 27,558 Mendelian disease variants, 20,964 complex disease variants, 5,809 cancer predisposing germline variants, and 43,364 recurrent cancer somatic mutations. Compared against nine different types of regulatory regions from FANTOM5 and ENCODE projects, we found that different types of disease variants show distinctive propensity for particular regulatory elements. Mendelian disease variants and recurrent cancer somatic mutations are 22-fold and 10- fold significantly enriched in promoter regions respectively (q<0.001), compared with allele-frequency-matched genomic background. Separate from these two categories, cancer predisposing germline variants are 27-fold enriched in histone modification regions (q<0.001), 10-fold enriched in chromatin physical interaction regions (q<0.001), and 6-fold enriched in transcription promoters (q<0.001). Furthermore, Mendelian disease variants and recurrent cancer somatic mutations share very similar distribution across types of functional effects. We further found that regulatory regions are located within over 50% coding exon regions. Transcription promoters, methylation regions, and transcription insulators have the highest density of disease variants, with 472, 239, and 72 disease variants per one million base pairs, respectively. Disease-associated variants in different disease categories are preferentially located in particular regulatory elements. These results will be useful for an overall understanding about the differences among the pathogenic mechanisms of various disease-associated variants.

Disease-associated variants in different categories of disease located in distinct regulatory elements

PubMed Central

2015-01-01

Background The invention of high throughput sequencing technologies has led to the discoveries of hundreds of thousands of genetic variants associated with thousands of human diseases. Many of these genetic variants are located outside the protein coding regions, and as such, it is challenging to interpret the function of these genetic variants by traditional genetic approaches. Recent genome-wide functional genomics studies, such as FANTOM5 and ENCODE have uncovered a large number of regulatory elements across hundreds of different tissues or cell lines in the human genome. These findings provide an opportunity to study the interaction between regulatory elements and disease-associated genetic variants. Identifying these diseased-related regulatory elements will shed light on understanding the mechanisms of how these variants regulate gene expression and ultimately result in disease formation and progression. Results In this study, we curated and categorized 27,558 Mendelian disease variants, 20,964 complex disease variants, 5,809 cancer predisposing germline variants, and 43,364 recurrent cancer somatic mutations. Compared against nine different types of regulatory regions from FANTOM5 and ENCODE projects, we found that different types of disease variants show distinctive propensity for particular regulatory elements. Mendelian disease variants and recurrent cancer somatic mutations are 22-fold and 10- fold significantly enriched in promoter regions respectively (q<0.001), compared with allele-frequency-matched genomic background. Separate from these two categories, cancer predisposing germline variants are 27-fold enriched in histone modification regions (q<0.001), 10-fold enriched in chromatin physical interaction regions (q<0.001), and 6-fold enriched in transcription promoters (q<0.001). Furthermore, Mendelian disease variants and recurrent cancer somatic mutations share very similar distribution across types of functional effects. We further found that regulatory regions are located within over 50% coding exon regions. Transcription promoters, methylation regions, and transcription insulators have the highest density of disease variants, with 472, 239, and 72 disease variants per one million base pairs, respectively. Conclusions Disease-associated variants in different disease categories are preferentially located in particular regulatory elements. These results will be useful for an overall understanding about the differences among the pathogenic mechanisms of various disease-associated variants. PMID:26110593

Epidemiology and Characteristics of Metallo-β-Lactamase-Producing Pseudomonas aeruginosa

PubMed Central

Bae, Il Kwon; Jang, In-Ho; Kang, Hyun-Kyung; Lee, Kyungwon

2015-01-01

Metallo-β-lactamase-producing Pseudomonas aeruginosa (MPPA) is an important nosocomial pathogen that shows resistance to all β-lactam antibiotics except monobactams. There are various types of metallo-β-lactamases (MBLs) in carbapenem-resistant P. aeruginosa including Imipenemase (IMP), Verona integron-encoded metallo-β-lactamase (VIM), Sao Paulo metallo-β-lactamase (SPM), Germany imipenemase (GIM), New Delhi metallo-β-lactamase (NDM), Florence imipenemase (FIM). Each MBL gene is located on specific genetic elements including integrons, transposons, plasmids, or on the chromosome, in which they carry genes encoding determinants of resistance to carbapenems and other antibiotics, conferring multidrug resistance to P. aeruginosa. In addition, these genetic elements are transferable to other Gram-negative species, increasing the antimicrobial resistance rate and complicating the treatment of infected patients. Therefore, it is essential to understand the epidemiology, resistance mechanism, and molecular characteristics of MPPA for infection control and prevention of a possible global health crisis. Here, we highlight the characteristics of MPPA. PMID:26157586

A Locus Encoding Variable Defense Systems against Invading DNA Identified in Streptococcus suis

PubMed Central

Okura, Masatoshi; Nozawa, Takashi; Watanabe, Takayasu; Murase, Kazunori; Nakagawa, Ichiro; Takamatsu, Daisuke; Osaki, Makoto; Sekizaki, Tsutomu; Gottschalk, Marcelo; Hamada, Shigeyuki

2017-01-01

Streptococcus suis, an important zoonotic pathogen, is known to have an open pan-genome and to develop a competent state. In S. suis, limited genetic lineages are suggested to be associated with zoonosis. However, little is known about the evolution of diversified lineages and their respective phenotypic or ecological characteristics. In this study, we performed comparative genome analyses of S. suis, with a focus on the competence genes, mobile genetic elements, and genetic elements related to various defense systems against exogenous DNAs (defense elements) that are associated with gene gain/loss/exchange mediated by horizontal DNA movements and their restrictions. Our genome analyses revealed a conserved competence-inducing peptide type (pherotype) of the competence system and large-scale genome rearrangements in certain clusters based on the genome phylogeny of 58 S. suis strains. Moreover, the profiles of the defense elements were similar or identical to each other among the strains belonging to the same genomic clusters. Our findings suggest that these genetic characteristics of each cluster might exert specific effects on the phenotypic or ecological differences between the clusters. We also found certain loci that shift several types of defense elements in S. suis. Of note, one of these loci is a previously unrecognized variable region in bacteria, at which strains of distinct clusters code for different and various defense elements. This locus might represent a novel defense mechanism that has evolved through an arms race between bacteria and invading DNAs, mediated by mobile genetic elements and genetic competence. PMID:28379509

Construction of Nef-positive doxycycline-dependent HIV-1 variants using bicistronic expression elements

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

Velden, Yme U. van der; Kleibeuker, Wendy; Harwig, Alex

Conditionally replicating HIV-1 variants that can be switched on and off at will are attractive tools for HIV research. We previously developed a genetically modified HIV-1 variant that replicates exclusively when doxycycline (dox) is administered. The nef gene in this HIV-rtTA variant was replaced with the gene encoding the dox-dependent rtTA transcriptional activator. Because loss of Nef expression compromises virus replication in primary cells and precludes studies on Nef function, we tested different approaches to restore Nef production in HIV-rtTA. Strategies that involved translation via an EMCV or synthetic internal ribosome entry site (IRES) failed because these elements were incompatiblemore » with efficient virus replication. Fusion protein approaches with the FMDV 2A peptide and human ubiquitin were successful and resulted in genetically-stable Nef-expressing HIV-rtTA strains that replicate more efficiently in primary T-cells and human immune system (HIS) mice than Nef-deficient variants, thus confirming the positive effect of Nef on in vivo virus replication. - Highlights: • Different approaches to encode additional proteins in the HIV-1 genome were tested. • IRES translation elements are incompatible with efficient HIV-1 replication. • Ubiquitin and 2A fusion protein approaches allow efficient HIV-1 replication. • Doxycycline-controlled HIV-1 variants that encode all viral proteins were developed. • Nef stimulates HIV-rtTA replication in primary cells and human immune system mice.« less

The ethylene response pathway in Arabidopsis

NASA Technical Reports Server (NTRS)

Kieber, J. J.; Evans, M. L. (Principal Investigator)

1997-01-01

The simple gas ethylene influences a diverse array of plant growth and developmental processes including germination, senescence, cell elongation, and fruit ripening. This review focuses on recent molecular genetic studies, principally in Arabidopsis, in which components of the ethylene response pathway have been identified. The isolation and characterization of two of these genes has revealed that ethylene sensing involves a protein kinase cascade. One of these genes encodes a protein with similarity to the ubiquitous Raf family of Ser/Thr protein kinases. A second gene shows similarity to the prokaryotic two-component histidine kinases and most likely encodes an ethylene receptor. Additional elements involved in ethylene signaling have only been identified genetically. The characterization of these genes and mutants will be discussed.

Bacteriophages encode factors required for protection in a symbiotic mutualism.

PubMed

Oliver, Kerry M; Degnan, Patrick H; Hunter, Martha S; Moran, Nancy A

2009-08-21

Bacteriophages are known to carry key virulence factors for pathogenic bacteria, but their roles in symbiotic bacteria are less well understood. The heritable symbiont Hamiltonella defensa protects the aphid Acyrthosiphon pisum from attack by the parasitoid Aphidius ervi by killing developing wasp larvae. In a controlled genetic background, we show that a toxin-encoding bacteriophage is required to produce the protective phenotype. Phage loss occurs repeatedly in laboratory-held H. defensa-infected aphid clonal lines, resulting in increased susceptibility to parasitism in each instance. Our results show that these mobile genetic elements can endow a bacterial symbiont with benefits that extend to the animal host. Thus, phages vector ecologically important traits, such as defense against parasitoids, within and among symbiont and animal host lineages.

Nucleases Encoded by Integrated Elements CJIE2 and CJIE4 Inhibit Natural Transformation of Campylobacter jejuni

USDA-ARS?s Scientific Manuscript database

The species Campylobacter jejuni displays huge genetic diversity, and is naturally competent for DNA uptake. Nevertheless, not every strain is able to acquire foreign DNA since nonnaturally transformable strains do exist. Previously we showed that many nonnaturally transformable C. jejuni strains ex...

Draft genome sequences of 50 MRSA ST5 isolates obtained from a U.S. hospital

USDA-ARS?s Scientific Manuscript database

Methicillin resistant Staphylococcus aureus (MRSA) can be a commensal or pathogen in humans. Pathogenicity and disease are related to the acquisition of mobile genetic elements encoding virulence and antimicrobial resistance genes. Here, we report draft genome sequences for 50 clinical MRSA isolates...

Transfer of scarlet fever-associated elements into the group A Streptococcus M1T1 clone.

PubMed

Ben Zakour, Nouri L; Davies, Mark R; You, Yuanhai; Chen, Jonathan H K; Forde, Brian M; Stanton-Cook, Mitchell; Yang, Ruifu; Cui, Yujun; Barnett, Timothy C; Venturini, Carola; Ong, Cheryl-lynn Y; Tse, Herman; Dougan, Gordon; Zhang, Jianzhong; Yuen, Kwok-Yung; Beatson, Scott A; Walker, Mark J

2015-11-02

The group A Streptococcus (GAS) M1T1 clone emerged in the 1980s as a leading cause of epidemic invasive infections worldwide, including necrotizing fasciitis and toxic shock syndrome. Horizontal transfer of mobile genetic elements has played a central role in the evolution of the M1T1 clone, with bacteriophage-encoded determinants DNase Sda1 and superantigen SpeA2 contributing to enhanced virulence and colonization respectively. Outbreaks of scarlet fever in Hong Kong and China in 2011, caused primarily by emm12 GAS, led to our investigation of the next most common cause of scarlet fever, emm1 GAS. Genomic analysis of 18 emm1 isolates from Hong Kong and 16 emm1 isolates from mainland China revealed the presence of mobile genetic elements associated with the expansion of emm12 scarlet fever clones in the M1T1 genomic background. These mobile genetic elements confer expression of superantigens SSA and SpeC, and resistance to tetracycline, erythromycin and clindamycin. Horizontal transfer of mobile DNA conferring multi-drug resistance and expression of a new superantigen repertoire in the M1T1 clone should trigger heightened public health awareness for the global dissemination of these genetic elements.

Transfer of scarlet fever-associated elements into the group A Streptococcus M1T1 clone

PubMed Central

Ben Zakour, Nouri L.; Davies, Mark R.; You, Yuanhai; Chen, Jonathan H. K.; Forde, Brian M.; Stanton-Cook, Mitchell; Yang, Ruifu; Cui, Yujun; Barnett, Timothy C.; Venturini, Carola; Ong, Cheryl-lynn Y.; Tse, Herman; Dougan, Gordon; Zhang, Jianzhong; Yuen, Kwok-Yung; Beatson, Scott A.; Walker, Mark J.

2015-01-01

The group A Streptococcus (GAS) M1T1 clone emerged in the 1980s as a leading cause of epidemic invasive infections worldwide, including necrotizing fasciitis and toxic shock syndrome123. Horizontal transfer of mobile genetic elements has played a central role in the evolution of the M1T1 clone45, with bacteriophage-encoded determinants DNase Sda16 and superantigen SpeA27 contributing to enhanced virulence and colonization respectively. Outbreaks of scarlet fever in Hong Kong and China in 2011, caused primarily by emm12 GAS8910, led to our investigation of the next most common cause of scarlet fever, emm1 GAS89. Genomic analysis of 18 emm1 isolates from Hong Kong and 16 emm1 isolates from mainland China revealed the presence of mobile genetic elements associated with the expansion of emm12 scarlet fever clones1011 in the M1T1 genomic background. These mobile genetic elements confer expression of superantigens SSA and SpeC, and resistance to tetracycline, erythromycin and clindamycin. Horizontal transfer of mobile DNA conferring multi-drug resistance and expression of a new superantigen repertoire in the M1T1 clone should trigger heightened public health awareness for the global dissemination of these genetic elements. PMID:26522788

Type VI secretion systems of human gut Bacteroidales segregate into three genetic architectures, two of which are contained on mobile genetic elements.

PubMed

Coyne, Michael J; Roelofs, Kevin G; Comstock, Laurie E

2016-01-15

Type VI secretion systems (T6SSs) are contact-dependent antagonistic systems employed by Gram negative bacteria to intoxicate other bacteria or eukaryotic cells. T6SSs were recently discovered in a few Bacteroidetes strains, thereby extending the presence of these systems beyond Proteobacteria. The present study was designed to analyze in a global nature the diversity, abundance, and properties of T6SSs in the Bacteroidales, the most predominant Gram negative bacterial order of the human gut. By performing extensive bioinformatics analyses and creating hidden Markov models for Bacteroidales Tss proteins, we identified 130 T6SS loci in 205 human gut Bacteroidales genomes. Of the 13 core T6SS proteins of Proteobacteria, human gut Bacteroidales T6SS loci encode orthologs of nine, and an additional five other core proteins not present in Proteobacterial T6SSs. The Bacteroidales T6SS loci segregate into three distinct genetic architectures with extensive DNA identity between loci of a given genetic architecture. We found that divergent DNA regions of a genetic architecture encode numerous types of effector and immunity proteins and likely include new classes of these proteins. TheT6SS loci of genetic architecture 1 are contained on highly similar integrative conjugative elements (ICEs), as are the T6SS loci of genetic architecture 2, whereas the T6SS loci of genetic architecture 3 are not and are confined to Bacteroides fragilis. Using collections of co-resident Bacteroidales strains from human subjects, we provide evidence for the transfer of genetic architecture 1 T6SS loci among co-resident Bacteroidales species in the human gut. However, we also found that established ecosystems can harbor strains with distinct T6SS of all genetic architectures. This is the first study to comprehensively analyze of the presence and diversity of T6SS loci within an order of bacteria and to analyze T6SSs of bacteria from a natural community. These studies demonstrate that more than half of our gut Bacteroidales, equivalent to about ¼ of the bacteria of this ecosystem, encode T6SSs. The data reveal several novel properties of these systems and suggest that antagonism between or distributed defense among these abundant intestinal bacteria may be common in established human gut communities.

Developmental rearrangement of cyanobacterial nif genes: nucleotide sequence, open reading frames, and cytochrome P-450 homology of the Anabaena sp. strain PCC 7120 nifD element.

PubMed Central

Lammers, P J; McLaughlin, S; Papin, S; Trujillo-Provencio, C; Ryncarz, A J

1990-01-01

An 11-kbp DNA element of unknown function interrupts the nifD gene in vegetative cells of Anabaena sp. strain PCC 7120. In developing heterocysts the nifD element excises from the chromosome via site-specific recombination between short repeat sequences that flank the element. The nucleotide sequence of the nifH-proximal half of the element was determined to elucidate the genetic potential of the element. Four open reading frames with the same relative orientation as the nifD element-encoded xisA gene were identified in the sequenced region. Each of the open reading frames was preceded by a reasonable ribosome-binding site and had biased codon utilization preferences consistent with low levels of expression. Open reading frame 3 was highly homologous with three cytochrome P-450 omega-hydroxylase proteins and showed regional homology to functionally significant domains common to the cytochrome P-450 superfamily. The sequence encoding open reading frame 2 was the most highly conserved portion of the sequenced region based on heterologous hybridization experiments with three genera of heterocystous cyanobacteria. Images PMID:2123860

Long interspersed element-1 protein expression is a hallmark of many human cancers.

PubMed

Rodić, Nemanja; Sharma, Reema; Sharma, Rajni; Zampella, John; Dai, Lixin; Taylor, Martin S; Hruban, Ralph H; Iacobuzio-Donahue, Christine A; Maitra, Anirban; Torbenson, Michael S; Goggins, Michael; Shih, Ie-Ming; Duffield, Amy S; Montgomery, Elizabeth A; Gabrielson, Edward; Netto, George J; Lotan, Tamara L; De Marzo, Angelo M; Westra, William; Binder, Zev A; Orr, Brent A; Gallia, Gary L; Eberhart, Charles G; Boeke, Jef D; Harris, Chris R; Burns, Kathleen H

2014-05-01

Cancers comprise a heterogeneous group of human diseases. Unifying characteristics include unchecked abilities of tumor cells to proliferate and spread anatomically, and the presence of clonal advantageous genetic changes. However, universal and highly specific tumor markers are unknown. Herein, we report widespread long interspersed element-1 (LINE-1) repeat expression in human cancers. We show that nearly half of all human cancers are immunoreactive for a LINE-1-encoded protein. LINE-1 protein expression is a common feature of many types of high-grade malignant cancers, is rarely detected in early stages of tumorigenesis, and is absent from normal somatic tissues. Studies have shown that LINE-1 contributes to genetic changes in cancers, with somatic LINE-1 insertions seen in selected types of human cancers, particularly colon cancer. We sought to correlate this observation with expression of the LINE-1-encoded protein, open reading frame 1 protein, and found that LINE-1 open reading frame 1 protein is a surprisingly broad, yet highly tumor-specific, antigen. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Transcriptional analysis of the R locus: Progress report, September 1986 through October 1987

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

Wessler, S.R.

1987-11-01

The R locus controls where, when and how much anthocyanins are expressed in at least 11 different tissues of the corn plant and seed. Enormous natural variation has been seen when the phenotypes of different R alleles are compared in a common genetic background. Some alleles have been shown to have a compound structure resulting from gene duplication and divergence. In these complex alleles, each member of the duplication (called R genic elements) has a unique pattern of expression. The function of the R locus is not known; genetic and biochemical analyses suggest that it may encode a protein thatmore » regulates other genes in the anthocyanin pathway. Over the past year we have determined that the genic elements (P), (S), and (Lc) all encode a very rare 2.8 kb transcript that is present in tissue displaying anthocyanin pigmentation. cDNA libraries have been constructed using mRNA isolated from tissues shown by Northern blots to be enriched for the R transcript. Full-length cDNAs will be sequenced and compared to each other.« less

An analysis of mobile genetic elements in three Plasmodium species and their potential impact on the nucleotide composition of the P. falciparum genome.

PubMed

Durand, Pierre M; Oelofse, Andries J; Coetzer, Theresa L

2006-11-04

The completed genome sequences of the malaria parasites P. falciparum, P. y. yoelii and P. vivax have revealed some unusual features. P. falciparum contains the most AT rich genome sequenced so far--over 90% in some regions. In comparison, P. y. yoelii is approximately 77% and P. vivax is approximately 55% AT rich. The evolutionary reasons for these findings are unknown. Mobile genetic elements have a considerable impact on genome evolution but a thorough investigation of these elements in Plasmodium has not been undertaken. We therefore performed a comprehensive genome analysis of these elements and their derivatives in the three Plasmodium species. Whole genome analysis was performed using bioinformatic methods. Forty potential protein encoding sequences with features of transposable elements were identified in P. vivax, eight in P. y. yoelii and only six in P. falciparum. Further investigation of the six open reading frames in P. falciparum revealed that only one is potentially an active mobile genetic element. Most of the open reading frames identified in all three species are hypothetical proteins. Some represent annotated host proteins such as the putative telomerase reverse transcriptase genes in P. y. yoelii and P. falciparum. One of the P. vivax open reading frames identified in this study demonstrates similarity to telomerase reverse transcriptase and we conclude it to be the orthologue of this gene. There is a divergence in the frequencies of mobile genetic elements in the three Plasmodium species investigated. Despite the limitations of whole genome analytical methods, it is tempting to speculate that mobile genetic elements might have been a driving force behind the compositional bias of the P. falciparum genome.

Aquaculture changes the profile of antibiotic resistance and mobile genetic element associated genes in Baltic Sea sediments.

PubMed

Muziasari, Windi I; Pärnänen, Katariina; Johnson, Timothy A; Lyra, Christina; Karkman, Antti; Stedtfeld, Robert D; Tamminen, Manu; Tiedje, James M; Virta, Marko

2016-04-01

Antibiotics are commonly used in aquaculture and they can change the environmental resistome by increasing antibiotic resistance genes (ARGs). Sediment samples were collected from two fish farms located in the Northern Baltic Sea, Finland, and from a site outside the farms (control). The sediment resistome was assessed by using a highly parallel qPCR array containing 295 primer sets to detect ARGs, mobile genetic elements and the 16S rRNA gene. The fish farm resistomes were enriched in transposon and integron associated genes and in ARGs encoding resistance to antibiotics which had been used to treat fish at the farms. Aminoglycoside resistance genes were also enriched in the farm sediments despite the farms not having used aminoglycosides. In contrast, the total relative abundance values of ARGs were higher in the control sediment resistome and they were mainly genes encoding efflux pumps followed by beta-lactam resistance genes, which are found intrinsically in many bacteria. This suggests that there is a natural Baltic sediment resistome. The resistome associated with fish farms can be from native ARGs enriched by antibiotic use at the farms and/or from ARGs and mobile elements that have been introduced by fish farming. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Type 3 Fimbriae Encoded on Plasmids Are Expressed from a Unique Promoter without Affecting Host Motility, Facilitating an Exceptional Phenotype That Enhances Conjugal Plasmid Transfer

PubMed Central

Madsen, Jonas Stenløkke; Riber, Leise; Kot, Witold; Basfeld, Alrun; Burmølle, Mette; Hansen, Lars Hestbjerg; Sørensen, Søren Johannes

2016-01-01

Horizontal gene transfer (HGT), the transmission of genetic material to a recipient that is not the progeny of the donor, is fundamental in bacterial evolution. HGT is often mediated by mobile genetic elements such as conjugative plasmids, which may be in conflict with the chromosomal elements of the genome because they are independent replicons that may petition their own evolutionary strategy. Here we study differences between type 3 fimbriae encoded on wild type plasmids and in chromosomes. Using known and newly characterized plasmids we show that the expression of type 3 fimbriae encoded on plasmids is systematically different, as MrkH, a c-di-GMP dependent transcriptional activator is not needed for strong expression of the fimbriae. MrkH is required for expression of type 3 fimbriae of the Klebsiella pneumoniae chromosome, wherefrom the fimbriae operon (mrkABCDF) of plasmids is believed to have originated. We find that mrkABCDFs of plasmids are highly expressed via a unique promoter that differs from the original Klebsiella promoter resulting in fundamental behavioral consequences. Plasmid associated mrkABCDFs did not influence the swimming behavior of the host, that hereby acquired an exceptional phenotype being able to both actively swim (planktonic behavior) and express biofilm associated fimbriae (sessile behavior). We show that this exceptional phenotype enhances the conjugal transfer of the plasmid. PMID:27627107

Extended spectrum β-lactamases, carbapenemases and mobile genetic elements responsible for antibiotics resistance in Gram-negative bacteria.

PubMed

El Salabi, Allaaeddin; Walsh, Timothey R; Chouchani, Chedly

2013-05-01

Infectious diseases due to Gram-negative bacteria are a leading cause of morbidity and mortality worldwide. Antimicrobial agents represent one major therapeutic tools implicated to treat these infections. The misuse of antimicrobial agents has resulted in the emergence of resistant strains of Gram-negatives in particular Enterobacteriaceae and non-fermenters; they have an effect not only on a human but on the public health when bacteria use the resistance mechanisms to spread in the hospital environment and to the community outside the hospitals by means of mobile genetic elements. Gram-negative bacteria have become increasingly resistant to antimicrobial agents. They have developed several mechanisms by which they can withstand to antimicrobials, these mechanisms include the production of Extended-spectrum β-lactamases (ESBLs) and carbapenemases, furthermore, Gram-negative bacteria are now capable of spreading such resistance between members of the family Enterobacteriaceae and non-fermenters using mobile genetic elements as vehicles for such resistance mechanisms rendering antibiotics useless. Therefore, addressing the issue of mechanisms of antimicrobial resistance is considered one of most urgent priorities. This review will help to illustrate different resistance mechanisms; ESBLs, carbapenemases encoded by genes carried by mobile genetic elements, which are used by Gram-negative bacteria to escape antimicrobial effect.

Novel Structure of Ty3 Reverse Transcriptase | Center for Cancer Research

Cancer.gov

Retrotransposons are mobile genetic elements that self amplify via a single-stranded RNA intermediate, which is converted to double-stranded DNA by an encoded reverse transcriptase (RT) with both DNA polymerase (pol) and ribonuclease H (RNase) activities. Categorized by whether they contain flanking long terminal repeat (LTR) sequences, retrotransposons play a critical role in

Disabling a Type I-E CRISPR-Cas Nuclease with a Bacteriophage-Encoded Anti-CRISPR Protein.

PubMed

Pawluk, April; Shah, Megha; Mejdani, Marios; Calmettes, Charles; Moraes, Trevor F; Davidson, Alan R; Maxwell, Karen L

2017-12-12

CRISPR (clustered regularly interspaced short palindromic repeat)-Cas adaptive immune systems are prevalent defense mechanisms in bacteria and archaea. They provide sequence-specific detection and neutralization of foreign nucleic acids such as bacteriophages and plasmids. One mechanism by which phages and other mobile genetic elements are able to overcome the CRISPR-Cas system is through the expression of anti-CRISPR proteins. Over 20 different families of anti-CRISPR proteins have been described, each of which inhibits a particular type of CRISPR-Cas system. In this work, we determined the structure of type I-E anti-CRISPR protein AcrE1 by X-ray crystallography. We show that AcrE1 binds to the CRISPR-associated helicase/nuclease Cas3 and that the C-terminal region of the anti-CRISPR protein is important for its inhibitory activity. We further show that AcrE1 can convert the endogenous type I-E CRISPR system into a programmable transcriptional repressor. IMPORTANCE The CRISPR-Cas immune system provides bacteria with resistance to invasion by potentially harmful viruses, plasmids, and other foreign mobile genetic elements. This study presents the first structural and mechanistic insight into a phage-encoded protein that inactivates the type I-E CRISPR-Cas system in Pseudomonas aeruginosa The interaction of this anti-CRISPR protein with the CRISPR-associated helicase/nuclease proteins Cas3 shuts down the CRISPR-Cas system and protects phages carrying this gene from destruction. This interaction also allows the repurposing of the endogenous type I-E CRISPR system into a programmable transcriptional repressor, providing a new biotechnological tool for genetic studies of bacteria encoding this type I-E CRISPR-Cas system. Copyright © 2017 Pawluk et al.

A Novel aadA Aminoglycoside Resistance Gene in Bovine and Porcine Pathogens.

PubMed

Cameron, Andrew; Klima, Cassidy L; Ha, Reuben; Gruninger, Robert J; Zaheer, Rahat; McAllister, Tim A

2018-01-01

A novel variant of the AAD(3″) class of aminoglycoside-modifying enzymes was discovered in fatal bovine respiratory disease-associated pathogens Pasteurella multocida and Histophilus somni . The aadA31 gene encodes a spectinomycin/streptomycin adenylyltransferase and was located in a variant of the integrative and conjugative element ICE Mh1 , a mobile genetic element transmissible among members of the family Pasteurellaceae . The gene was also detected in Mannheimia haemolytica from a case of porcine pneumonia and in Moraxella bovoculi from a case of keratoconjunctivitis. IMPORTANCE Aminoglycosides are important antimicrobials used worldwide for prophylaxis and/or therapy in multiple production animal species. The emergence of new resistance genes jeopardizes current pathogen detection and treatment methods. The risk of resistance gene transfer to other animal and human pathogens is elevated when resistance genes are carried by mobile genetic elements. This study identified a new variant of a spectinomycin/streptomycin resistance gene harbored in a self-transmissible mobile element. The gene was also present in four different bovine pathogen species.

A Novel aadA Aminoglycoside Resistance Gene in Bovine and Porcine Pathogens

PubMed Central

Cameron, Andrew; Klima, Cassidy L.; Ha, Reuben; Gruninger, Robert J.; Zaheer, Rahat

2018-01-01

ABSTRACT A novel variant of the AAD(3″) class of aminoglycoside-modifying enzymes was discovered in fatal bovine respiratory disease-associated pathogens Pasteurella multocida and Histophilus somni. The aadA31 gene encodes a spectinomycin/streptomycin adenylyltransferase and was located in a variant of the integrative and conjugative element ICEMh1, a mobile genetic element transmissible among members of the family Pasteurellaceae. The gene was also detected in Mannheimia haemolytica from a case of porcine pneumonia and in Moraxella bovoculi from a case of keratoconjunctivitis. IMPORTANCE Aminoglycosides are important antimicrobials used worldwide for prophylaxis and/or therapy in multiple production animal species. The emergence of new resistance genes jeopardizes current pathogen detection and treatment methods. The risk of resistance gene transfer to other animal and human pathogens is elevated when resistance genes are carried by mobile genetic elements. This study identified a new variant of a spectinomycin/streptomycin resistance gene harbored in a self-transmissible mobile element. The gene was also present in four different bovine pathogen species. PMID:29507894

Genome dynamics and its impact on evolution of Escherichia coli.

PubMed

Dobrindt, Ulrich; Chowdary, M Geddam; Krumbholz, G; Hacker, J

2010-08-01

The Escherichia coli genome consists of a conserved part, the so-called core genome, which encodes essential cellular functions and of a flexible, strain-specific part. Genes that belong to the flexible genome code for factors involved in bacterial fitness and adaptation to different environments. Adaptation includes increase in fitness and colonization capacity. Pathogenic as well as non-pathogenic bacteria carry mobile and accessory genetic elements such as plasmids, bacteriophages, genomic islands and others, which code for functions required for proper adaptation. Escherichia coli is a very good example to study the interdependency of genome architecture and lifestyle of bacteria. Thus, these species include pathogenic variants as well as commensal bacteria adapted to different host organisms. In Escherichia coli, various genetic elements encode for pathogenicity factors as well as factors, which increase the fitness of non-pathogenic bacteria. The processes of genome dynamics, such as gene transfer, genome reduction, rearrangements as well as point mutations contribute to the adaptation of the bacteria into particular environments. Using Escherichia coli model organisms, such as uropathogenic strain 536 or commensal strain Nissle 1917, we studied mechanisms of genome dynamics and discuss these processes in the light of the evolution of microbes.

Molecular characterization of the 2011 Hong Kong scarlet fever outbreak.

PubMed

Tse, Herman; Bao, Jessie Y J; Davies, Mark R; Maamary, Peter; Tsoi, Hoi-Wah; Tong, Amy H Y; Ho, Tom C C; Lin, Chi-Ho; Gillen, Christine M; Barnett, Timothy C; Chen, Jonathan H K; Lee, Mianne; Yam, Wing-Cheong; Wong, Chi-Kin; Ong, Cheryl-Lynn Y; Chan, Yee-Wai; Wu, Cheng-Wei; Ng, Tony; Lim, Wilina W L; Tsang, Thomas H F; Tse, Cindy W S; Dougan, Gordon; Walker, Mark J; Lok, Si; Yuen, Kwok-Yung

2012-08-01

A scarlet fever outbreak occurred in Hong Kong in 2011. The majority of cases resulted in the isolation of Streptococcus pyogenes emm12 with multiple antibiotic resistances. Phylogenetic analysis of 22 emm12 scarlet fever outbreak isolates, 7 temporally and geographically matched emm12 non-scarlet fever isolates, and 18 emm12 strains isolated during 2005-2010 indicated the outbreak was multiclonal. Genome sequencing of 2 nonclonal scarlet fever isolates (HKU16 and HKU30), coupled with diagnostic polymerase chain reaction assays, identified 2 mobile genetic elements distributed across the major lineages: a 64.9-kb integrative and conjugative element encoding tetracycline and macrolide resistance and a 46.4-kb prophage encoding superantigens SSA and SpeC and the DNase Spd1. Phenotypic comparison of HKU16 and HKU30 with the S. pyogenes M1T1 strain 5448 revealed that HKU16 displays increased adherence to HEp-2 human epithelial cells, whereas HKU16, HKU30, and 5448 exhibit equivalent resistance to neutrophils and virulence in a humanized plasminogen murine model. However, in contrast to M1T1, the virulence of HKU16 and HKU30 was not associated with covRS mutation. The multiclonal nature of the emm12 scarlet fever isolates suggests that factors such as mobile genetic elements, environmental factors, and host immune status may have contributed to the 2011 scarlet fever outbreak.

Molecular Characterization of the 2011 Hong Kong Scarlet Fever Outbreak

PubMed Central

Tse, Herman; Bao, Jessie Y. J.; Davies, Mark R.; Maamary, Peter; Tsoi, Hoi-Wah; Tong, Amy H. Y.; Ho, Tom C. C.; Lin, Chi-Ho; Gillen, Christine M.; Barnett, Timothy C.; Chen, Jonathan H. K.; Lee, Mianne; Yam, Wing-Cheong; Wong, Chi-Kin; Ong, Cheryl-lynn Y.; Chan, Yee-Wai; Wu, Cheng-Wei; Ng, Tony; Lim, Wilina W. L.; Tsang, Thomas H. F.; Tse, Cindy W. S.; Dougan, Gordon; Walker, Mark J.; Lok, Si; Yuen, Kwok-Yung

2012-01-01

A scarlet fever outbreak occurred in Hong Kong in 2011. The majority of cases resulted in the isolation of Streptococcus pyogenes emm12 with multiple antibiotic resistances. Phylogenetic analysis of 22 emm12 scarlet fever outbreak isolates, 7 temporally and geographically matched emm12 non–scarlet fever isolates, and 18 emm12 strains isolated during 2005–2010 indicated the outbreak was multiclonal. Genome sequencing of 2 nonclonal scarlet fever isolates (HKU16 and HKU30), coupled with diagnostic polymerase chain reaction assays, identified 2 mobile genetic elements distributed across the major lineages: a 64.9-kb integrative and conjugative element encoding tetracycline and macrolide resistance and a 46.4-kb prophage encoding superantigens SSA and SpeC and the DNase Spd1. Phenotypic comparison of HKU16 and HKU30 with the S. pyogenes M1T1 strain 5448 revealed that HKU16 displays increased adherence to HEp-2 human epithelial cells, whereas HKU16, HKU30, and 5448 exhibit equivalent resistance to neutrophils and virulence in a humanized plasminogen murine model. However, in contrast to M1T1, the virulence of HKU16 and HKU30 was not associated with covRS mutation. The multiclonal nature of the emm12 scarlet fever isolates suggests that factors such as mobile genetic elements, environmental factors, and host immune status may have contributed to the 2011 scarlet fever outbreak. PMID:22615319

The Regulatory Properties of Autonomous Subtelomeric P Elements Are Sensitive to a Suppressor of Variegation in Drosophila Melanogaster

PubMed Central

Ronsseray, S.; Lehmann, M.; Nouaud, D.; Anxolabehere, D.

1996-01-01

Genetic recombination was used in Drosophila melanogaster to isolate P elements, inserted at the telomeres of X chromosomes (cytological site 1A) from natural populations, in a genetic background devoid of other P elements. We show that complete maternally inherited P repression in the germline (P cytotype) can be elicited by only two autonomous P elements at 1A and that a single element at this site has partial regulatory properties. The analysis of the surrounding chromosomal regions of the P elements at 1A shows that in all cases these elements are flanked by Telomeric Associated Sequences, tandemly repetitive noncoding sequences that have properties of heterochromatin. In addition, we show that the regulatory properties of P elements at 1A can be inhibited by some of the mutant alleles of the Su(var)205 gene and by a deficiency of this gene. However, the regulatory properties of reference P strains (Harwich and Texas 007) are not impaired by Su(var)205 mutations. Su(var)205 encodes Heterochromatin Protein 1 (HP1). These results suggest that the HP1 dosage effect on the P element properties is site-dependent and could involve the structure of the chromatin. PMID:8844154

Staphylococcus aureus innate immune evasion is lineage-specific: a bioinfomatics study.

PubMed

McCarthy, Alex J; Lindsay, Jodi A

2013-10-01

Staphylococcus aureus is a major human pathogen, and is targeted by the host innate immune system. In response, S. aureus genomes encode dozens of secreted proteins that inhibit complement, chemotaxis and neutrophil activation resulting in successful evasion of innate immune responses. These proteins include immune evasion cluster proteins (IEC; Chp, Sak, Scn), staphylococcal superantigen-like proteins (SSLs), phenol soluble modulins (PSMs) and several leukocidins. Biochemical studies have indicated that genetic variants of these proteins can have unique functions. To ascertain the scale of genetic variation in secreted immune evasion proteins, whole genome sequences of 88 S. aureus isolates, representing 25 clonal complex (CC) lineages, in the public domain were analysed across 43 genes encoding 38 secreted innate immune evasion protein complexes. Twenty-three genes were variable, with between 2 and 15 variants, and the variants had lineage-specific distributions. They include genes encoding Eap, Ecb, Efb, Flipr/Flipr-like, Hla, Hld, Hlg, Sbi, Scin-B/C and 13 SSLs. Most of these protein complexes inhibit complement, chemotaxis and neutrophil activation suggesting that isolates from each S. aureus lineage respond to the innate immune system differently. In contrast, protein complexes that lyse neutrophils (LukSF-PVL, LukMF, LukED and PSMs) were highly conserved, but can be carried on mobile genetic elements (MGEs). MGEs also encode proteins with narrow host-specificities arguing that their acquisition has important roles in host/environmental adaptation. In conclusion, this data suggests that each lineage of S. aureus evades host immune responses differently, and that isolates can adapt to new host environments by acquiring MGEs and the immune evasion protein complexes that they encode. Cocktail therapeutics that targets multiple variant proteins may be the most appropriate strategy for controlling S. aureus infections. Copyright © 2013 Elsevier B.V. All rights reserved.

Enamel formation and amelogenesis imperfecta.

PubMed

Hu, Jan C-C; Chun, Yong-Hee P; Al Hazzazzi, Turki; Simmer, James P

2007-01-01

Dental enamel is the epithelial-derived hard tissue covering the crowns of teeth. It is the most highly mineralized and hardest tissue in the body. Dental enamel is acellular and has no physiological means of repair outside of the protective and remineralization potential provided by saliva. Enamel is comprised of highly organized hydroxyapatite crystals that form in a defined extracellular space, the contents of which are supplied and regulated by ameloblasts. The entire process is under genetic instruction. The genetic control of amelogenesis is poorly understood, but requires the activities of multiple components that are uniquely important for dental enamel formation. Amelogenesis imperfecta (AI) is a collective designation for the variety of inherited conditions displaying isolated enamel malformations, but the designation is also used to indicate the presence of an enamel phenotype in syndromes. Recently, genetic studies have demonstrated the importance of genes encoding enamel matrix proteins in the etiology of isolated AI. Here we review the essential elements of dental enamel formation and the results of genetic analyses that have identified disease-causing mutations in genes encoding enamel matrix proteins. In addition, we provide a fresh perspective on the roles matrix proteins play in catalyzing the biomineralization of dental enamel. Copyright 2007 S. Karger AG, Basel.

Identification of the Intragenomic Promoter Controlling Hepatitis E Virus Subgenomic RNA Transcription.

PubMed

Ding, Qiang; Nimgaonkar, Ila; Archer, Nicholas F; Bram, Yaron; Heller, Brigitte; Schwartz, Robert E; Ploss, Alexander

2018-05-08

Approximately 20 million hepatitis E virus (HEV) infections occur annually in both developing and industrialized countries. Most infections are self-limiting, but they can lead to chronic infections and cirrhosis in immunocompromised patients, and death in pregnant women. The mechanisms of HEV replication remain incompletely understood due to scarcity of adequate experimental platforms. HEV undergoes asymmetric genome replication, but it produces an additional subgenomic (SG) RNA encoding the viral capsid and a viroporin in partially overlapping open reading frames. Using a novel transcomplementation system, we mapped the intragenomic subgenomic promoter regulating SG RNA synthesis. This cis -acting element is highly conserved across all eight HEV genotypes, and when the element is mutated, it abrogates particle assembly and release. Our work defines previously unappreciated viral regulatory elements and provides the first in-depth view of the intracellular genome dynamics of this emerging human pathogen. IMPORTANCE HEV is an emerging pathogen causing severe liver disease. The genetic information of HEV is encoded in RNA. The genomic RNA is initially copied into a complementary, antigenomic RNA that is a template for synthesis of more genomic RNA and for so-called subgenomic RNA. In this study, we identified the precise region within the HEV genome at which the synthesis of the subgenomic RNA is initiated. The nucleotides within this region are conserved across genetically distinct variants of HEV, highlighting the general importance of this segment for the virus. To identify this regulatory element, we developed a new experimental system that is a powerful tool with broad utility to mechanistically dissect many other poorly understood functional elements of HEV. Copyright © 2018 Ding et al.

Regulatory activities of transposable elements: from conflicts to benefits

PubMed Central

Chuong, Edward B.; Elde, Nels C.; Feschotte, Cédric

2017-01-01

Transposable elements (TEs) are a prolific source of tightly regulated, biochemically active non-coding elements, such as transcription factor binding sites and non-coding RNAs. A wealth of recent studies reinvigorates the idea that these elements are pervasively co-opted for the regulation of host genes. We argue that the inherent genetic properties of TEs and conflicting relationships with their hosts facilitate their recruitment for regulatory functions in diverse genomes. We review recent findings supporting the long-standing hypothesis that the waves of TE invasions endured by organisms for eons have catalyzed the evolution of gene regulatory networks. We also discuss the challenges of dissecting and interpreting the phenotypic impact of regulatory activities encoded by TEs in health and disease. PMID:27867194

Identification of functional elements and regulatory circuits by Drosophila modENCODE

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

Roy, Sushmita; Ernst, Jason; Kharchenko, Peter V.

2010-12-22

To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- andmore » tissue-specific regulators, and enables gene-expression prediction. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation. Several years after the complete genetic sequencing of many species, it is still unclear how to translate genomic information into a functional map of cellular and developmental programs. The Encyclopedia of DNA Elements (ENCODE) (1) and model organism ENCODE (modENCODE) (2) projects use diverse genomic assays to comprehensively annotate the Homo sapiens (human), Drosophila melanogaster (fruit fly), and Caenorhabditis elegans (worm) genomes, through systematic generation and computational integration of functional genomic data sets. Previous genomic studies in flies have made seminal contributions to our understanding of basic biological mechanisms and genome functions, facilitated by genetic, experimental, computational, and manual annotation of the euchromatic and heterochromatic genome (3), small genome size, short life cycle, and a deep knowledge of development, gene function, and chromosome biology. The functions of {approx}40% of the protein and nonprotein-coding genes [FlyBase 5.12 (4)] have been determined from cDNA collections (5, 6), manual curation of gene models (7), gene mutations and comprehensive genome-wide RNA interference screens (8-10), and comparative genomic analyses (11, 12). The Drosophila modENCODE project has generated more than 700 data sets that profile transcripts, histone modifications and physical nucleosome properties, general and specific transcription factors (TFs), and replication programs in cell lines, isolated tissues, and whole organisms across several developmental stages (Fig. 1). Here, we computationally integrate these data sets and report (i) improved and additional genome annotations, including full-length proteincoding genes and peptides as short as 21 amino acids; (ii) noncoding transcripts, including 132 candidate structural RNAs and 1608 nonstructural transcripts; (iii) additional Argonaute (Ago)-associated small RNA genes and pathways, including new microRNAs (miRNAs) encoded within protein-coding exons and endogenous small interfering RNAs (siRNAs) from 3-inch untranslated regions; (iv) chromatin 'states' defined by combinatorial patterns of 18 chromatin marks that are associated with distinct functions and properties; (v) regions of high TF occupancy and replication activity with likely epigenetic regulation; (vi)mixed TF and miRNA regulatory networks with hierarchical structure and enriched feed-forward loops; (vii) coexpression- and co-regulation-based functional annotations for nearly 3000 genes; (viii) stage- and tissue-specific regulators; and (ix) predictive models of gene expression levels and regulator function.« less

Plasmids of Legionella Species.

DTIC Science & Technology

1982-06-18

LEGIONELLA SPECIES *PERRY MIKESELL, CPT GREGORY B. KNUDSON, PhD U.S. ARMY MEDICAL RESEARCH INSTITUTE OF INFECTIOUS DISEASES FORT DETRICK, FREDERICK, MARYLAND...sponsible for metabolism, resistan to metals and fertility factors , plasmids also encode for druKg_-sistance factors . These latter genetic elements are an...3). The etiological agent was identified as a fastidious, aerobic, gram-nega- tive bacterium and given the name Legionella pneumophila. The number of

Nonlinear inversion of potential-field data using a hybrid-encoding genetic algorithm

USGS Publications Warehouse

Chen, C.; Xia, J.; Liu, J.; Feng, G.

2006-01-01

Using a genetic algorithm to solve an inverse problem of complex nonlinear geophysical equations is advantageous because it does not require computer gradients of models or "good" initial models. The multi-point search of a genetic algorithm makes it easier to find the globally optimal solution while avoiding falling into a local extremum. As is the case in other optimization approaches, the search efficiency for a genetic algorithm is vital in finding desired solutions successfully in a multi-dimensional model space. A binary-encoding genetic algorithm is hardly ever used to resolve an optimization problem such as a simple geophysical inversion with only three unknowns. The encoding mechanism, genetic operators, and population size of the genetic algorithm greatly affect search processes in the evolution. It is clear that improved operators and proper population size promote the convergence. Nevertheless, not all genetic operations perform perfectly while searching under either a uniform binary or a decimal encoding system. With the binary encoding mechanism, the crossover scheme may produce more new individuals than with the decimal encoding. On the other hand, the mutation scheme in a decimal encoding system will create new genes larger in scope than those in the binary encoding. This paper discusses approaches of exploiting the search potential of genetic operations in the two encoding systems and presents an approach with a hybrid-encoding mechanism, multi-point crossover, and dynamic population size for geophysical inversion. We present a method that is based on the routine in which the mutation operation is conducted in the decimal code and multi-point crossover operation in the binary code. The mix-encoding algorithm is called the hybrid-encoding genetic algorithm (HEGA). HEGA provides better genes with a higher probability by a mutation operator and improves genetic algorithms in resolving complicated geophysical inverse problems. Another significant result is that final solution is determined by the average model derived from multiple trials instead of one computation due to the randomness in a genetic algorithm procedure. These advantages were demonstrated by synthetic and real-world examples of inversion of potential-field data. ?? 2005 Elsevier Ltd. All rights reserved.

Crosstalk between vertical and horizontal gene transfer: plasmid replication control by a conjugative relaxase

PubMed Central

Lorenzo-Díaz, Fabián; Fernández-López, Cris; Lurz, Rudi

2017-01-01

Abstract Horizontal gene transfer is a key process in the evolution of bacteria and also represents a source of genetic variation in eukaryotes. Among elements participating in gene transfer, thousands of small (<10 kb) mobile bacterial plasmids that replicate by the rolling circle mechanism represent a driving force in the spread of antibiotic resistances. In general, these plasmids are built as genetic modules that encode a replicase, an antibiotic-resistance determinant, and a relaxase that participates in their conjugative mobilization. Further, they control their relatively high copy number (∼30 copies per genome equivalent) by antisense RNAs alone or combined with a repressor protein. We report here that the MobM conjugative relaxase encoded by the promiscuous plasmid pMV158 participates in regulation of the plasmid copy number by transcriptional repression of the antisense RNA, thus increasing the number of plasmid molecules ready to be horizontally transferred (mobilization) and/or vertically inherited (replication). This type of crosstalk between genetic modules involved in vertical and horizontal gene flow has not been reported before. PMID:28525572

Plant development. Arabidopsis NAC45/86 direct sieve element morphogenesis culminating in enucleation.

PubMed

Furuta, Kaori Miyashima; Yadav, Shri Ram; Lehesranta, Satu; Belevich, Ilya; Miyashima, Shunsuke; Heo, Jung-ok; Vatén, Anne; Lindgren, Ove; De Rybel, Bert; Van Isterdael, Gert; Somervuo, Panu; Lichtenberger, Raffael; Rocha, Raquel; Thitamadee, Siripong; Tähtiharju, Sari; Auvinen, Petri; Beeckman, Tom; Jokitalo, Eija; Helariutta, Ykä

2014-08-22

Photoassimilates such as sugars are transported through phloem sieve element cells in plants. Adapted for effective transport, sieve elements develop as enucleated living cells. We used electron microscope imaging and three-dimensional reconstruction to follow sieve element morphogenesis in Arabidopsis. We show that sieve element differentiation involves enucleation, in which the nuclear contents are released and degraded in the cytoplasm at the same time as other organelles are rearranged and the cytosol is degraded. These cellular reorganizations are orchestrated by the genetically redundant NAC domain-containing transcription factors, NAC45 and NAC86 (NAC45/86). Among the NAC45/86 targets, we identified a family of genes required for enucleation that encode proteins with nuclease domains. Thus, sieve elements differentiate through a specialized autolysis mechanism. Copyright © 2014, American Association for the Advancement of Science.

High-level generation of polyclonal antibodies by genetic immunization.

PubMed

Chambers, Ross S; Johnston, Stephen Albert

2003-09-01

Antibodies are important tools for investigating the proteome, but current methods for producing them have become a rate-limiting step. A primary obstacle in most methods for generating antibodies or antibody-like molecules is the requirement for at least microgram quantities of purified protein. We have developed a technology for producing antibodies using genetic immunization. Genetic immunization-based antibody production offers several advantages, including high throughput and high specificity. Moreover, antibodies produced from genetically immunized animals are more likely to recognize the native protein. Here we show that a genetic immunization-based system can be used to efficiently raise useful antibodies to a wide range of antigens. We accomplished this by linking the antigen gene to various elements that enhance antigenicity and by codelivering plasmids encoding genetic adjuvants. Our system, which was tested by immunizing mice with >130 antigens, has shown a final success rate of 84%.

Strain Variation in the Transcriptome of the Dengue Fever Vector, Aedes aegypti.

PubMed

Bonizzoni, Mariangela; Dunn, W Augustine; Campbell, Corey L; Olson, Ken E; Marinotti, Osvaldo; James, Anthony A

2012-01-01

Studies of transcriptome dynamics provide a basis for understanding functional elements of the genome and the complexity of gene regulation. The dengue vector mosquito, Aedes aegypti, exhibits great adaptability to diverse ecological conditions, is phenotypically polymorphic, and shows variation in vectorial capacity to arboviruses. Previous genome sequencing showed richness in repetitive DNA and transposable elements that can contribute to genome plasticity. Population genetic studies revealed a varying degree of worldwide genetic polymorphism. However, the extent of functional genetic polymorphism across strains is unknown. The transcriptomes of three Ae. aegypti strains, Chetumal (CTM), Rexville D-Puerto Rico (Rex-D) and Liverpool (LVP), were compared. CTM is more susceptible than Rex- D to infection by dengue virus serotype 2. A total of 4188 transcripts exhibit either no or small variation (<2-fold) among sugar-fed samples of the three strains and between sugar- and blood-fed samples within each strain, corresponding most likely to genes encoding products necessary for vital functions. Transcripts enriched in blood-fed mosquitoes encode proteins associated with catalytic activities, molecular transport, metabolism of lipids, carbohydrates and amino acids, and functions related to blood digestion and the progression of the gonotropic cycle. Significant qualitative and quantitative differences were found in individual transcripts among strains including differential representation of paralogous gene products. The majority of immunity-associated transcripts decreased in accumulation after a bloodmeal and the results are discussed in relation to the different susceptibility of CTM and Rex-D mosquitoes to DENV2 infection.

Differential patterns of acquired virulence genes distinguish Salmonella strains

PubMed Central

Conner, Christopher P.; Heithoff, Douglas M.; Julio, Steven M.; Sinsheimer, Robert L.; Mahan, Michael J.

1998-01-01

Analysis of several Salmonella typhimurium in vivo-induced genes located in regions of atypical base composition has uncovered acquired genetic elements that cumulatively engender pathogenicity. Many of these regions are associated with mobile elements, encode predicted adhesin and invasin-like functions, and are required for full virulence. Some of these regions distinguish broad host range from host-adapted Salmonella serovars and may contribute to inherent differences in host specificity, tissue tropism, and disease manifestation. Maintenance of this archipelago of acquired sequence by selection in specific hosts reveals a fossil record of the evolution of pathogenic species. PMID:9539791

A hobo transgene that encodes the P-element transposase in Drosophila melanogaster: autoregulation and cytotype control of transposase activity.

PubMed Central

Simmons, Michael J; Haley, Kevin J; Grimes, Craig D; Raymond, John D; Niemi, Jarad B

2002-01-01

Drosophila were genetically transformed with a hobo transgene that contains a terminally truncated but otherwise complete P element fused to the promoter from the Drosophila hsp70 gene. Insertions of this H(hsp/CP) transgene on either of the major autosomes produced the P transposase in both the male and female germlines, but not in the soma. Heat-shock treatments significantly increased transposase activity in the female germline; in the male germline, these treatments had little effect. The transposase activity of two insertions of the H(hsp/CP) transgene was not significantly greater than their separate activities, and one insertion of this transgene reduced the transposase activity of P(ry(+), Delta2-3)99B, a stable P transgene, in the germline as well as in the soma. These observations suggest that, through alternate splicing, the H(hsp/CP) transgene produces a repressor that feeds back negatively to regulate transposase expression or function in both the somatic and germline tissues. The H(hsp/CP) transgenes are able to induce gonadal dysgenesis when the transposase they encode has P-element targets to attack. However, this ability and the ability to induce P-element excisions are repressed by the P cytotype, a chromosomal/cytoplasmic state that regulates P elements in the germline. PMID:12019234

Surveying DNA Elements within Functional Genes of Heterocyst-Forming Cyanobacteria

PubMed Central

Hilton, Jason A.; Meeks, John C.; Zehr, Jonathan P.

2016-01-01

Some cyanobacteria are capable of differentiating a variety of cell types in response to environmental factors. For instance, in low nitrogen conditions, some cyanobacteria form heterocysts, which are specialized for N2 fixation. Many heterocyst-forming cyanobacteria have DNA elements interrupting key N2 fixation genes, elements that are excised during heterocyst differentiation. While the mechanism for the excision of the element has been well-studied, many questions remain regarding the introduction of the elements into the cyanobacterial lineage and whether they have been retained ever since or have been lost and reintroduced. To examine the evolutionary relationships and possible function of DNA sequences that interrupt genes of heterocyst-forming cyanobacteria, we identified and compared 101 interruption element sequences within genes from 38 heterocyst-forming cyanobacterial genomes. The interruption element lengths ranged from about 1 kb (the minimum able to encode the recombinase responsible for element excision), up to nearly 1 Mb. The recombinase gene sequences served as genetic markers that were common across the interruption elements and were used to track element evolution. Elements were found that interrupted 22 different orthologs, only five of which had been previously observed to be interrupted by an element. Most of the newly identified interrupted orthologs encode proteins that have been shown to have heterocyst-specific activity. However, the presence of interruption elements within genes with no known role in N2 fixation, as well as in three non-heterocyst-forming cyanobacteria, indicates that the processes that trigger the excision of elements may not be limited to heterocyst development or that the elements move randomly within genomes. This comprehensive analysis provides the framework to study the history and behavior of these unique sequences, and offers new insight regarding the frequency and persistence of interruption elements in heterocyst-forming cyanobacteria. PMID:27206019

Surveying DNA Elements within Functional Genes of Heterocyst-Forming Cyanobacteria.

PubMed

Hilton, Jason A; Meeks, John C; Zehr, Jonathan P

2016-01-01

Some cyanobacteria are capable of differentiating a variety of cell types in response to environmental factors. For instance, in low nitrogen conditions, some cyanobacteria form heterocysts, which are specialized for N2 fixation. Many heterocyst-forming cyanobacteria have DNA elements interrupting key N2 fixation genes, elements that are excised during heterocyst differentiation. While the mechanism for the excision of the element has been well-studied, many questions remain regarding the introduction of the elements into the cyanobacterial lineage and whether they have been retained ever since or have been lost and reintroduced. To examine the evolutionary relationships and possible function of DNA sequences that interrupt genes of heterocyst-forming cyanobacteria, we identified and compared 101 interruption element sequences within genes from 38 heterocyst-forming cyanobacterial genomes. The interruption element lengths ranged from about 1 kb (the minimum able to encode the recombinase responsible for element excision), up to nearly 1 Mb. The recombinase gene sequences served as genetic markers that were common across the interruption elements and were used to track element evolution. Elements were found that interrupted 22 different orthologs, only five of which had been previously observed to be interrupted by an element. Most of the newly identified interrupted orthologs encode proteins that have been shown to have heterocyst-specific activity. However, the presence of interruption elements within genes with no known role in N2 fixation, as well as in three non-heterocyst-forming cyanobacteria, indicates that the processes that trigger the excision of elements may not be limited to heterocyst development or that the elements move randomly within genomes. This comprehensive analysis provides the framework to study the history and behavior of these unique sequences, and offers new insight regarding the frequency and persistence of interruption elements in heterocyst-forming cyanobacteria.

Exploring dynamics of molybdate in living animal cells by a genetically encoded FRET nanosensor.

PubMed

Nakanishi, Yoichi; Iida, Syuntaro; Ueoka-Nakanishi, Hanayo; Niimi, Tomoaki; Tomioka, Rie; Maeshima, Masayoshi

2013-01-01

Molybdenum (Mo) is an essential trace element for almost all living organisms including animals. Mo is used as a catalytic center of molybdo-enzymes for oxidation/reduction reactions of carbon, nitrogen, and sulfur metabolism. Whilst living cells are known to import inorganic molybdate oxyanion from the surrounding environment, the in vivo dynamics of cytosolic molybdate remain poorly understood as no appropriate indicator is available for this trace anion. We here describe a genetically encoded Förester-resonance-energy-transfer (FRET)-based nanosensor composed of CFP, YFP and the bacterial molybdate-sensor protein ModE. The nanosensor MolyProbe containing an optimized peptide-linker responded to nanomolar-range molybdate selectively, and increased YFP:CFP fluorescence intensity ratio by up to 109%. By introduction of the nanosensor, we have been able to successfully demonstrate the real-time dynamics of molybdate in living animal cells. Furthermore, time course analyses of the dynamics suggest that novel oxalate-sensitive- and sulfate-resistant- transporter(s) uptake molybdate in a model culture cell.

Disproportionate Contributions of Select Genomic Compartments and Cell Types to Genetic Risk for Coronary Artery Disease

PubMed Central

Won, Hong-Hee; Natarajan, Pradeep; Dobbyn, Amanda; Jordan, Daniel M.; Roussos, Panos; Lage, Kasper; Raychaudhuri, Soumya

2015-01-01

Large genome-wide association studies (GWAS) have identified many genetic loci associated with risk for myocardial infarction (MI) and coronary artery disease (CAD). Concurrently, efforts such as the National Institutes of Health (NIH) Roadmap Epigenomics Project and the Encyclopedia of DNA Elements (ENCODE) Consortium have provided unprecedented data on functional elements of the human genome. In the present study, we systematically investigate the biological link between genetic variants associated with this complex disease and their impacts on gene function. First, we examined the heritability of MI/CAD according to genomic compartments. We observed that single nucleotide polymorphisms (SNPs) residing within nearby regulatory regions show significant polygenicity and contribute between 59–71% of the heritability for MI/CAD. Second, we showed that the polygenicity and heritability explained by these SNPs are enriched in histone modification marks in specific cell types. Third, we found that a statistically higher number of 45 MI/CAD-associated SNPs that have been identified from large-scale GWAS studies reside within certain functional elements of the genome, particularly in active enhancer and promoter regions. Finally, we observed significant heterogeneity of this signal across cell types, with strong signals observed within adipose nuclei, as well as brain and spleen cell types. These results suggest that the genetic etiology of MI/CAD is largely explained by tissue-specific regulatory perturbation within the human genome. PMID:26509271

Novel Structure of Ty3 Reverse Transcriptase | Center for Cancer Research

Cancer.gov

Retrotransposons are mobile genetic elements that self amplify via a single-stranded RNA intermediate, which is converted to double-stranded DNA by an encoded reverse transcriptase (RT) with both DNA polymerase (pol) and ribonuclease H (RNase) activities. Categorized by whether they contain flanking long terminal repeat (LTR) sequences, retrotransposons play a critical role in the architecture of eukaryotic genomes and are the evolutionary origin of retroviruses, including human immunodeficiency virus (HIV).

SXT/R391 integrative and conjugative elements in Proteus species reveal abundant genetic diversity and multidrug resistance

PubMed Central

Li, Xinyue; Du, Yu; Du, Pengcheng; Dai, Hang; Fang, Yujie; Li, Zhenpeng; Lv, Na; Zhu, Baoli; Kan, Biao; Wang, Duochun

2016-01-01

SXT/R391 integrative and conjugative elements (ICEs) are self-transmissible mobile genetic elements that are found in most members of Enterobacteriaceae. Here, we determined fifteen SXT/R391 ICEs carried by Proteus isolates from food (4.2%) and diarrhoea patients (17.3%). BLASTn searches against GenBank showed that the fifteen SXT/R391 ICEs were closely related to that from different Enterobacteriaceae species, including Proteus mirabilis. Using core gene phylogenetic analysis, the fifteen SXT/R391 ICEs were grouped into six distinct clusters, including a dominant cluster and three clusters that have not been previously reported in Proteus isolates. The SXT/R391 ICEs shared a common structure with a set of conserved genes, five hotspots and two variable regions, which contained more foreign genes, including drug-resistance genes. Notably, a class A β-lactamase gene was identified in nine SXT/R391 ICEs. Collectively, the ICE-carrying isolates carried resistance genes for 20 tested drugs. Six isolates were resistant to chloramphenicol, kanamycin, streptomycin, trimethoprim-sulfamethoxazole, sulfisoxazole and tetracycline, which are drug resistances commonly encoded by ICEs. Our results demonstrate abundant genetic diversity and multidrug resistance of the SXT/R391 ICEs carried by Proteus isolates, which may have significance for public health. It is therefore necessary to continuously monitor the antimicrobial resistance and related mobile elements among Proteus isolates. PMID:27892525

SZDB: A Database for Schizophrenia Genetic Research

PubMed Central

Wu, Yong; Yao, Yong-Gang

2017-01-01

Abstract Schizophrenia (SZ) is a debilitating brain disorder with a complex genetic architecture. Genetic studies, especially recent genome-wide association studies (GWAS), have identified multiple variants (loci) conferring risk to SZ. However, how to efficiently extract meaningful biological information from bulk genetic findings of SZ remains a major challenge. There is a pressing need to integrate multiple layers of data from various sources, eg, genetic findings from GWAS, copy number variations (CNVs), association and linkage studies, gene expression, protein–protein interaction (PPI), co-expression, expression quantitative trait loci (eQTL), and Encyclopedia of DNA Elements (ENCODE) data, to provide a comprehensive resource to facilitate the translation of genetic findings into SZ molecular diagnosis and mechanism study. Here we developed the SZDB database (http://www.szdb.org/), a comprehensive resource for SZ research. SZ genetic data, gene expression data, network-based data, brain eQTL data, and SNP function annotation information were systematically extracted, curated and deposited in SZDB. In-depth analyses and systematic integration were performed to identify top prioritized SZ genes and enriched pathways. Multiple types of data from various layers of SZ research were systematically integrated and deposited in SZDB. In-depth data analyses and integration identified top prioritized SZ genes and enriched pathways. We further showed that genes implicated in SZ are highly co-expressed in human brain and proteins encoded by the prioritized SZ risk genes are significantly interacted. The user-friendly SZDB provides high-confidence candidate variants and genes for further functional characterization. More important, SZDB provides convenient online tools for data search and browse, data integration, and customized data analyses. PMID:27451428

Versatile Cas9-Driven Subpopulation Selection Toolbox for Lactococcus lactis.

PubMed

van der Els, Simon; James, Jennelle K; Kleerebezem, Michiel; Bron, Peter A

2018-04-15

CRISPR-Cas9 technology has been exploited for the removal or replacement of genetic elements in a wide range of prokaryotes and eukaryotes. Here, we describe the extension of the Cas9 application toolbox to the industrially important dairy species Lactococcus lactis The Cas9 expression vector pLABTarget, encoding the Streptocccus pyogenes Cas9 under the control of a constitutive promoter, was constructed, allowing plug and play introduction of short guide RNA (sgRNA) sequences to target specific genetic loci. Introduction of a pepN -targeting derivative of pLABTarget into L. lactis strain MG1363 led to a strong reduction in the number of transformants obtained, which did not occur in a pepN deletion derivative of the same strain, demonstrating the specificity and lethality of the Cas9-mediated double-strand breaks in the lactococcal chromosome. Moreover, the same pLABTarget derivative allowed the selection of a pepN deletion subpopulation from its corresponding single-crossover plasmid integrant precursor, accelerating the construction and selection of gene-specific deletion derivatives in L. lactis Finally, pLABTarget, which contained sgRNAs designed to target mobile genetic elements, allowed the effective curing of plasmids, prophages, and integrative conjugative elements (ICEs). These results establish that pLABTarget enables the effective exploitation of Cas9 targeting in L. lactis , while the broad-host-range vector used suggests that this toolbox could readily be expanded to other Gram-positive bacteria. IMPORTANCE Mobile genetic elements in Lactococcus lactis and other lactic acid bacteria (LAB) play an important role in dairy fermentation, having both positive and detrimental effects during the production of fermented dairy products. The pLABTarget vector offers an efficient cloning platform for Cas9 application in lactic acid bacteria. Targeting Cas9 toward mobile genetic elements enabled their effective curing, which is of particular interest in the context of potentially problematic prophages present in a strain. Moreover, Cas9 targeting of other mobile genetic elements enables the deciphering of their contribution to dairy fermentation processes and further establishment of their importance for product characteristics. Copyright © 2018 American Society for Microbiology.

Evolutionary Optimization of Yagi-Uda Antennas

NASA Technical Reports Server (NTRS)

Lohn, Jason D.; Kraus, William F.; Linden, Derek S.; Colombano, Silvano P.

2001-01-01

Yagi-Uda antennas are known to be difficult to design and optimize due to their sensitivity at high gain, and the inclusion of numerous parasitic elements. We present a genetic algorithm-based automated antenna optimization system that uses a fixed Yagi-Uda topology and a byte-encoded antenna representation. The fitness calculation allows the implicit relationship between power gain and sidelobe/backlobe loss to emerge naturally, a technique that is less complex than previous approaches. The genetic operators used are also simpler. Our results include Yagi-Uda antennas that have excellent bandwidth and gain properties with very good impedance characteristics. Results exceeded previous Yagi-Uda antennas produced via evolutionary algorithms by at least 7.8% in mainlobe gain. We also present encouraging preliminary results where a coevolutionary genetic algorithm is used.

H-NS-like nucleoid-associated proteins, mobile genetic elements and horizontal gene transfer in bacteria.

PubMed

Dorman, Charles J

2014-09-01

Horizontal gene transfer plays an important role in the evolution of bacterial species, conferring new genetic traits on the recipient bacterium that extend its range of phenotypes and plasmids make important contributions to this process. However, the inappropriate expression of newly acquired genes may lead to a loss of competitive fitness, resulting in the elimination of the new gene-bacterium combination. It is thought that transcriptional silencing of horizontally acquired genes offers a route out of this dilemma and that nucleoid-associated proteins, especially those related to the H-NS protein, play a particularly important role in the silencing process. The discovery that many plasmids express orthologues of nucleoid-associated proteins adds an interesting dimension to current models of regulatory integration following lateral transfer of DNA. Other horizontally acquired genetic elements, such as genomic islands, also express nucleoid-associated proteins of their own. Here the interactions of H-NS-like nucleoid-associated proteins encoded by the core genome, genomic islands and plasmids are described. Copyright © 2014 Elsevier Inc. All rights reserved.

Loss-of-function of a ubiquitin-related modifier promotes the mobilization of the active MITE mPing.

PubMed

Tsukiyama, Takuji; Teramoto, Shota; Yasuda, Kanako; Horibata, Akira; Mori, Nanako; Okumoto, Yutaka; Teraishi, Masayoshi; Saito, Hiroki; Onishi, Akiko; Tamura, Kanako; Tanisaka, Takatoshi

2013-05-01

Miniature inverted-repeat transposable elements (MITEs) are widespread in both prokaryotic and eukaryotic genomes, where their copy numbers can attain several thousands. Little is known, however, about the genetic factor(s) affecting their transpositions. Here, we show that disruption of a gene encoding ubiquitin-like protein markedly enhances the transposition activity of a MITE mPing in intact rice plants without any exogenous stresses. We found that the transposition activity of mPing is far higher in the lines harboring a non-functional allele at the Rurm1 (Rice ubiquitin-related modifier-1) locus than in the wild-type line. Although the alteration of cytosine methylation pattern triggers the activation of transposable elements under exogenous stress conditions, the methylation degrees in the whole genome, the mPing-body region, and the mPing-flanking regions of the non-functional Rurm1 line were unchanged. This study provides experimental evidence for one of the models of genome shock theory that genetic accidents within cells enhance the transposition activities of transposable elements.

Inhibition of Exotoxin Production by Mobile Genetic Element SCCmec-Encoded psm-mec RNA Is Conserved in Staphylococcal Species

PubMed Central

Saito, Yuki; Mao, Han; Sekimizu, Kazuhisa; Kaito, Chikara

2014-01-01

Staphylococcal species acquire antibiotic resistance by incorporating the mobile-genetic element SCCmec. We previously found that SCCmec-encoded psm-mec RNA suppresses exotoxin production as a regulatory RNA, and the psm-mec translation product increases biofilm formation in Staphylococcus aureus. Here, we examined whether the regulatory role of psm-mec on host bacterial virulence properties is conserved among other staphylococcal species, S. epidermidis and S. haemolyticus, both of which are important causes of nosocomial infections. In S. epidermidis, introduction of psm-mec decreased the production of cytolytic toxins called phenol-soluble modulins (PSMs) and increased biofilm formation. Introduction of psm-mec with a stop-codon mutation that did not express PSM-mec protein but did express psm-mec RNA also decreased PSM production, but did not increase biofilm formation. Thus, the psm-mec RNA inhibits PSM production, whereas the PSM-mec protein increases biofilm formation in S. epidermidis. In S. haemolyticus, introduction of psm-mec decreased PSM production, but did not affect biofilm formation. The mutated psm-mec with a stop-codon also caused the same effect. Thus, the psm-mec RNA also inhibits PSM production in S. haemolyticus. These findings suggest that the inhibitory role of psm-mec RNA on exotoxin production is conserved among staphylococcal species, although the stimulating effect of the psm-mec gene on biofilm formation is not conserved. PMID:24926994

Type II Toxin–Antitoxin Systems in the Unicellular Cyanobacterium Synechocystis sp. PCC 6803

PubMed Central

Kopfmann, Stefan; Roesch, Stefanie K.; Hess, Wolfgang R.

2016-01-01

Bacterial toxin–antitoxin (TA) systems are genetic elements, which are encoded by plasmid as well as chromosomal loci. They mediate plasmid and genomic island maintenance through post-segregational killing mechanisms but may also have milder effects, acting as mobile stress response systems that help certain cells of a population in persisting adverse growth conditions. Very few cyanobacterial TA system have been characterized thus far. In this work, we focus on the cyanobacterium Synechocystis 6803, a widely used model organism. We expand the number of putative Type II TA systems from 36 to 69 plus seven stand-alone components. Forty-seven TA pairs are located on the chromosome and 22 are plasmid-located. Different types of toxins are associated with various antitoxins in a mix and match principle. According to protein domains and experimental data, 81% of all toxins in Synechocystis 6803 likely exhibit RNase activity, suggesting extensive potential for toxicity-related RNA degradation and toxin-mediated transcriptome remodeling. Of particular interest is the Ssr8013–Slr8014 system encoded on plasmid pSYSG, which is part of a larger defense island or the pSYSX system Slr6056–Slr6057, which is linked to a bacterial ubiquitin-like system. Consequently, Synechocystis 6803 is one of the most prolific sources of new information about these genetic elements. PMID:27455323

Involvement of the VDE homing endonuclease and rapamycin in regulation of the Saccharomyces cerevisiae GSH11 gene encoding the high affinity glutathione transporter.

PubMed

Miyake, Tsuyoshi; Hiraishi, Hiroyuki; Sammoto, Hiroyuki; Ono, Bun-Ichiro

2003-10-10

The Saccharomyces cerevisiae gene HGT1/GSH11 encodes the high affinity glutathione transporter and is repressed by cysteine added to the culture medium. It has been found previously that a 5'-upstream cis-element, CCGCCACAC, is responsible for regulating GSH11 expression and that several proteins bind to this element (Miyake, T., Kanayama, M., Sammoto, H., and Ono, B. (2002) Mol. Genet. Genomics 266, 1004-1011). In this report we present evidence that the most prominent of these proteins is VDE, known previously as the homing endonuclease encoded by VMA1. We show also that GSH11 is not expressed in a VDE-deleted strain and that inability to express the GSH11 of this strain is overcome by introduction of the coding region of VDE or the entire VMA1 gene. It is also found that VDE does not cut DNA in the vicinity of the GSH11 cis-element. Rapamycin, an inhibitor of the target of rapamycin (TOR) signal-transduction system, is found to enhance expression of GSH11 in a VDE-dependent manner under conditions of sulfur starvation. These results indicate that GSH11 is regulated by a system sensitive to sulfur starvation (presumably via cysteine depletion) and a more general system involving the nutritional starvation signal mediated by the TOR system. Both systems need to be operational (inhibition of TOR and sulfur starvation) for full expression of GSH11.

Distribution and regulation of the mobile genetic element-encoded phenol-soluble modulin PSM-mec in methicillin-resistant Staphylococcus aureus.

PubMed

Chatterjee, Som S; Chen, Liang; Joo, Hwang-Soo; Cheung, Gordon Y C; Kreiswirth, Barry N; Otto, Michael

2011-01-01

The phenol-soluble modulin PSM-mec is the only known staphylococcal toxin that is encoded on a mobile antibiotic resistance determinant, namely the staphylococcal cassette chromosome (SCC) element mec encoding resistance to methicillin. Here we show that the psm-mec gene is found frequently among methicillin-resistant Staphylococcus aureus (MRSA) strains of SCCmec types II, III, and VIII, and is a conserved part of the class A mec gene complex. Controlled expression of AgrA versus RNAIII in agr mutants of all 3 psm-mec-positive SCCmec types demonstrated that expression of psm-mec, which is highly variable, is controlled by AgrA in an RNAIII-independent manner. Furthermore, psm-mec isogenic deletion mutants showed only minor changes in PSMα peptide production and unchanged (or, as previously described, diminished) virulence compared to the corresponding wild-type strains in a mouse model of skin infection. This indicates that the recently reported regulatory impact of the psm-mec locus on MRSA virulence, which is opposite to that of the PSM-mec peptide and likely mediated by a regulatory RNA, is minor when analyzed in the original strain background. Our study gives new insight in the distribution, regulation, and role in virulence of the PSM-mec peptide and the psm-mec gene locus.

Detection of β-lactamase encoding genes in feces, soil and water from a Brazilian pig farm.

PubMed

Furlan, João Pedro Rueda; Stehling, Eliana Guedes

2018-01-10

β-lactam antibiotics are widely used for the treatment of different types of infections worldwide and the resistance to these antibiotics has grown sharply, which is of great concern. Resistance to β-lactams in gram-negative bacteria is mainly due to the production of β-lactamases, which are classified according to their functional activities. The aim of this study was to verify the presence of β-lactamases encoding genes in feces, soil, and water from a Brazilian pig farm. Different β-lactamases encoding genes were found, including bla CTX-M-Gp1 , bla CTX-M-Gp9 , bla SHV , bla OXA-1-like , bla GES , and bla VEB . The bla SHV and bla CTX-M-Gp1 genes have been detected in all types of samples, indicating the spread of β-lactam resistant bacteria among farm pigs and the environment around them. These results indicate that β-lactamase encoding genes belonging to the cloxacillinase, ESBL, and carbapenemase and they have high potential to spread in different sources, due to the fact that genes are closely related to mobile genetic elements, especially plasmids.

Molecular characterization of long direct repeat (LDR) sequences expressing a stable mRNA encoding for a 35-amino-acid cell-killing peptide and a cis-encoded small antisense RNA in Escherichia coli.

PubMed

Kawano, Mitsuoki; Oshima, Taku; Kasai, Hiroaki; Mori, Hirotada

2002-07-01

Genome sequence analyses of Escherichia coli K-12 revealed four copies of long repetitive elements. These sequences are designated as long direct repeat (LDR) sequences. Three of the repeats (LDR-A, -B, -C), each approximately 500 bp in length, are located as tandem repeats at 27.4 min on the genetic map. Another copy (LDR-D), 450 bp in length and nearly identical to LDR-A, -B and -C, is located at 79.7 min, a position that is directly opposite the position of LDR-A, -B and -C. In this study, we demonstrate that LDR-D encodes a 35-amino-acid peptide, LdrD, the overexpression of which causes rapid cell killing and nucleoid condensation of the host cell. Northern blot and primer extension analysis showed constitutive transcription of a stable mRNA (approximately 370 nucleotides) encoding LdrD and an unstable cis-encoded antisense RNA (approximately 60 nucleotides), which functions as a trans-acting regulator of ldrD translation. We propose that LDR encodes a toxin-antitoxin module. LDR-homologous sequences are not pre-sent on any known plasmids but are conserved in Salmonella and other enterobacterial species.

Arabidopsis and the Genetic Potential for the Phytoremediation of Toxic Elemental and Organic Pollutants

PubMed Central

Cobbett, Christopher S.; Meagher, Richard B.

2002-01-01

In a process called phytoremediation, plants can be used to extract, detoxify, and/or sequester toxic pollutants from soil, water, and air. Phytoremediation may become an essential tool in cleaning the environment and reducing human and animal exposure to potential carcinogens and other toxins. Arabidopsis has provided useful information about the genetic, physiological, and biochemical mechanisms behind phytoremediation, and it is an excellent model genetic organism to test foreign gene expression. This review focuses on Arabidopsis studies concerning: 1) the remediation of elemental pollutants; 2) the remediation of organic pollutants; and 3) the phytoremediation genome. Elemental pollutants include heavy metals and metalloids (e.g., mercury, lead, cadmium, arsenic) that are immutable. The general goal of phytoremediation is to extract, detoxify, and hyperaccumulate elemental pollutants in above-ground plant tissues for later harvest. A few dozen Arabidopsis genes and proteins that play direct roles in the remediation of elemental pollutants are discussed. Organic pollutants include toxic chemicals such as benzene, benzo(a)pyrene, polychlorinated biphenyls, trichloroethylene, trinitrotoluene, and dichlorodiphenyltrichloroethane. Phytoremediation of organic pollutants is focused on their complete mineralization to harmless products, however, less is known about the potential of plants to act on complex organic chemicals. A preliminary survey of the Arabidopsis genome suggests that as many as 700 genes encode proteins that have the capacity to act directly on environmental pollutants or could be modified to do so. The potential of the phytoremediation proteome to be used to reduce human exposure to toxic pollutants appears to be enormous and untapped. PMID:22303204

Staphylococcus aureus toxin gene hitchhikes on a transferable antibiotic resistance element.

PubMed

Otto, Michael

2010-01-01

Virulence and antibiotic resistance of the dangerous human pathogen Staphylococcus aureus are to large extent determined by the acquisition of mobile genetic elements (MGEs). Up to now, these elements were known to comprise either resistance or virulence determinants, but not a mixture of the two. Queck et al. now found a cytolysin gene of the phenol-soluble modulin (PSM) family within SCCmec elements, which contain methicillin resistance genes and are largely responsible for the spread of methicillin-resistant S. aureus (MRSA). The novel gene, called psm-mec, had a significant impact on virulence in MRSA strains that do not produce high levels of genome-encoded PSMs. This first example of a combination of toxin and resistance genes on one staphylococcal MGE shows that such bundling is possible and may lead to an even faster acquisition of toxin and resistance genes by S. aureus and other staphylococcal pathogens.

Diagnostic use of computational retrotransposon detection: Successful definition of pathogenetic mechanism in a ciliopathy phenotype.

PubMed

Takenouchi, Toshiki; Kuchikata, Tomu; Yoshihashi, Hiroshi; Fujiwara, Mineko; Uehara, Tomoko; Miyama, Sahoko; Yamada, Shiro; Kosaki, Kenjiro

2017-05-01

Among more than 5,000 human monogenic disorders with known causative genes, transposable element insertion of a Long Interspersed Nuclear Element 1 (LINE1, L1) is known as the mechanistic basis in only 13 genetic conditions. Meckel-Gruber syndrome is a rare ciliopathy characterized by occipital encephalocele and cystic kidney disease. Here, we document a boy with occipital encephalocele, post-axial polydactyly, and multicystic renal disease. A medical exome analysis detected a heterozygous frameshift mutation, c.4582_4583delCG p.(Arg1528Serfs*17) in CC2D2A in the maternally derived allele. The further use of a dedicated bioinformatics algorithm for detecting retrotransposon insertions led to the detection of an L1 insertion affecting exon 7 in the paternally derived allele. The complete sequencing and sequence homology analysis of the inserted L1 element showed that the L1 element was classified as L1HS (L1 human specific) and that the element had intact open reading frames in the two L1-encoded proteins. This observation ranks Meckel-Gruber syndrome as only the 14th disorder to be caused by an L1 insertion among more than 5,000 known human genetic disorders. Although a transposable element detection algorithm is not included in the current best-practice next-generation sequencing analysis, the present observation illustrates the utility of such an algorithm, which would require modest computational time and resources. Whether the seemingly infrequent recognition of L1 insertion in the pathogenesis of human genetic diseases might simply reflect a lack of appropriate detection methods remains to be seen. © 2017 Wiley Periodicals, Inc.

Ribosomal frameshifting and dual-target antiactivation restrict quorum-sensing-activated transfer of a mobile genetic element.

PubMed

Ramsay, Joshua P; Tester, Laura G L; Major, Anthony S; Sullivan, John T; Edgar, Christina D; Kleffmann, Torsten; Patterson-House, Jackson R; Hall, Drew A; Tate, Warren P; Hynes, Michael F; Ronson, Clive W

2015-03-31

Symbiosis islands are integrative and conjugative mobile genetic elements that convert nonsymbiotic rhizobia into nitrogen-fixing symbionts of leguminous plants. Excision of the Mesorhizobium loti symbiosis island ICEMlSym(R7A) is indirectly activated by quorum sensing through TraR-dependent activation of the excisionase gene rdfS. Here we show that a +1 programmed ribosomal frameshift (PRF) fuses the coding sequences of two TraR-activated genes, msi172 and msi171, producing an activator of rdfS expression named Frameshifted excision activator (FseA). Mass-spectrometry and mutational analyses indicated that the PRF occurred through +1 slippage of the tRNA(phe) from UUU to UUC within a conserved msi172-encoded motif. FseA activated rdfS expression in the absence of ICEMlSym(R7A), suggesting that it directly activated rdfS transcription, despite being unrelated to any characterized DNA-binding proteins. Bacterial two-hybrid and gene-reporter assays demonstrated that FseA was also bound and inhibited by the ICEMlSym(R7A)-encoded quorum-sensing antiactivator QseM. Thus, activation of ICEMlSym(R7A) excision is counteracted by TraR antiactivation, ribosomal frameshifting, and FseA antiactivation. This robust suppression likely dampens the inherent biological noise present in the quorum-sensing autoinduction circuit and ensures that ICEMlSym(R7A) transfer only occurs in a subpopulation of cells in which both qseM expression is repressed and FseA is translated. The architecture of the ICEMlSym(R7A) transfer regulatory system provides an example of how a set of modular components have assembled through evolution to form a robust genetic toggle that regulates gene transcription and translation at both single-cell and cell-population levels.

Ribosomal frameshifting and dual-target antiactivation restrict quorum-sensing–activated transfer of a mobile genetic element

PubMed Central

Ramsay, Joshua P.; Tester, Laura G. L.; Major, Anthony S.; Sullivan, John T.; Edgar, Christina D.; Kleffmann, Torsten; Patterson-House, Jackson R.; Hall, Drew A.; Tate, Warren P.; Hynes, Michael F.; Ronson, Clive W.

2015-01-01

Symbiosis islands are integrative and conjugative mobile genetic elements that convert nonsymbiotic rhizobia into nitrogen-fixing symbionts of leguminous plants. Excision of the Mesorhizobium loti symbiosis island ICEMlSymR7A is indirectly activated by quorum sensing through TraR-dependent activation of the excisionase gene rdfS. Here we show that a +1 programmed ribosomal frameshift (PRF) fuses the coding sequences of two TraR-activated genes, msi172 and msi171, producing an activator of rdfS expression named Frameshifted excision activator (FseA). Mass-spectrometry and mutational analyses indicated that the PRF occurred through +1 slippage of the tRNAphe from UUU to UUC within a conserved msi172-encoded motif. FseA activated rdfS expression in the absence of ICEMlSymR7A, suggesting that it directly activated rdfS transcription, despite being unrelated to any characterized DNA-binding proteins. Bacterial two-hybrid and gene-reporter assays demonstrated that FseA was also bound and inhibited by the ICEMlSymR7A-encoded quorum-sensing antiactivator QseM. Thus, activation of ICEMlSymR7A excision is counteracted by TraR antiactivation, ribosomal frameshifting, and FseA antiactivation. This robust suppression likely dampens the inherent biological noise present in the quorum-sensing autoinduction circuit and ensures that ICEMlSymR7A transfer only occurs in a subpopulation of cells in which both qseM expression is repressed and FseA is translated. The architecture of the ICEMlSymR7A transfer regulatory system provides an example of how a set of modular components have assembled through evolution to form a robust genetic toggle that regulates gene transcription and translation at both single-cell and cell-population levels. PMID:25787256

The CRISPR conundrum: evolve and maybe die, or survive and risk stagnation

PubMed Central

García-Martínez, Jesús; Maldonado, Rafael D.; Guzmán, Noemí M.; Mojica, Francisco J. M.

2018-01-01

CRISPR-Cas represents a prokaryotic defense mechanism against invading genetic elements. Although there is a diversity of CRISPR-Cas systems, they all share similar, essential traits. In general, a CRISPR-Cas system consists of one or more groups of DNA repeats named CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), regularly separated by unique sequences referred to as spacers, and a set of functionally associated cas (CRISPR associated) genes typically located next to one of the repeat arrays. The origin of spacers is in many cases unknown but, when ascertained, they usually match foreign genetic molecules. The proteins encoded by some of the cas genes are in charge of the incorporation of new spacers upon entry of a genetic element. Other Cas proteins participate in generating CRISPR-spacer RNAs and perform the task of destroying nucleic acid molecules carrying sequences similar to the spacer. In this way, CRISPR-Cas provides protection against genetic intruders that could substantially affect the cell viability, thus acting as an adaptive immune system. However, this defensive action also hampers the acquisition of potentially beneficial, horizontally transferred genes, undermining evolution. Here we cover how the model bacterium Escherichia coli deals with CRISPR-Cas to tackle this major dilemma, evolution versus survival. PMID:29850463

[The role of integrons in dissemination of antibiotic resistance].

PubMed

Ploy, M C; Lambert, T; Gassama, A; Denis, F

2000-01-01

Bacteria can transfer genetic information to get protection against most antibiotics. The acquisition of resistance genes involves genetic mobile elements such as plasmids and transposons. Another genetic structures, named integrons, have been described and contain one or more gene cassettes located at a specific site. Integrons contain an intI gene encoding a site-specific recombinase belonging to the integrase family and a recombination site attI. A gene cassette includes an open reading frame and, at the 3'-end, a recombination site attC. Integration or excision of cassettes occurs by a site-specific recombination mechanism catalyzed by the integrase. However, insertion can rarely occur, at non-specific sites leading to a stable situation for the cassette. Cassettes are transcribed from a common promoter located in the 5'-conserved segment and expression of distal genes is reduced by the presence of upstream cassettes. Most gene cassettes encode antibiotic resistant determinants but antiseptic resistant genes have also been described. Integrons seem to have a major role in the spread of multidrug resistance in Gram-negative bacteria but integrons in Gram-positive bacteria have been recently described. Moreover, the finding of super-integrons with gene cassettes coding for other determinants (biochemical functions, virulence factors) in different Gram negative bacteria suggests that integrons are probably implied in bacterial genome evolution.

The Influence of LINE-1 and SINE Retrotransposons on Mammalian Genomes.

PubMed

Richardson, Sandra R; Doucet, Aurélien J; Kopera, Huira C; Moldovan, John B; Garcia-Perez, José Luis; Moran, John V

2015-04-01

Transposable elements have had a profound impact on the structure and function of mammalian genomes. The retrotransposon Long INterspersed Element-1 (LINE-1 or L1), by virtue of its replicative mobilization mechanism, comprises ∼17% of the human genome. Although the vast majority of human LINE-1 sequences are inactive molecular fossils, an estimated 80-100 copies per individual retain the ability to mobilize by a process termed retrotransposition. Indeed, LINE-1 is the only active, autonomous retrotransposon in humans and its retrotransposition continues to generate both intra-individual and inter-individual genetic diversity. Here, we briefly review the types of transposable elements that reside in mammalian genomes. We will focus our discussion on LINE-1 retrotransposons and the non-autonomous Short INterspersed Elements (SINEs) that rely on the proteins encoded by LINE-1 for their mobilization. We review cases where LINE-1-mediated retrotransposition events have resulted in genetic disease and discuss how the characterization of these mutagenic insertions led to the identification of retrotransposition-competent LINE-1s in the human and mouse genomes. We then discuss how the integration of molecular genetic, biochemical, and modern genomic technologies have yielded insight into the mechanism of LINE-1 retrotransposition, the impact of LINE-1-mediated retrotransposition events on mammalian genomes, and the host cellular mechanisms that protect the genome from unabated LINE-1-mediated retrotransposition events. Throughout this review, we highlight unanswered questions in LINE-1 biology that provide exciting opportunities for future research. Clearly, much has been learned about LINE-1 and SINE biology since the publication of Mobile DNA II thirteen years ago. Future studies should continue to yield exciting discoveries about how these retrotransposons contribute to genetic diversity in mammalian genomes.

The Zinc-Finger Antiviral Protein ZAP Inhibits LINE and Alu Retrotransposition

PubMed Central

Moldovan, John B.; Moran, John V.

2015-01-01

Long INterspersed Element-1 (LINE-1 or L1) is the only active autonomous retrotransposon in the human genome. To investigate the interplay between the L1 retrotransposition machinery and the host cell, we used co-immunoprecipitation in conjunction with liquid chromatography and tandem mass spectrometry to identify cellular proteins that interact with the L1 first open reading frame-encoded protein, ORF1p. We identified 39 ORF1p-interacting candidate proteins including the zinc-finger antiviral protein (ZAP or ZC3HAV1). Here we show that the interaction between ZAP and ORF1p requires RNA and that ZAP overexpression in HeLa cells inhibits the retrotransposition of engineered human L1 and Alu elements, an engineered mouse L1, and an engineered zebrafish LINE-2 element. Consistently, siRNA-mediated depletion of endogenous ZAP in HeLa cells led to a ~2-fold increase in human L1 retrotransposition. Fluorescence microscopy in cultured human cells demonstrated that ZAP co-localizes with L1 RNA, ORF1p, and stress granule associated proteins in cytoplasmic foci. Finally, molecular genetic and biochemical analyses indicate that ZAP reduces the accumulation of full-length L1 RNA and the L1-encoded proteins, yielding mechanistic insight about how ZAP may inhibit L1 retrotransposition. Together, these data suggest that ZAP inhibits the retrotransposition of LINE and Alu elements. PMID:25951186

Detection of different β-lactamases encoding genes, including blaNDM, and plasmid-mediated quinolone resistance genes in different water sources from Brazil.

PubMed

Sanchez, Danilo Garcia; de Melo, Fernanda Maciel; Savazzi, Eduardo Angelino; Stehling, Eliana Guedes

2018-06-16

Bacterial resistance occurs by spontaneous mutations or horizontal gene transfer mediated by mobile genetic elements, which represents a great concern. Resistance to β-lactam antibiotics is mainly due to the production of β-lactamases, and an important mechanism of fluoroquinolone resistance is the acquisition plasmid determinants. The aim of this study was to verify the presence of β-lactamase-encoding genes and plasmid-mediated quinolone resistance genes in different water samples obtained from São Paulo state, Brazil. A high level of these resistance genes was detected, being the bla SHV , bla GES , and qnr the most prevalent. Besides that, the bla NDM gene, which codify an important and hazardous metallo-β-lactamase, was detected.

A Mobile Element in mutS Drives Hypermutation in a Marine Vibrio

PubMed Central

Chu, Nathaniel D.; Clarke, Sean A.; Timberlake, Sonia; Polz, Martin F.; Grossman, Alan D.

2017-01-01

ABSTRACT Bacteria face a trade-off between genetic fidelity, which reduces deleterious mistakes in the genome, and genetic innovation, which allows organisms to adapt. Evidence suggests that many bacteria balance this trade-off by modulating their mutation rates, but few mechanisms have been described for such modulation. Following experimental evolution and whole-genome resequencing of the marine bacterium Vibrio splendidus 12B01, we discovered one such mechanism, which allows this bacterium to switch to an elevated mutation rate. This switch is driven by the excision of a mobile element residing in mutS, which encodes a DNA mismatch repair protein. When integrated within the bacterial genome, the mobile element provides independent promoter and translation start sequences for mutS—different from the bacterium’s original mutS promoter region—which allow the bacterium to make a functional mutS gene product. Excision of this mobile element rejoins the mutS gene with host promoter and translation start sequences but leaves a 2-bp deletion in the mutS sequence, resulting in a frameshift and a hypermutator phenotype. We further identified hundreds of clinical and environmental bacteria across Betaproteobacteria and Gammaproteobacteria that possess putative mobile elements within the same amino acid motif in mutS. In a subset of these bacteria, we detected excision of the element but not a frameshift mutation; the mobile elements leave an intact mutS coding sequence after excision. Our findings reveal a novel mechanism by which one bacterium alters its mutation rate and hint at a possible evolutionary role for mobile elements within mutS in other bacteria. PMID:28174306

[ENCODE apophenia or a panglossian analysis of the human genome].

PubMed

Casane, Didier; Fumey, Julien; Laurenti, Patrick

2015-01-01

In September 2012, a batch of more than 30 articles presenting the results of the ENCODE (Encyclopaedia of DNA Elements) project was released. Many of these articles appeared in Nature and Science, the two most prestigious interdisciplinary scientific journals. Since that time, hundreds of other articles dedicated to the further analyses of the Encode data have been published. The time of hundreds of scientists and hundreds of millions of dollars were not invested in vain since this project had led to an apparent paradigm shift: contrary to the classical view, 80% of the human genome is not junk DNA, but is functional. This hypothesis has been criticized by evolutionary biologists, sometimes eagerly, and detailed refutations have been published in specialized journals with impact factors far below those that published the main contribution of the Encode project to our understanding of genome architecture. In 2014, the Encode consortium released a new batch of articles that neither suggested that 80% of the genome is functional nor commented on the disappearance of their 2012 scientific breakthrough. Unfortunately, by that time many biologists had accepted the idea that 80% of the genome is functional, or at least, that this idea is a valid alternative to the long held evolutionary genetic view that it is not. In order to understand the dynamics of the genome, it is necessary to re-examine the basics of evolutionary genetics because, not only are they well established, they also will allow us to avoid the pitfall of a panglossian interpretation of Encode. Actually, the architecture of the genome and its dynamics are the product of trade-offs between various evolutionary forces, and many structural features are not related to functional properties. In other words, evolution does not produce the best of all worlds, not even the best of all possible worlds, but only one possible world. © 2015 médecine/sciences – Inserm.

Staphylococcal food poisoning caused by Staphylococcus argenteus harboring staphylococcal enterotoxin genes.

PubMed

Wakabayashi, Yuki; Umeda, Kaoru; Yonogi, Shinya; Nakamura, Hiromi; Yamamoto, Kaori; Kumeda, Yuko; Kawatsu, Kentaro

2018-01-16

Staphylococcal food poisoning (SFP) is caused by staphylococcal enterotoxins (SEs) preformed in food materials. SE genes are encoded on mobile genetic elements and are widely found across Staphylococcus species including S. argenteus, although most SFP cases are caused by S. aureus. S. argenteus, recently discriminated from S. aureus as a novel species, are non-pigmented staphylococci phenotypically related to S. aureus. In 2014 and 2015, two independent food poisoning cases occurred in Osaka, Japan, in which non-pigmented staphylococci were predominantly isolated. Several enterotoxin genes (seb, seg, sei, sem, sen, seo, and selu2) were found in their genome and the production of SEB was confirmed by reverse passive agglutination tests. The non-pigmented isolates from patients, food handlers, food, and cooking utensils all produced the same pulsed-field gel electrophoresis pattern. These non-pigmented isolates were coagulase-positive and biochemically identical to S. aureus. We performed further genetic analysis using nucA sequencing and multi-locus sequence typing, and identified these isolates as S. argenteus. We also found that seb was encoded on the Staphylococcus aureus pathogenicity island, while seg, sei, sem, sen, seo, and selu2 were encoded on the enterotoxin gene cluster. From these results, we concluded that the two food poisoning outbreaks were SFP cases caused by S. argenteus harboring SE genes. Copyright © 2017 Elsevier B.V. All rights reserved.

Human Peripheral Blood Antibodies with Long HCDR3s Are Established Primarily at Original Recombination Using a Limited Subset of Germline Genes

PubMed Central

Briney, Bryan S.; Willis, Jordan R.; Crowe, James E.

2012-01-01

A number of antibodies that efficiently neutralize microbial targets contain long heavy chain complementarity determining region 3 (HCDR3) loops. For HIV, several of the most broad and potently neutralizing antibodies have exceptionally long HCDR3s. Two broad potently neutralizing HIV-specific antibodies, PG9 and PG16, exhibit secondary structure. Two other long HCDR3 antibodies, 2F5 and 4E10, protect against mucosal challenge with SHIV. Induction of such long HCDR3 antibodies may be critical to the design of an effective vaccine strategy for HIV and other pathogens, however it is unclear at present how to induce such antibodies. Here, we present genetic evidence that human peripheral blood antibodies containing long HCDR3s are not primarily generated by insertions introduced during the somatic hypermutation process. Instead, they are typically formed by processes occurring as part of the original recombination event. Thus, the response of B cells encoding antibodies with long HCDR3s results from selection of unusual clones from the naïve repertoire rather than through accumulation of insertions. These antibodies typically use a small subset of D and J gene segments that are particularly suited to encoding long HCDR3s, resulting in the incorporation of highly conserved genetic elements in the majority of antibody sequences encoding long HCDR3s. PMID:22590602

Temperate Phages Acquire DNA from Defective Prophages by Relaxed Homologous Recombination: The Role of Rad52-Like Recombinases

PubMed Central

De Paepe, Marianne; Hutinet, Geoffrey; Son, Olivier; Amarir-Bouhram, Jihane; Schbath, Sophie; Petit, Marie-Agnès

2014-01-01

Bacteriophages (or phages) dominate the biosphere both numerically and in terms of genetic diversity. In particular, genomic comparisons suggest a remarkable level of horizontal gene transfer among temperate phages, favoring a high evolution rate. Molecular mechanisms of this pervasive mosaicism are mostly unknown. One hypothesis is that phage encoded recombinases are key players in these horizontal transfers, thanks to their high efficiency and low fidelity. Here, we associate two complementary in vivo assays and a bioinformatics analysis to address the role of phage encoded recombinases in genomic mosaicism. The first assay allowed determining the genetic determinants of mosaic formation between lambdoid phages and Escherichia coli prophage remnants. In the second assay, recombination was monitored between sequences on phage λ, and allowed to compare the performance of three different Rad52-like recombinases on the same substrate. We also addressed the importance of homologous recombination in phage evolution by a genomic comparison of 84 E. coli virulent and temperate phages or prophages. We demonstrate that mosaics are mainly generated by homology-driven mechanisms that tolerate high substrate divergence. We show that phage encoded Rad52-like recombinases act independently of RecA, and that they are relatively more efficient when the exchanged fragments are divergent. We also show that accessory phage genes orf and rap contribute to mosaicism. A bioinformatics analysis strengthens our experimental results by showing that homologous recombination left traces in temperate phage genomes at the borders of recently exchanged fragments. We found no evidence of exchanges between virulent and temperate phages of E. coli. Altogether, our results demonstrate that Rad52-like recombinases promote gene shuffling among temperate phages, accelerating their evolution. This mechanism may prove to be more general, as other mobile genetic elements such as ICE encode Rad52-like functions, and play an important role in bacterial evolution itself. PMID:24603854

Flipping chromosomes in deep-sea archaea

PubMed Central

Catchpole, Ryan; Gadelle, Danièle; Marguet, Evelyne; Barbe, Valérie; Forterre, Patrick

2017-01-01

One of the major mechanisms driving the evolution of all organisms is genomic rearrangement. In hyperthermophilic Archaea of the order Thermococcales, large chromosomal inversions occur so frequently that even closely related genomes are difficult to align. Clearly not resulting from the native homologous recombination machinery, the causative agent of these inversions has remained elusive. We present a model in which genomic inversions are catalyzed by the integrase enzyme encoded by a family of mobile genetic elements. We characterized the integrase from Thermococcus nautili plasmid pTN3 and showed that besides canonical site-specific reactions, it catalyzes low sequence specificity recombination reactions with the same outcome as homologous recombination events on DNA segments as short as 104bp both in vitro and in vivo, in contrast to other known tyrosine recombinases. Through serial culturing, we showed that the integrase-mediated divergence of T. nautili strains occurs at an astonishing rate, with at least four large-scale genomic inversions appearing within 60 generations. Our results and the ubiquitous distribution of pTN3-like integrated elements suggest that a major mechanism of evolution of an entire order of Archaea results from the activity of a selfish mobile genetic element. PMID:28628615

Genetic evidence for conserved non-coding element function across species–the ears have it

PubMed Central

Turner, Eric E.; Cox, Timothy C.

2014-01-01

Comparison of genomic sequences from diverse vertebrate species has revealed numerous highly conserved regions that do not appear to encode proteins or functional RNAs. Often these “conserved non-coding elements,” or CNEs, can direct gene expression to specific tissues in transgenic models, demonstrating they have regulatory function. CNEs are frequently found near “developmental” genes, particularly transcription factors, implying that these elements have essential regulatory roles in development. However, actual examples demonstrating CNE regulatory functions across species have been few, and recent loss-of-function studies of several CNEs in mice have shown relatively minor effects. In this Perspectives article, we discuss new findings in “fancy” rats and Highland cattle demonstrating that function of a CNE near the Hmx1 gene is crucial for normal external ear development and when disrupted can mimic loss-of function Hmx1 coding mutations in mice and humans. These findings provide important support for conserved developmental roles of CNEs in divergent species, and reinforce the concept that CNEs should be examined systematically in the ongoing search for genetic causes of human developmental disorders in the era of genome-scale sequencing. PMID:24478720

Carbon source-dependent expansion of the genetic code in bacteria

PubMed Central

Prat, Laure; Heinemann, Ilka U.; Aerni, Hans R.; Rinehart, Jesse; O’Donoghue, Patrick; Söll, Dieter

2012-01-01

Despite the fact that the genetic code is known to vary between organisms in rare cases, it is believed that in the lifetime of a single cell the code is stable. We found Acetohalobium arabaticum cells grown on pyruvate genetically encode 20 amino acids, but in the presence of trimethylamine (TMA), A. arabaticum dynamically expands its genetic code to 21 amino acids including pyrrolysine (Pyl). A. arabaticum is the only known organism that modulates the size of its genetic code in response to its environment and energy source. The gene cassette pylTSBCD, required to biosynthesize and genetically encode UAG codons as Pyl, is present in the genomes of 24 anaerobic archaea and bacteria. Unlike archaeal Pyl-decoding organisms that constitutively encode Pyl, we observed that A. arabaticum controls Pyl encoding by down-regulating transcription of the entire Pyl operon under growth conditions lacking TMA, to the point where no detectable Pyl-tRNAPyl is made in vivo. Pyl-decoding archaea adapted to an expanded genetic code by minimizing TAG codon frequency to typically ∼5% of ORFs, whereas Pyl-decoding bacteria (∼20% of ORFs contain in-frame TAGs) regulate Pyl-tRNAPyl formation and translation of UAG by transcriptional deactivation of genes in the Pyl operon. We further demonstrate that Pyl encoding occurs in a bacterium that naturally encodes the Pyl operon, and identified Pyl residues by mass spectrometry in A. arabaticum proteins including two methylamine methyltransferases. PMID:23185002

Interplay of a non-conjugative integrative element and a conjugative plasmid in the spread of antibiotic resistance via suicidal plasmid transfer from an aquaculture Vibrio isolate.

PubMed

Nonaka, Lisa; Yamamoto, Tatsuya; Maruyama, Fumito; Hirose, Yuu; Onishi, Yuki; Kobayashi, Takeshi; Suzuki, Satoru; Nomura, Nobuhiko; Masuda, Michiaki; Yano, Hirokazu

2018-01-01

The capture of antimicrobial resistance genes (ARGs) by mobile genetic elements (MGEs) plays a critical role in resistance acquisition for human-associated bacteria. Although aquaculture environments are recognized as important reservoirs of ARGs, intra- and intercellular mobility of MGEs discovered in marine organisms is poorly characterized. Here, we show a new pattern of interspecies ARGs transfer involving a 'non-conjugative' integrative element. To identify active MGEs in a Vibrio ponticus isolate, we conducted whole-genome sequencing of a transconjugant obtained by mating between Escherichia coli and Vibrio ponticus. This revealed integration of a plasmid (designated pSEA1) into the chromosome, consisting of a self-transmissible plasmid backbone of the MOBH group, ARGs, and a 13.8-kb integrative element Tn6283. Molecular genetics analysis suggested a two-step gene transfer model. First, Tn6283 integrates into the recipient chromosome during suicidal plasmid transfer, followed by homologous recombination between the Tn6283 copy in the chromosome and that in the newly transferred pSEA1. Tn6283 is unusual among integrative elements in that it apparently does not encode transfer function and its excision barely generates unoccupied donor sites. Thus, its movement is analogous to the transposition of insertion sequences rather than to that of canonical integrative and conjugative elements. Overall, this study reveals the presence of a previously unrecognized type of MGE in a marine organism, highlighting diversity in the mode of interspecies gene transfer.

Two-photon calcium imaging from head-fixed Drosophila during optomotor walking behavior.

PubMed

Seelig, Johannes D; Chiappe, M Eugenia; Lott, Gus K; Dutta, Anirban; Osborne, Jason E; Reiser, Michael B; Jayaraman, Vivek

2010-07-01

Drosophila melanogaster is a model organism rich in genetic tools to manipulate and identify neural circuits involved in specific behaviors. Here we present a technique for two-photon calcium imaging in the central brain of head-fixed Drosophila walking on an air-supported ball. The ball's motion is tracked at high resolution and can be treated as a proxy for the fly's own movements. We used the genetically encoded calcium sensor, GCaMP3.0, to record from important elements of the motion-processing pathway, the horizontal-system lobula plate tangential cells (LPTCs) in the fly optic lobe. We presented motion stimuli to the tethered fly and found that calcium transients in horizontal-system neurons correlated with robust optomotor behavior during walking. Our technique allows both behavior and physiology in identified neurons to be monitored in a genetic model organism with an extensive repertoire of walking behaviors.

Adaptation of genetically monomorphic bacteria: evolution of copper resistance through multiple horizontal gene transfers of complex and versatile mobile genetic elements.

PubMed

Richard, D; Ravigné, V; Rieux, A; Facon, B; Boyer, C; Boyer, K; Grygiel, P; Javegny, S; Terville, M; Canteros, B I; Robène, I; Vernière, C; Chabirand, A; Pruvost, O; Lefeuvre, P

2017-04-01

Copper-based antimicrobial compounds are widely used to control plant bacterial pathogens. Pathogens have adapted in response to this selective pressure. Xanthomonas citri pv. citri, a major citrus pathogen causing Asiatic citrus canker, was first reported to carry plasmid-encoded copper resistance in Argentina. This phenotype was conferred by the copLAB gene system. The emergence of resistant strains has since been reported in Réunion and Martinique. Using microsatellite-based genotyping and copLAB PCR, we demonstrated that the genetic structure of the copper-resistant strains from these three regions was made up of two distant clusters and varied for the detection of copLAB amplicons. In order to investigate this pattern more closely, we sequenced six copper-resistant X. citri pv. citri strains from Argentina, Martinique and Réunion, together with reference copper-resistant Xanthomonas and Stenotrophomonas strains using long-read sequencing technology. Genes involved in copper resistance were found to be strain dependent with the novel identification in X. citri pv. citri of copABCD and a cus heavy metal efflux resistance-nodulation-division system. The genes providing the adaptive trait were part of a mobile genetic element similar to Tn3-like transposons and included in a conjugative plasmid. This indicates the system's great versatility. The mining of all available bacterial genomes suggested that, within the bacterial community, the spread of copper resistance associated with mobile elements and their plasmid environments was primarily restricted to the Xanthomonadaceae family. © 2017 John Wiley & Sons Ltd.

Rye B chromosomes encode a functional Argonaute-like protein with in vitro slicer activities similar to its A chromosome paralog.

PubMed

Ma, Wei; Gabriel, Tobias Sebastian; Martis, Mihaela Maria; Gursinsky, Torsten; Schubert, Veit; Vrána, Jan; Doležel, Jaroslav; Grundlach, Heidrun; Altschmied, Lothar; Scholz, Uwe; Himmelbach, Axel; Behrens, Sven-Erik; Banaei-Moghaddam, Ali Mohammad; Houben, Andreas

2017-01-01

B chromosomes (Bs) are supernumerary, dispensable parts of the nuclear genome, which appear in many different species of eukaryote. So far, Bs have been considered to be genetically inert elements without any functional genes. Our comparative transcriptome analysis and the detection of active RNA polymerase II (RNAPII) in the proximity of B chromatin demonstrate that the Bs of rye (Secale cereale) contribute to the transcriptome. In total, 1954 and 1218 B-derived transcripts with an open reading frame were expressed in generative and vegetative tissues, respectively. In addition to B-derived transposable element transcripts, a high percentage of short transcripts without detectable similarity to known proteins and gene fragments from A chromosomes (As) were found, suggesting an ongoing gene erosion process. In vitro analysis of the A- and B-encoded AGO4B protein variants demonstrated that both possess RNA slicer activity. These data demonstrate unambiguously the presence of a functional AGO4B gene on Bs and that these Bs carry both functional protein coding genes and pseudogene copies. Thus, B-encoded genes may provide an additional level of gene control and complexity in combination with their related A-located genes. Hence, physiological effects, associated with the presence of Bs, may partly be explained by the activity of B-located (pseudo)genes. © 2016 IPK Gatersleben. New Phytologist © 2016 New Phytologist Trust.

A Conserved Helicase Processivity Factor Is Needed for Conjugation and Replication of an Integrative and Conjugative Element

PubMed Central

Thomas, Jacob; Lee, Catherine A.; Grossman, Alan D.

2013-01-01

Integrative and conjugative elements (ICEs) are agents of horizontal gene transfer and have major roles in evolution and acquisition of new traits, including antibiotic resistances. ICEs are found integrated in a host chromosome and can excise and transfer to recipient bacteria via conjugation. Conjugation involves nicking of the ICE origin of transfer (oriT) by the ICE–encoded relaxase and transfer of the nicked single strand of ICE DNA. For ICEBs1 of Bacillus subtilis, nicking of oriT by the ICEBs1 relaxase NicK also initiates rolling circle replication. This autonomous replication of ICEBs1 is critical for stability of the excised element in growing cells. We found a conserved and previously uncharacterized ICE gene that is required for conjugation and replication of ICEBs1. Our results indicate that this gene, helP (formerly ydcP), encodes a helicase processivity factor that enables the host-encoded helicase PcrA to unwind the double-stranded ICEBs1 DNA. HelP was required for both conjugation and replication of ICEBs1, and HelP and NicK were the only ICEBs1 proteins needed for replication from ICEBs1 oriT. Using chromatin immunoprecipitation, we measured association of HelP, NicK, PcrA, and the host-encoded single-strand DNA binding protein Ssb with ICEBs1. We found that NicK was required for association of HelP and PcrA with ICEBs1 DNA. HelP was required for association of PcrA and Ssb with ICEBs1 regions distal, but not proximal, to oriT, indicating that PcrA needs HelP to progress beyond nicked oriT and unwind ICEBs1. In vitro, HelP directly stimulated the helicase activity of the PcrA homologue UvrD. Our findings demonstrate that HelP is a helicase processivity factor needed for efficient unwinding of ICEBs1 for conjugation and replication. Homologues of HelP and PcrA-type helicases are encoded on many known and putative ICEs. We propose that these factors are essential for ICE conjugation, replication, and genetic stability. PMID:23326247

Fast Kinetics of Calcium Signaling and Sensor Design

PubMed Central

Tang, Shen; Reddish, Florence; Zhuo, You; Yang, Jenny J.

2015-01-01

Fast calcium signaling is regulated by numerous calcium channels exhibiting high spatiotemporal profiles which are currently measured by fluorescent calcium sensors. There is still a strong need to improve the kinetics of genetically encoded calcium indicators (sensors) to capture calcium dynamics in the millisecond time frame. In this review, we summarize several major fast calcium signaling pathways and discuss the recent developments and application of genetically encoded calcium indicators to detect these pathways. A new class of genetically encoded calcium indicators designed with site-directed mutagenesis on the surface of beta-barrel fluorescent proteins to form a pentagonal bipyramidal-like calcium binding domain dramatically accelerates calcium binding kinetics. Furthermore, novel genetically encoded calcium indicators with significantly increased fluorescent lifetime change are advantageous in deep-field imaging with high light-scattering and notable morphology change. PMID:26151819

A mobile element in mutS drives hypermutation in a marine Vibrio

DOE PAGES

Chu, Nathaniel D.; Clarke, Sean A.; Timberlake, Sonia; ...

2017-02-07

Bacteria face a trade-off between genetic fidelity, which reduces deleterious mistakes in the genome, and genetic innovation, which allows organisms to adapt. Evidence suggests that many bacteria balance this trade-off by modulating their mutation rates, but few mechanisms have been described for such modulation. Following experimental evolution and whole-genome resequencing of the marine bacterium Vibrio splendidus 12B01, we discovered one such mechanism, which allows this bacterium to switch to an elevated mutation rate. This switch is driven by the excision of a mobile element residing in mutS, which encodes a DNA mismatch repair protein. When integrated within the bacterial genome,more » the mobile element provides independent promoter and translation start sequences for mutS—different from the bacterium’s original mutS promoter region—which allow the bacterium to make a functional mutS gene product. Excision of this mobile element rejoins the mutS gene with host promoter and translation start sequences but leaves a 2-bp deletion in the mutS sequence, resulting in a frameshift and a hypermutator phenotype. We further identified hundreds of clinical and environmental bacteria across Betaproteobacteria and Gammaproteobacteria that possess putative mobile elements within the same amino acid motif in mutS. In a subset of these bacteria, we detected excision of the element but not a frameshift mutation; the mobile elements leave an intact mutS coding sequence after excision. Finally, our findings reveal a novel mechanism by which one bacterium alters its mutation rate and hint at a possible evolutionary role for mobile elements within mutS in other bacteria.« less

A mobile element in mutS drives hypermutation in a marine Vibrio

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

Chu, Nathaniel D.; Clarke, Sean A.; Timberlake, Sonia

Bacteria face a trade-off between genetic fidelity, which reduces deleterious mistakes in the genome, and genetic innovation, which allows organisms to adapt. Evidence suggests that many bacteria balance this trade-off by modulating their mutation rates, but few mechanisms have been described for such modulation. Following experimental evolution and whole-genome resequencing of the marine bacterium Vibrio splendidus 12B01, we discovered one such mechanism, which allows this bacterium to switch to an elevated mutation rate. This switch is driven by the excision of a mobile element residing in mutS, which encodes a DNA mismatch repair protein. When integrated within the bacterial genome,more » the mobile element provides independent promoter and translation start sequences for mutS—different from the bacterium’s original mutS promoter region—which allow the bacterium to make a functional mutS gene product. Excision of this mobile element rejoins the mutS gene with host promoter and translation start sequences but leaves a 2-bp deletion in the mutS sequence, resulting in a frameshift and a hypermutator phenotype. We further identified hundreds of clinical and environmental bacteria across Betaproteobacteria and Gammaproteobacteria that possess putative mobile elements within the same amino acid motif in mutS. In a subset of these bacteria, we detected excision of the element but not a frameshift mutation; the mobile elements leave an intact mutS coding sequence after excision. Finally, our findings reveal a novel mechanism by which one bacterium alters its mutation rate and hint at a possible evolutionary role for mobile elements within mutS in other bacteria.« less

An Island Grouping Genetic Algorithm for Fuzzy Partitioning Problems

PubMed Central

Salcedo-Sanz, S.; Del Ser, J.; Geem, Z. W.

2014-01-01

This paper presents a novel fuzzy clustering technique based on grouping genetic algorithms (GGAs), which are a class of evolutionary algorithms especially modified to tackle grouping problems. Our approach hinges on a GGA devised for fuzzy clustering by means of a novel encoding of individuals (containing elements and clusters sections), a new fitness function (a superior modification of the Davies Bouldin index), specially tailored crossover and mutation operators, and the use of a scheme based on a local search and a parallelization process, inspired from an island-based model of evolution. The overall performance of our approach has been assessed over a number of synthetic and real fuzzy clustering problems with different objective functions and distance measures, from which it is concluded that the proposed approach shows excellent performance in all cases. PMID:24977235

Hybrid concatenated codes and iterative decoding

NASA Technical Reports Server (NTRS)

Divsalar, Dariush (Inventor); Pollara, Fabrizio (Inventor)

2000-01-01

Several improved turbo code apparatuses and methods. The invention encompasses several classes: (1) A data source is applied to two or more encoders with an interleaver between the source and each of the second and subsequent encoders. Each encoder outputs a code element which may be transmitted or stored. A parallel decoder provides the ability to decode the code elements to derive the original source information d without use of a received data signal corresponding to d. The output may be coupled to a multilevel trellis-coded modulator (TCM). (2) A data source d is applied to two or more encoders with an interleaver between the source and each of the second and subsequent encoders. Each of the encoders outputs a code element. In addition, the original data source d is output from the encoder. All of the output elements are coupled to a TCM. (3) At least two data sources are applied to two or more encoders with an interleaver between each source and each of the second and subsequent encoders. The output may be coupled to a TCM. (4) At least two data sources are applied to two or more encoders with at least two interleavers between each source and each of the second and subsequent encoders. (5) At least one data source is applied to one or more serially linked encoders through at least one interleaver. The output may be coupled to a TCM. The invention includes a novel way of terminating a turbo coder.

The cost of copy number in a selfish genetic element: the 2-μm plasmid of Saccharomyces cerevisiae.

PubMed

Harrison, Ellie; Koufopanou, V; Burt, A; MacLean, R C

2012-11-01

Many autonomously replicating genetic elements exist as multiple copies within the cell. The copy number of these elements is often assumed to have important fitness consequences for both element and host, yet the forces shaping its evolution are not well understood. The 2 μm is a multicopy plasmid of Saccharomyces yeasts, encoding just four genes that are solely involved in plasmid replication. One simple model for the fitness relationship between yeasts and 2 μm is that plasmid copy number evolves as a trade-off between selection for increased vertical transmission, favouring high copy number, and selection for decreased virulence, favouring low copy number. To test this model, we experimentally manipulated the copy number of the plasmid and directly measured the fitness cost, in terms of growth rate reduction, associated with high plasmid copy number. We find that the fitness burden imposed by the 2 μm increases with plasmid copy number, such that each copy imposes a fitness burden of 0.17% (± 0.008%), greatly exceeding the cost expected for it to be stably maintained in yeast populations. Our results demonstrate the crucial importance of copy number in the evolution of yeast per 2 μm associations and pave the way for future studies examining how selection can shape the cost of multicopy elements. © 2012 The Authors. Journal of Evolutionary Biology © 2012 European Society For Evolutionary Biology.

oriTfinder: a web-based tool for the identification of origin of transfers in DNA sequences of bacterial mobile genetic elements.

PubMed

Li, Xiaobin; Xie, Yingzhou; Liu, Meng; Tai, Cui; Sun, Jingyong; Deng, Zixin; Ou, Hong-Yu

2018-05-04

oriTfinder is a web server that facilitates the rapid identification of the origin of transfer site (oriT) of a conjugative plasmid or chromosome-borne integrative and conjugative element. The utilized back-end database oriTDB was built upon more than one thousand known oriT regions of bacterial mobile genetic elements (MGEs) as well as the known MGE-encoding relaxases and type IV coupling proteins (T4CP). With a combination of similarity searches for the oriTDB-archived oriT nucleotide sequences and the co-localization of the flanking relaxase homologous genes, the oriTfinder can predict the oriT region with high accuracy in the DNA sequence of a bacterial plasmid or chromosome in minutes. The server also detects the other transfer-related modules, including the potential relaxase gene, T4CP gene and the type IV secretion system gene cluster, and the putative genes coding for virulence factors and acquired antibiotic resistance determinants. oriTfinder may contribute to meeting the increasing demands of re-annotations for bacterial conjugative, mobilizable or non-transferable elements and aid in the rapid risk accession of disease-relevant trait dissemination in pathogenic bacteria of interest. oriTfinder is freely available to all users without any login requirement at http://bioinfo-mml.sjtu.edu.cn/oriTfinder.

Investigation of Clostridium botulinum group III's mobilome content.

PubMed

Woudstra, Cédric; Le Maréchal, Caroline; Souillard, Rozenn; Anniballi, Fabrizio; Auricchio, Bruna; Bano, Luca; Bayon-Auboyer, Marie-Hélène; Koene, Miriam; Mermoud, Isabelle; Brito, Roseane B; Lobato, Francisco C F; Silva, Rodrigo O S; Dorner, Martin B; Fach, Patrick

2018-02-01

Clostridium botulinum group III is mainly responsible for botulism in animals. It could lead to high animal mortality rates and, therefore, represents a major environmental and economic concern. Strains of this group harbor the botulinum toxin locus on an unstable bacteriophage. Since the release of the first complete C. botulinum group III genome sequence (strain BKT015925), strains have been found to contain others mobile elements encoding for toxin components. In this study, seven assays targeting toxin genes present on the genetic mobile elements of C. botulinum group III were developed with the objective to better characterize C. botulinum group III strains. The investigation of 110 C. botulinum group III strains and 519 naturally contaminated samples collected during botulism outbreaks in Europe showed alpha-toxin and C2-I/C2-II markers to be systematically associated with type C/D bont-positive samples, which may indicate an important role of these elements in the pathogenicity mechanisms. On the contrary, bont type D/C strains and the related positive samples appeared to contain almost none of the markers tested. Interestingly, 31 bont-negative samples collected on farms after a botulism outbreak revealed to be positive for some of the genetic mobile elements tested. This suggests loss of the bont phage, either in farm environment after the outbreak or during laboratory handling. Copyright © 2017 Elsevier Ltd. All rights reserved.

Virulence determinants, drug resistance and mobile genetic elements of Laribacter hongkongensis: a genome-wide analysis

PubMed Central

2011-01-01

Background Laribacter hongkongensis is associated with community-acquired gastroenteritis and traveler's diarrhea. In this study, we performed an in-depth annotation of the genes in its genome related to the various steps in the infective process, drug resistance and mobile genetic elements. Results For acid and bile resistance, L. hongkongensis possessed a urease gene cassette, two arc gene clusters and bile salt efflux systems. For intestinal colonization, it possessed a putative adhesin of the autotransporter family homologous to those of diffusely adherent Escherichia coli (E. coli) and enterotoxigenic E. coli. To evade from host defense, it possessed superoxide dismutase and catalases. For lipopolysaccharide biosynthesis, it possessed the same set of genes that encode enzymes for synthesizing lipid A, two Kdo units and heptose units as E. coli, but different genes for its symmetrical acylation pattern, and nine genes for polysaccharide side chains biosynthesis. It contained a number of CDSs that encode putative cell surface acting (RTX toxin and hemolysins) and intracellular cytotoxins (patatin-like proteins) and enzymes for invasion (outer membrane phospholipase A). It contained a broad variety of antibiotic resistance-related genes, including genes related to β-lactam (n = 10) and multidrug efflux (n = 54). It also contained eight prophages, 17 other phage-related CDSs and 26 CDSs for transposases. Conclusions The L. hongkongensis genome possessed genes for acid and bile resistance, intestinal mucosa colonization, evasion of host defense and cytotoxicity and invasion. A broad variety of antibiotic resistance or multidrug resistance genes, a high number of prophages, other phage-related CDSs and CDSs for transposases, were also identified. PMID:21711902

CRISPR/Cas9 and active genetics-based trans-species replacement of the endogenous Drosophila kni-L2 CRM reveals unexpected complexity.

PubMed

Xu, Xiang-Ru Shannon; Gantz, Valentino Matteo; Siomava, Natalia; Bier, Ethan

2017-12-23

The knirps ( kni ) locus encodes transcription factors required for induction of the L2 wing vein in Drosophila . Here, we employ diverse CRISPR/Cas9 genome editing tools to generate a series of targeted lesions within the endogenous cis-regulatory module (CRM) required for kni expression in the L2 vein primordium. Phenotypic analysis of these ' in locus ' mutations based on both expression of Kni protein and adult wing phenotypes, reveals novel unexpected features of L2-CRM function including evidence for a chromosome pairing-dependent process that promotes transcription. We also demonstrate that self-propagating active genetic elements (CopyCat elements) can efficiently delete and replace the L2-CRM with orthologous sequences from other divergent fly species. Wing vein phenotypes resulting from these trans-species enhancer replacements parallel features of the respective donor fly species. This highly sensitive phenotypic readout of enhancer function in a native genomic context reveals novel features of CRM function undetected by traditional reporter gene analysis. © 2017, Xu et al.

CRISPR/Cas9 and active genetics-based trans-species replacement of the endogenous Drosophila kni-L2 CRM reveals unexpected complexity

PubMed Central

Siomava, Natalia

2017-01-01

The knirps (kni) locus encodes transcription factors required for induction of the L2 wing vein in Drosophila. Here, we employ diverse CRISPR/Cas9 genome editing tools to generate a series of targeted lesions within the endogenous cis-regulatory module (CRM) required for kni expression in the L2 vein primordium. Phenotypic analysis of these ‘in locus’ mutations based on both expression of Kni protein and adult wing phenotypes, reveals novel unexpected features of L2-CRM function including evidence for a chromosome pairing-dependent process that promotes transcription. We also demonstrate that self-propagating active genetic elements (CopyCat elements) can efficiently delete and replace the L2-CRM with orthologous sequences from other divergent fly species. Wing vein phenotypes resulting from these trans-species enhancer replacements parallel features of the respective donor fly species. This highly sensitive phenotypic readout of enhancer function in a native genomic context reveals novel features of CRM function undetected by traditional reporter gene analysis. PMID:29274230

Targeted gene insertion for molecular medicine.

PubMed

Voigt, Katrin; Izsvák, Zsuzsanna; Ivics, Zoltán

2008-11-01

Genomic insertion of a functional gene together with suitable transcriptional regulatory elements is often required for long-term therapeutical benefit in gene therapy for several genetic diseases. A variety of integrating vectors for gene delivery exist. Some of them exhibit random genomic integration, whereas others have integration preferences based on attributes of the targeted site, such as primary DNA sequence and physical structure of the DNA, or through tethering to certain DNA sequences by host-encoded cellular factors. Uncontrolled genomic insertion bears the risk of the transgene being silenced due to chromosomal position effects, and can lead to genotoxic effects due to mutagenesis of cellular genes. None of the vector systems currently used in either preclinical experiments or clinical trials displays sufficient preferences for target DNA sequences that would ensure appropriate and reliable expression of the transgene and simultaneously prevent hazardous side effects. We review in this paper the advantages and disadvantages of both viral and non-viral gene delivery technologies, discuss mechanisms of target site selection of integrating genetic elements (viruses and transposons), and suggest distinct molecular strategies for targeted gene delivery.

Endoribonuclease type II toxin-antitoxin systems: functional or selfish?

PubMed

Ramisetty, Bhaskar Chandra Mohan; Santhosh, Ramachandran Sarojini

2017-07-01

Most bacterial genomes have multiple type II toxin-antitoxin systems (TAs) that encode two proteins which are referred to as a toxin and an antitoxin. Toxins inhibit a cellular process, while the interaction of the antitoxin with the toxin attenuates the toxin's activity. Endoribonuclease-encoding TAs cleave RNA in a sequence-dependent fashion, resulting in translational inhibition. To account for their prevalence and retention by bacterial genomes, TAs are credited with clinically significant phenomena, such as bacterial programmed cell death, persistence, biofilms and anti-addiction to plasmids. However, the programmed cell death and persistence hypotheses have been challenged because of conceptual, methodological and/or strain issues. In an alternative view, chromosomal TAs seem to be retained by virtue of addiction at two levels: via a poison-antidote combination (TA proteins) and via transcriptional reprogramming of the downstream core gene (due to integration). Any perturbation in the chromosomal TA operons could cause fitness loss due to polar effects on the downstream genes and hence be detrimental under natural conditions. The endoribonucleases encoding chromosomal TAs are most likely selfish DNA as they are retained by bacterial genomes, even though TAs do not confer a direct advantage via the TA proteins. TAs are likely used by various replicons as 'genetic arms' that allow the maintenance of themselves and associated genetic elements. TAs seem to be the 'selfish arms' that make the best use of the 'arms race' between bacterial genomes and plasmids.

Coordinated regulation of accessory genetic elements produces cyclic di-nucleotides for V. cholerae virulence.

PubMed

Davies, Bryan W; Bogard, Ryan W; Young, Travis S; Mekalanos, John J

2012-04-13

The function of the Vibrio 7(th) pandemic island-1 (VSP-1) in cholera pathogenesis has remained obscure. Utilizing chromatin immunoprecipitation sequencing and RNA sequencing to map the regulon of the master virulence regulator ToxT, we identify a TCP island-encoded small RNA that reduces the expression of a previously unrecognized VSP-1-encoded transcription factor termed VspR. VspR modulates the expression of several VSP-1 genes including one that encodes a novel class of di-nucleotide cyclase (DncV), which preferentially synthesizes a previously undescribed hybrid cyclic AMP-GMP molecule. We show that DncV is required for efficient intestinal colonization and downregulates V. cholerae chemotaxis, a phenotype previously associated with hyperinfectivity. This pathway couples the actions of previously disparate genomic islands, defines VSP-1 as a pathogenicity island in V. cholerae, and implicates its occurrence in 7(th) pandemic strains as a benefit for host adaptation through the production of a regulatory cyclic di-nucleotide. Copyright © 2012 Elsevier Inc. All rights reserved.

Transgenic mouse models in the study of reproduction: insights into GATA protein function.

PubMed

Tevosian, Sergei G

2014-07-01

For the past 2 decades, transgenic technology in mice has allowed for an unprecedented insight into the transcriptional control of reproductive development and function. The key factor among the mouse genetic tools that made this rapid advance possible is a conditional transgenic approach, a particularly versatile method of creating gene deletions and substitutions in the mouse genome. A centerpiece of this strategy is an enzyme, Cre recombinase, which is expressed from defined DNA regulatory elements that are active in the tissue of choice. The regulatory DNA element (either genetically engineered or natural) assures Cre expression only in predetermined cell types, leading to the guided deletion of genetically modified (flanked by loxP or 'floxed' by loxP) gene loci. This review summarizes and compares the studies in which genes encoding GATA family transcription factors were targeted either globally or by Cre recombinases active in the somatic cells of ovaries and testes. The conditional gene loss experiments require detailed knowledge of the spatial and temporal expression of Cre activity, and the challenges in interpreting the outcomes are highlighted. These studies also expose the complexity of GATA-dependent regulation of gonadal gene expression and suggest that gene function is highly context dependent. © 2014 Society for Reproduction and Fertility.

E622, a miniature, virulence-associated mobile element.

PubMed

Stavrinides, John; Kirzinger, Morgan W B; Beasley, Federico C; Guttman, David S

2012-01-01

Miniature inverted terminal repeat elements (MITEs) are nonautonomous mobile elements that have a significant impact on bacterial evolution. Here we characterize E622, a 611-bp virulence-associated MITE from Pseudomonas syringae, which contains no coding region but has almost perfect 168-bp inverted repeats. Using an antibiotic coupling assay, we show that E622 is transposable and can mobilize an antibiotic resistance gene contained between its borders. Its predicted parent element, designated TnE622, has a typical transposon structure with a three-gene operon, consisting of resolvase, integrase, and exeA-like genes, which is bounded by the same terminal inverted repeats as E622. A broader genome level survey of the E622/TnE622 inverted repeats identified homologs in Pseudomonas, Salmonella, Shewanella, Erwinia, Pantoea, and the cyanobacteria Nostoc and Cyanothece, many of which appear to encompass known virulence genes, including genes encoding toxins, enzymes, and type III secreted effectors. Its association with niche-specific genetic determinants, along with its persistence and evolutionary diversification, indicates that this mobile element family has played a prominent role in the evolution of many agriculturally and clinically relevant pathogenic bacteria.

A DNase encoded by integrated element CJIE1 inhibits natural transformation of Campylobacter jejuni.

PubMed

Gaasbeek, Esther J; Wagenaar, Jaap A; Guilhabert, Magalie R; Wösten, Marc M S M; van Putten, Jos P M; van der Graaf-van Bloois, Linda; Parker, Craig T; van der Wal, Fimme J

2009-04-01

The species Campylobacter jejuni is considered naturally competent for DNA uptake and displays strong genetic diversity. Nevertheless, nonnaturally transformable strains and several relatively stable clonal lineages exist. In the present study, the molecular mechanism responsible for the nonnatural transformability of a subset of C. jejuni strains was investigated. Comparative genome hybridization indicated that C. jejuni Mu-like prophage integrated element 1 (CJIE1) was more abundant in nonnaturally transformable C. jejuni strains than in naturally transformable strains. Analysis of CJIE1 indicated the presence of dns (CJE0256), which is annotated as a gene encoding an extracellular DNase. DNase assays using a defined dns mutant and a dns-negative strain expressing Dns from a plasmid indicated that Dns is an endogenous DNase. The DNA-hydrolyzing activity directly correlated with the natural transformability of the knockout mutant and the dns-negative strain expressing Dns from a plasmid. Analysis of a broader set of strains indicated that the majority of nonnaturally transformable strains expressed DNase activity, while all naturally competent strains lacked this activity. The inhibition of natural transformation in C. jejuni via endogenous DNase activity may contribute to the formation of stable lineages in the C. jejuni population.

The Genome of the Western Clawed Frog Xenopus tropicalis

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

Hellsten, Uffe; Harland, Richard M.; Gilchrist, Michael J.

2009-10-01

The western clawed frog Xenopus tropicalis is an important model for vertebrate development that combines experimental advantages of the African clawed frog Xenopus laevis with more tractable genetics. Here we present a draft genome sequence assembly of X. tropicalis. This genome encodes over 20,000 protein-coding genes, including orthologs of at least 1,700 human disease genes. Over a million expressed sequence tags validated the annotation. More than one-third of the genome consists of transposable elements, with unusually prevalent DNA transposons. Like other tetrapods, the genome contains gene deserts enriched for conserved non-coding elements. The genome exhibits remarkable shared synteny with humanmore » and chicken over major parts of large chromosomes, broken by lineage-specific chromosome fusions and fissions, mainly in the mammalian lineage.« less

Natural history of the ERVWE1 endogenous retroviral locus

PubMed Central

Bonnaud, Bertrand; Beliaeff, Jean; Bouton, Olivier; Oriol, Guy; Duret, Laurent; Mallet, François

2005-01-01

Background The human HERV-W multicopy family includes a unique proviral locus, termed ERVWE1, whose full-length envelope ORF was preserved through evolution by the action of a selective pressure. The encoded Env protein (Syncytin) is involved in hominoid placental physiology. Results In order to infer the natural history of this domestication process, a comparative genomic analysis of the human 7q21.2 syntenic regions in eutherians was performed. In primates, this region was progressively colonized by LTR-elements, leading to two different evolutionary pathways in Cercopithecidae and Hominidae, a genetic drift versus a domestication, respectively. Conclusion The preservation in Hominoids of a genomic structure consisting in the juxtaposition of a retrotransposon-derived MaLR LTR and the ERVWE1 provirus suggests a functional link between both elements. PMID:16176588

Dissection of affinity captured LINE-1 macromolecular complexes

PubMed Central

Mita, Paolo; Jiang, Hua; Adney, Emily M; Wudzinska, Aleksandra; Badri, Sana; Ischenko, Dmitry; Eng, George; Burns, Kathleen H; Fenyö, David; Chait, Brian T; Alexeev, Dmitry; Rout, Michael P; Boeke, Jef D

2018-01-01

Long Interspersed Nuclear Element-1 (LINE-1, L1) is a mobile genetic element active in human genomes. L1-encoded ORF1 and ORF2 proteins bind L1 RNAs, forming ribonucleoproteins (RNPs). These RNPs interact with diverse host proteins, some repressive and others required for the L1 lifecycle. Using differential affinity purifications, quantitative mass spectrometry, and next generation RNA sequencing, we have characterized the proteins and nucleic acids associated with distinctive, enzymatically active L1 macromolecular complexes. Among them, we describe a cytoplasmic intermediate that we hypothesize to be the canonical ORF1p/ORF2p/L1-RNA-containing RNP, and we describe a nuclear population containing ORF2p, but lacking ORF1p, which likely contains host factors participating in target-primed reverse transcription. PMID:29309035

Breast cancer risk variants at 6q25 display different phenotype associations and regulate ESR1, RMND1 and CCDC170.

PubMed

Dunning, Alison M; Michailidou, Kyriaki; Kuchenbaecker, Karoline B; Thompson, Deborah; French, Juliet D; Beesley, Jonathan; Healey, Catherine S; Kar, Siddhartha; Pooley, Karen A; Lopez-Knowles, Elena; Dicks, Ed; Barrowdale, Daniel; Sinnott-Armstrong, Nicholas A; Sallari, Richard C; Hillman, Kristine M; Kaufmann, Susanne; Sivakumaran, Haran; Moradi Marjaneh, Mahdi; Lee, Jason S; Hills, Margaret; Jarosz, Monika; Drury, Suzie; Canisius, Sander; Bolla, Manjeet K; Dennis, Joe; Wang, Qin; Hopper, John L; Southey, Melissa C; Broeks, Annegien; Schmidt, Marjanka K; Lophatananon, Artitaya; Muir, Kenneth; Beckmann, Matthias W; Fasching, Peter A; Dos-Santos-Silva, Isabel; Peto, Julian; Sawyer, Elinor J; Tomlinson, Ian; Burwinkel, Barbara; Marme, Frederik; Guénel, Pascal; Truong, Thérèse; Bojesen, Stig E; Flyger, Henrik; González-Neira, Anna; Perez, Jose I A; Anton-Culver, Hoda; Eunjung, Lee; Arndt, Volker; Brenner, Hermann; Meindl, Alfons; Schmutzler, Rita K; Brauch, Hiltrud; Hamann, Ute; Aittomäki, Kristiina; Blomqvist, Carl; Ito, Hidemi; Matsuo, Keitaro; Bogdanova, Natasha; Dörk, Thilo; Lindblom, Annika; Margolin, Sara; Kosma, Veli-Matti; Mannermaa, Arto; Tseng, Chiu-Chen; Wu, Anna H; Lambrechts, Diether; Wildiers, Hans; Chang-Claude, Jenny; Rudolph, Anja; Peterlongo, Paolo; Radice, Paolo; Olson, Janet E; Giles, Graham G; Milne, Roger L; Haiman, Christopher A; Henderson, Brian E; Goldberg, Mark S; Teo, Soo H; Yip, Cheng Har; Nord, Silje; Borresen-Dale, Anne-Lise; Kristensen, Vessela; Long, Jirong; Zheng, Wei; Pylkäs, Katri; Winqvist, Robert; Andrulis, Irene L; Knight, Julia A; Devilee, Peter; Seynaeve, Caroline; Figueroa, Jonine; Sherman, Mark E; Czene, Kamila; Darabi, Hatef; Hollestelle, Antoinette; van den Ouweland, Ans M W; Humphreys, Keith; Gao, Yu-Tang; Shu, Xiao-Ou; Cox, Angela; Cross, Simon S; Blot, William; Cai, Qiuyin; Ghoussaini, Maya; Perkins, Barbara J; Shah, Mitul; Choi, Ji-Yeob; Kang, Daehee; Lee, Soo Chin; Hartman, Mikael; Kabisch, Maria; Torres, Diana; Jakubowska, Anna; Lubinski, Jan; Brennan, Paul; Sangrajrang, Suleeporn; Ambrosone, Christine B; Toland, Amanda E; Shen, Chen-Yang; Wu, Pei-Ei; Orr, Nick; Swerdlow, Anthony; McGuffog, Lesley; Healey, Sue; Lee, Andrew; Kapuscinski, Miroslav; John, Esther M; Terry, Mary Beth; Daly, Mary B; Goldgar, David E; Buys, Saundra S; Janavicius, Ramunas; Tihomirova, Laima; Tung, Nadine; Dorfling, Cecilia M; van Rensburg, Elizabeth J; Neuhausen, Susan L; Ejlertsen, Bent; Hansen, Thomas V O; Osorio, Ana; Benitez, Javier; Rando, Rachel; Weitzel, Jeffrey N; Bonanni, Bernardo; Peissel, Bernard; Manoukian, Siranoush; Papi, Laura; Ottini, Laura; Konstantopoulou, Irene; Apostolou, Paraskevi; Garber, Judy; Rashid, Muhammad Usman; Frost, Debra; Izatt, Louise; Ellis, Steve; Godwin, Andrew K; Arnold, Norbert; Niederacher, Dieter; Rhiem, Kerstin; Bogdanova-Markov, Nadja; Sagne, Charlotte; Stoppa-Lyonnet, Dominique; Damiola, Francesca; Sinilnikova, Olga M; Mazoyer, Sylvie; Isaacs, Claudine; Claes, Kathleen B M; De Leeneer, Kim; de la Hoya, Miguel; Caldes, Trinidad; Nevanlinna, Heli; Khan, Sofia; Mensenkamp, Arjen R; Hooning, Maartje J; Rookus, Matti A; Kwong, Ava; Olah, Edith; Diez, Orland; Brunet, Joan; Pujana, Miquel Angel; Gronwald, Jacek; Huzarski, Tomasz; Barkardottir, Rosa B; Laframboise, Rachel; Soucy, Penny; Montagna, Marco; Agata, Simona; Teixeira, Manuel R; Park, Sue Kyung; Lindor, Noralane; Couch, Fergus J; Tischkowitz, Marc; Foretova, Lenka; Vijai, Joseph; Offit, Kenneth; Singer, Christian F; Rappaport, Christine; Phelan, Catherine M; Greene, Mark H; Mai, Phuong L; Rennert, Gad; Imyanitov, Evgeny N; Hulick, Peter J; Phillips, Kelly-Anne; Piedmonte, Marion; Mulligan, Anna Marie; Glendon, Gord; Bojesen, Anders; Thomassen, Mads; Caligo, Maria A; Yoon, Sook-Yee; Friedman, Eitan; Laitman, Yael; Borg, Ake; von Wachenfeldt, Anna; Ehrencrona, Hans; Rantala, Johanna; Olopade, Olufunmilayo I; Ganz, Patricia A; Nussbaum, Robert L; Gayther, Simon A; Nathanson, Katherine L; Domchek, Susan M; Arun, Banu K; Mitchell, Gillian; Karlan, Beth Y; Lester, Jenny; Maskarinec, Gertraud; Woolcott, Christy; Scott, Christopher; Stone, Jennifer; Apicella, Carmel; Tamimi, Rulla; Luben, Robert; Khaw, Kay-Tee; Helland, Åslaug; Haakensen, Vilde; Dowsett, Mitch; Pharoah, Paul D P; Simard, Jacques; Hall, Per; García-Closas, Montserrat; Vachon, Celine; Chenevix-Trench, Georgia; Antoniou, Antonis C; Easton, Douglas F; Edwards, Stacey L

2016-04-01

We analyzed 3,872 common genetic variants across the ESR1 locus (encoding estrogen receptor α) in 118,816 subjects from three international consortia. We found evidence for at least five independent causal variants, each associated with different phenotype sets, including estrogen receptor (ER(+) or ER(-)) and human ERBB2 (HER2(+) or HER2(-)) tumor subtypes, mammographic density and tumor grade. The best candidate causal variants for ER(-) tumors lie in four separate enhancer elements, and their risk alleles reduce expression of ESR1, RMND1 and CCDC170, whereas the risk alleles of the strongest candidates for the remaining independent causal variant disrupt a silencer element and putatively increase ESR1 and RMND1 expression.

Breast cancer risk variants at 6q25 display different phenotype associations and regulate ESR1, RMND1 and CCDC170

PubMed Central

Dunning, Alison M; Michailidou, Kyriaki; Kuchenbaecker, Karoline B; Thompson, Deborah; French, Juliet D; Beesley, Jonathan; Healey, Catherine S; Kar, Siddhartha; Pooley, Karen A; Lopez-Knowles, Elena; Dicks, Ed; Barrowdale, Daniel; Sinnott-Armstrong, Nicholas A; Sallari, Richard C; Hillman, Kristine M; Kaufmann, Susanne; Sivakumaran, Haran; Marjaneh, Mahdi Moradi; Lee, Jason S; Hills, Margaret; Jarosz, Monika; Drury, Suzie; Canisius, Sander; Bolla, Manjeet K; Dennis, Joe; Wang, Qin; Hopper, John L; Southey, Melissa C; Broeks, Annegien; Schmidt, Marjanka K; Lophatananon, Artitaya; Muir, Kenneth; Beckmann, Matthias W; Fasching, Peter A; dos-Santos-Silva, Isabel; Peto, Julian; Sawyer, Elinor J; Tomlinson, Ian; Burwinkel, Barbara; Marme, Frederik; Guénel, Pascal; Truong, Thérèse; Bojesen, Stig E; Flyger, Henrik; González-Neira, Anna; Perez, Jose I A; Anton-Culver, Hoda; Eunjung, Lee; Arndt, Volker; Brenner, Hermann; Meindl, Alfons; Schmutzler, Rita K; Brauch, Hiltrud; Hamann, Ute; Aittomäki, Kristiina; Blomqvist, Carl; Ito, Hidemi; Matsuo, Keitaro; Bogdanova, Natasha; Dörk, Thilo; Lindblom, Annika; Margolin, Sara; Kosma, Veli-Matti; Mannermaa, Arto; Tseng, Chiu-chen; Wu, Anna H; Lambrechts, Diether; Wildiers, Hans; Chang-Claude, Jenny; Rudolph, Anja; Peterlongo, Paolo; Radice, Paolo; Olson, Janet E; Giles, Graham G; Milne, Roger L; Haiman, Christopher A; Henderson, Brian E; Goldberg, Mark S; Teo, Soo H; Yip, Cheng Har; Nord, Silje; Borresen-Dale, Anne-Lise; Kristensen, Vessela; Long, Jirong; Zheng, Wei; Pylkäs, Katri; Winqvist, Robert; Andrulis, Irene L; Knight, Julia A; Devilee, Peter; Seynaeve, Caroline; Figueroa, Jonine; Sherman, Mark E; Czene, Kamila; Darabi, Hatef; Hollestelle, Antoinette; van den Ouweland, Ans M W; Humphreys, Keith; Gao, Yu-Tang; Shu, Xiao-Ou; Cox, Angela; Cross, Simon S; Blot, William; Cai, Qiuyin; Ghoussaini, Maya; Perkins, Barbara J; Shah, Mitul; Choi, Ji-Yeob; Kang, Daehee; Lee, Soo Chin; Hartman, Mikael; Kabisch, Maria; Torres, Diana; Jakubowska, Anna; Lubinski, Jan; Brennan, Paul; Sangrajrang, Suleeporn; Ambrosone, Christine B; Toland, Amanda E; Shen, Chen-Yang; Wu, Pei-Ei; Orr, Nick; Swerdlow, Anthony; McGuffog, Lesley; Healey, Sue; Lee, Andrew; Kapuscinski, Miroslav; John, Esther M; Terry, Mary Beth; Daly, Mary B; Goldgar, David E; Buys, Saundra S; Janavicius, Ramunas; Tihomirova, Laima; Tung, Nadine; Dorfling, Cecilia M; van Rensburg, Elizabeth J; Neuhausen, Susan L; Ejlertsen, Bent; Hansen, Thomas V O; Osorio, Ana; Benitez, Javier; Rando, Rachel; Weitzel, Jeffrey N; Bonanni, Bernardo; Peissel, Bernard; Manoukian, Siranoush; Papi, Laura; Ottini, Laura; Konstantopoulou, Irene; Apostolou, Paraskevi; Garber, Judy; Rashid, Muhammad Usman; Frost, Debra; Izatt, Louise; Ellis, Steve; Godwin, Andrew K; Arnold, Norbert; Niederacher, Dieter; Rhiem, Kerstin; Bogdanova-Markov, Nadja; Sagne, Charlotte; Stoppa-Lyonnet, Dominique; Damiola, Francesca; Sinilnikova, Olga M; Mazoyer, Sylvie; Isaacs, Claudine; Claes, Kathleen B M; De Leeneer, Kim; de la Hoya, Miguel; Caldes, Trinidad; Nevanlinna, Heli; Khan, Sofia; Mensenkamp, Arjen R; Hooning, Maartje J; Rookus, Matti A; Kwong, Ava; Olah, Edith; Diez, Orland; Brunet, Joan; Pujana, Miquel Angel; Gronwald, Jacek; Huzarski, Tomasz; Barkardottir, Rosa B; Laframboise, Rachel; Soucy, Penny; Montagna, Marco; Agata, Simona; Teixeira, Manuel R; Park, Sue Kyung; Lindor, Noralane; Couch, Fergus J; Tischkowitz, Marc; Foretova, Lenka; Vijai, Joseph; Offit, Kenneth; Singer, Christian F; Rappaport, Christine; Phelan, Catherine M; Greene, Mark H; Mai, Phuong L; Rennert, Gad; Imyanitov, Evgeny N; Hulick, Peter J; Phillips, Kelly-Anne; Piedmonte, Marion; Mulligan, Anna Marie; Glendon, Gord; Bojesen, Anders; Thomassen, Mads; Caligo, Maria A; Yoon, Sook-Yee; Friedman, Eitan; Laitman, Yael; Borg, Ake; von Wachenfeldt, Anna; Ehrencrona, Hans; Rantala, Johanna; Olopade, Olufunmilayo I; Ganz, Patricia A; Nussbaum, Robert L; Gayther, Simon A; Nathanson, Katherine L; Domchek, Susan M; Arun, Banu K; Mitchell, Gillian; Karlan, Beth Y; Lester, Jenny; Maskarinec, Gertraud; Woolcott, Christy; Scott, Christopher; Stone, Jennifer; Apicella, Carmel; Tamimi, Rulla; Luben, Robert; Khaw, Kay-Tee; Helland, Åslaug; Haakensen, Vilde; Dowsett, Mitch; Pharoah, Paul D P; Simard, Jacques; Hall, Per; García-Closas, Montserrat; Vachon, Celine; Chenevix-Trench, Georgia; Antoniou, Antonis C; Easton, Douglas F; Edwards, Stacey L

2016-01-01

We analyzed 3,872 common genetic variants across the ESR1 locus (encoding estrogen receptor α) in 118,816 subjects from three international consortia. We found evidence for at least five independent causal variants, each associated with different phenotype sets, including estrogen receptor (ER+ or ER−) and human ERBB2 (HER2+ or HER2−) tumor subtypes, mammographic density and tumor grade. The best candidate causal variants for ER− tumors lie in four separate enhancer elements, and their risk alleles reduce expression of ESR1, RMND1 and CCDC170, whereas the risk alleles of the strongest candidates for the remaining independent causal variant disrupt a silencer element and putatively increase ESR1 and RMND1 expression. PMID:26928228

Localization of a bacterial group II intron-encoded protein in human cells.

PubMed

Reinoso-Colacio, Mercedes; García-Rodríguez, Fernando Manuel; García-Cañadas, Marta; Amador-Cubero, Suyapa; García Pérez, José Luis; Toro, Nicolás

2015-08-05

Group II introns are mobile retroelements that self-splice from precursor RNAs to form ribonucleoparticles (RNP), which can invade new specific genomic DNA sites. This specificity can be reprogrammed, for insertion into any desired DNA site, making these introns useful tools for bacterial genetic engineering. However, previous studies have suggested that these elements may function inefficiently in eukaryotes. We investigated the subcellular distribution, in cultured human cells, of the protein encoded by the group II intron RmInt1 (IEP) and several mutants. We created fusions with yellow fluorescent protein (YFP) and with a FLAG epitope. We found that the IEP was localized in the nucleus and nucleolus of the cells. Remarkably, it also accumulated at the periphery of the nuclear matrix. We were also able to identify spliced lariat intron RNA, which co-immunoprecipitated with the IEP, suggesting that functional RmInt1 RNPs can be assembled in cultured human cells.

Localization of a bacterial group II intron-encoded protein in human cells

PubMed Central

Reinoso-Colacio, Mercedes; García-Rodríguez, Fernando Manuel; García-Cañadas, Marta; Amador-Cubero, Suyapa; Pérez, José Luis García; Toro, Nicolás

2015-01-01

Group II introns are mobile retroelements that self-splice from precursor RNAs to form ribonucleoparticles (RNP), which can invade new specific genomic DNA sites. This specificity can be reprogrammed, for insertion into any desired DNA site, making these introns useful tools for bacterial genetic engineering. However, previous studies have suggested that these elements may function inefficiently in eukaryotes. We investigated the subcellular distribution, in cultured human cells, of the protein encoded by the group II intron RmInt1 (IEP) and several mutants. We created fusions with yellow fluorescent protein (YFP) and with a FLAG epitope. We found that the IEP was localized in the nucleus and nucleolus of the cells. Remarkably, it also accumulated at the periphery of the nuclear matrix. We were also able to identify spliced lariat intron RNA, which co-immunoprecipitated with the IEP, suggesting that functional RmInt1 RNPs can be assembled in cultured human cells. PMID:26244523

The group II intron maturase: a reverse transcriptase and splicing factor go hand in hand.

PubMed

Zhao, Chen; Pyle, Anna Marie

2017-12-01

The splicing of group II introns in vivo requires the assistance of a multifunctional intron encoded protein (IEP, or maturase). Each IEP is also a reverse-transcriptase enzyme that enables group II introns to behave as mobile genetic elements. During splicing or retro-transposition, each group II intron forms a tight, specific complex with its own encoded IEP, resulting in a highly reactive holoenzyme. This review focuses on the structural basis for IEP function, as revealed by recent crystal structures of an IEP reverse transcriptase domain and cryo-EM structures of an IEP-intron complex. These structures explain how the same IEP scaffold is utilized for intron recognition, splicing and reverse transcription, while providing a physical basis for understanding the evolutionary transformation of the IEP into the eukaryotic splicing factor Prp8. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mechanisms of viral mutation.

PubMed

Sanjuán, Rafael; Domingo-Calap, Pilar

2016-12-01

The remarkable capacity of some viruses to adapt to new hosts and environments is highly dependent on their ability to generate de novo diversity in a short period of time. Rates of spontaneous mutation vary amply among viruses. RNA viruses mutate faster than DNA viruses, single-stranded viruses mutate faster than double-strand virus, and genome size appears to correlate negatively with mutation rate. Viral mutation rates are modulated at different levels, including polymerase fidelity, sequence context, template secondary structure, cellular microenvironment, replication mechanisms, proofreading, and access to post-replicative repair. Additionally, massive numbers of mutations can be introduced by some virus-encoded diversity-generating elements, as well as by host-encoded cytidine/adenine deaminases. Our current knowledge of viral mutation rates indicates that viral genetic diversity is determined by multiple virus- and host-dependent processes, and that viral mutation rates can evolve in response to specific selective pressures.

Two novel families of plasmids from hyperthermophilic archaea encoding new families of replication proteins

PubMed Central

Soler, Nicolas; Marguet, Evelyne; Cortez, Diego; Desnoues, Nicole; Keller, Jenny; van Tilbeurgh, Herman; Sezonov, Guennadi; Forterre, Patrick

2010-01-01

Thermococcales (phylum Euryarchaeota) are model organisms for physiological and molecular studies of hyperthermophiles. Here we describe three new plasmids from Thermococcales that could provide new tools and model systems for genetic and molecular studies in Archaea. The plasmids pTN2 from Thermococcus nautilus sp. 30-1 and pP12-1 from Pyrococcus sp. 12-1 belong to the same family. They have similar size (∼12 kb) and share six genes, including homologues of genes encoded by the virus PAV1 from Pyrococcus abyssi. The plasmid pT26-2 from Thermococcus sp. 26-2 (21.5 kb), that corresponds to another plasmid family, encodes many proteins having homologues in virus-like elements integrated in several genomes of Thermococcales and Methanococcales. Our analyses confirm that viruses and plasmids are evolutionary related and co-evolve with their hosts. Whereas all plasmids previously isolated from Thermococcales replicate by the rolling circle mechanism, the three plasmids described here probably replicate by the theta mechanism. The plasmids pTN2 and pP12-1 encode a putative helicase of the SFI superfamily and a new family of DNA polymerase, whose activity was demonstrated in vitro, whereas pT26-2 encodes a putative new type of helicase. This strengthens the idea that plasmids and viruses are a reservoir of novel protein families involved in DNA replication. PMID:20403814

Extraordinarily Adaptive Properties of the Genetically Encoded Amino Acids

PubMed Central

Ilardo, Melissa; Meringer, Markus; Freeland, Stephen; Rasulev, Bakhtiyor; Cleaves II, H. James

2015-01-01

Using novel advances in computational chemistry, we demonstrate that the set of 20 genetically encoded amino acids, used nearly universally to construct all coded terrestrial proteins, has been highly influenced by natural selection. We defined an adaptive set of amino acids as one whose members thoroughly cover relevant physico-chemical properties, or “chemistry space.” Using this metric, we compared the encoded amino acid alphabet to random sets of amino acids. These random sets were drawn from a computationally generated compound library containing 1913 alternative amino acids that lie within the molecular weight range of the encoded amino acids. Sets that cover chemistry space better than the genetically encoded alphabet are extremely rare and energetically costly. Further analysis of more adaptive sets reveals common features and anomalies, and we explore their implications for synthetic biology. We present these computations as evidence that the set of 20 amino acids found within the standard genetic code is the result of considerable natural selection. The amino acids used for constructing coded proteins may represent a largely global optimum, such that any aqueous biochemistry would use a very similar set. PMID:25802223

Comparison of the gentamicin resistance transposon Tn5281 with regions encoding gentamicin resistance in Enterococcus faecalis isolates from diverse geographic locations.

PubMed Central

Hodel-Christian, S L; Murray, B E

1992-01-01

The genetic determinant encoding gentamicin resistance (Gmr) on the beta-lactamase encoding plasmid pBEM10 of Enterococcus faecalis HH22 is carried on a transposon, termed Tn5281, that is highly related to the staphylococcal Gmr transposons Tn4001 found in Australian isolates of Staphylococcus aureus and Tn4031 found in United States isolates of Staphylococcus epidermidis. We have now studied plasmid DNA from Gmr strains of E. faecalis isolated from diverse geographical locations (Houston, Pennsylvania, Thailand, and Chile) by using restriction endonuclease analysis and DNA-DNA hybridization to determine whether other Gmr E. faecalis carry Tn5281 or a similar type of element. We also compared these enterococci to several United States isolates of Staphylococcus aureus with nonmobile Gmr determinants. Three E. faecalis isolates (from Houston and Chile) carried Tn5281-like elements, whereas two isolates (from Houston and Pennsylvania) had restriction endonuclease and DNA-DNA hybridization patterns more similar to those of the Tn4001-IS257 hybrid found in the nonmobile Gmr determinants in United States isolates of S. aureus. A strain from Thailand had a third pattern unrelated to either Tn5281 or the nonmobile Gmr determinants present in United States isolates of S. aureus. Our results demonstrate that there is both similarity and diversity between the Gmr determinant of strains of E. faecalis isolated in diverse geographic locations. Images PMID:1332593

The Obscure World of Integrative and Mobilizable Elements, Highly Widespread Elements that Pirate Bacterial Conjugative Systems

PubMed Central

Guédon, Gérard; Libante, Virginie; Coluzzi, Charles; Payot, Sophie

2017-01-01

Conjugation is a key mechanism of bacterial evolution that involves mobile genetic elements. Recent findings indicated that the main actors of conjugative transfer are not the well-known conjugative or mobilizable plasmids but are the integrated elements. This paper reviews current knowledge on “integrative and mobilizable elements” (IMEs) that have recently been shown to be highly diverse and highly widespread but are still rarely described. IMEs encode their own excision and integration and use the conjugation machinery of unrelated co-resident conjugative element for their own transfer. Recent studies revealed a much more complex and much more diverse lifecycle than initially thought. Besides their main transmission as integrated elements, IMEs probably use plasmid-like strategies to ensure their maintenance after excision. Their interaction with conjugative elements reveals not only harmless hitchhikers but also hunters that use conjugative elements as target for their integration or harmful parasites that subvert the conjugative apparatus of incoming elements to invade cells that harbor them. IMEs carry genes conferring various functions, such as resistance to antibiotics, that can enhance the fitness of their hosts and that contribute to their maintenance in bacterial populations. Taken as a whole, IMEs are probably major contributors to bacterial evolution. PMID:29165361

Cytotype Control of Drosophila Melanogaster P Element Transposition: Genomic Position Determines Maternal Repression

PubMed Central

Misra, S.; Buratowski, R. M.; Ohkawa, T.; Rio, D. C.

1993-01-01

P element transposition in Drosophila is controlled by the cytotype regulatory state: in P cytotype, transposition is repressed, whereas in M cytotype, transposition can occur. P cytotype is determined by a combination of maternally inherited factors and chromosomal P elements in the zygote. Transformant strains containing single elements that encoded the 66-kD P element protein zygotically repressed transposition, but did not display the maternal repression characteristic of P cytotype. Upon mobilization to new genomic positions, some of these repressor elements showed significant maternal repression of transposition in genetic assays, involving a true maternal effect. Thus, the genomic position of repressor elements can determine the maternal vs. zygotic inheritance of P cytotype. Immunoblotting experiments indicate that this genomic position effect does not operate solely by controlling the expression level of the 66-kD repressor protein during oogenesis. Likewise, P element derivatives containing the hsp26 maternal regulator sequence expressed high levels of the 66-kD protein during oogenesis, but showed no detectable maternal repression. These data suggest that the location of a repressor element in the genome may determine maternal inheritance of P cytotype by a mechanism involving more than the overall level of expression of the 66-kD protein in the ovary. PMID:8293979

Genetic programs can be compressed and autonomously decompressed in live cells

NASA Astrophysics Data System (ADS)

Lapique, Nicolas; Benenson, Yaakov

2018-04-01

Fundamental computer science concepts have inspired novel information-processing molecular systems in test tubes1-13 and genetically encoded circuits in live cells14-21. Recent research has shown that digital information storage in DNA, implemented using deep sequencing and conventional software, can approach the maximum Shannon information capacity22 of two bits per nucleotide23. In nature, DNA is used to store genetic programs, but the information content of the encoding rarely approaches this maximum24. We hypothesize that the biological function of a genetic program can be preserved while reducing the length of its DNA encoding and increasing the information content per nucleotide. Here we support this hypothesis by describing an experimental procedure for compressing a genetic program and its subsequent autonomous decompression and execution in human cells. As a test-bed we choose an RNAi cell classifier circuit25 that comprises redundant DNA sequences and is therefore amenable for compression, as are many other complex gene circuits15,18,26-28. In one example, we implement a compressed encoding of a ten-gene four-input AND gate circuit using only four genetic constructs. The compression principles applied to gene circuits can enable fitting complex genetic programs into DNA delivery vehicles with limited cargo capacity, and storing compressed and biologically inert programs in vivo for on-demand activation.

Genetically Encoded Catalytic Hairpin Assembly for Sensitive RNA Imaging in Live Cells.

PubMed

Mudiyanselage, Aruni P K K Karunanayake; Yu, Qikun; Leon-Duque, Mark A; Zhao, Bin; Wu, Rigumula; You, Mingxu

2018-06-26

DNA and RNA nanotechnology has been used for the development of dynamic molecular devices. In particular, programmable enzyme-free nucleic acid circuits, such as catalytic hairpin assembly, have been demonstrated as useful tools for bioanalysis and to scale up system complexity to an extent beyond current cellular genetic circuits. However, the intracellular functions of most synthetic nucleic acid circuits have been hindered by challenges in the biological delivery and degradation. On the other hand, genetically encoded and transcribed RNA circuits emerge as alternative powerful tools for long-term embedded cellular analysis and regulation. Herein, we reported a genetically encoded RNA-based catalytic hairpin assembly circuit for sensitive RNA imaging inside living cells. The split version of Broccoli, a fluorogenic RNA aptamer, was used as the reporter. One target RNA can catalytically trigger the fluorescence from tens-to-hundreds of Broccoli. As a result, target RNAs can be sensitively detected. We have further engineered our circuit to allow easy programming to image various target RNA sequences. This design principle opens the arena for developing a large variety of genetically encoded RNA circuits for cellular applications.

On a biologically inspired topology optimization method

NASA Astrophysics Data System (ADS)

Kobayashi, Marcelo H.

2010-03-01

This work concerns the development of a biologically inspired methodology for the study of topology optimization in engineering and natural systems. The methodology is based on L systems and its turtle interpretation for the genotype-phenotype modeling of the topology development. The topology is analyzed using the finite element method, and optimized using an evolutionary algorithm with the genetic encoding of the L system and its turtle interpretation, as well as, body shape and physical characteristics. The test cases considered in this work clearly show the suitability of the proposed method for the study of engineering and natural complex systems.

Rubinstein-Taybi Syndrome and Epigenetic Alterations.

PubMed

Korzus, Edward

2017-01-01

Rubinstein-Taybi syndrome (RSTS) is a rare genetic disorder in humans characterized by growth and psychomotor delay, abnormal gross anatomy, and mild to severe mental retardation (Rubinstein and Taybi, Am J Dis Child 105:588-608, 1963, Hennekam et al., Am J Med Genet Suppl 6:56-64, 1990). RSTS is caused by de novo mutations in epigenetics-associated genes, including the cAMP response element-binding protein (CREBBP), the gene-encoding protein referred to as CBP, and the EP300 gene, which encodes the p300 protein, a CBP homologue. Recent studies of the epigenetic mechanisms underlying cognitive functions in mice provide direct evidence for the involvement of nuclear factors (e.g., CBP) in the control of higher cognitive functions. In fact, a role for CBP in higher cognitive function is suggested by the finding that RSTS is caused by heterozygous mutations at the CBP locus (Petrij et al., Nature 376:348-351, 1995). CBP was demonstrated to possess an intrinsic histone acetyltransferase activity (Ogryzko et al., Cell 87:953-959, 1996) that is required for CREB-mediated gene expression (Korzus et al., Science 279:703-707, 1998). The intrinsic protein acetyltransferase activity in CBP might directly destabilize promoter-bound nucleosomes, facilitating the activation of transcription. Due to the complexity of developmental abnormalities and the possible genetic compensation associated with this congenital disorder, however, it is difficult to establish a direct role for CBP in cognitive function in the adult brain. Although aspects of the clinical presentation in RSTS cases have been extensively studied, a spectrum of symptoms found in RSTS patients can be accessed only after birth, and, thus, prenatal genetic tests for this extremely rare genetic disorder are seldom considered. Even though there has been intensive research on the genetic and epigenetic function of the CREBBP gene in rodents, the etiology of this devastating congenital human disorder is largely unknown.

CRISPR/cas Loci of Type II Propionibacterium acnes Confer Immunity against Acquisition of Mobile Elements Present in Type I P. acnes

PubMed Central

Brüggemann, Holger; Lomholt, Hans B.; Tettelin, Hervé; Kilian, Mogens

2012-01-01

Propionibacterium acnes is a skin commensal that occasionally acts as an opportunistic pathogen. The population structure of this species shows three main lineages (I–III). While type I strains are mainly associated with sebaceous follicles of human skin and inflammatory acne, types II and III strains are more often associated with deep tissue infections. We investigated the occurrence and distribution of the clustered regularly interspaced short palindromic repeats (CRISPR) in P. acnes, assessed their immunological memory, and addressed the question if such a system could account for type-specific properties of the species. A collection of 108 clinical isolates covering all known phylotypes of P. acnes was screened for the existence of CRISPR/cas loci. We found that CRISPR loci are restricted to type II P. acnes strains. Sequence analyses of the CRISPR spacers revealed that the system confers immunity to P. acnes-specific phages and to two mobile genetic elements. These elements are found almost exclusively in type I P. acnes strains. Genome sequencing of a type I P. acnes isolate revealed that one element, 54 kb in size, encodes a putative secretion/tight adherence (TAD) system. Thus, CRISPR/cas loci in P. acnes recorded the exposure of type II strains to mobile genetic elements of type I strains. The CRISPR/cas locus is deleted in type I strains, which conceivably accounts for their ability to horizontally acquire fitness or virulence traits and might indicate that type I strains constitute a younger subpopulation of P. acnes. PMID:22479553

Behavior of restriction–modification systems as selfish mobile elements and their impact on genome evolution

PubMed Central

Kobayashi, Ichizo

2001-01-01

Restriction–modification (RM) systems are composed of genes that encode a restriction enzyme and a modification methylase. RM systems sometimes behave as discrete units of life, like viruses and transposons. RM complexes attack invading DNA that has not been properly modified and thus may serve as a tool of defense for bacterial cells. However, any threat to their maintenance, such as a challenge by a competing genetic element (an incompatible plasmid or an allelic homologous stretch of DNA, for example) can lead to cell death through restriction breakage in the genome. This post-segregational or post-disturbance cell killing may provide the RM complexes (and any DNA linked with them) with a competitive advantage. There is evidence that they have undergone extensive horizontal transfer between genomes, as inferred from their sequence homology, codon usage bias and GC content difference. They are often linked with mobile genetic elements such as plasmids, viruses, transposons and integrons. The comparison of closely related bacterial genomes also suggests that, at times, RM genes themselves behave as mobile elements and cause genome rearrangements. Indeed some bacterial genomes that survived post-disturbance attack by an RM gene complex in the laboratory have experienced genome rearrangements. The avoidance of some restriction sites by bacterial genomes may result from selection by past restriction attacks. Both bacteriophages and bacteria also appear to use homologous recombination to cope with the selfish behavior of RM systems. RM systems compete with each other in several ways. One is competition for recognition sequences in post-segregational killing. Another is super-infection exclusion, that is, the killing of the cell carrying an RM system when it is infected with another RM system of the same regulatory specificity but of a different sequence specificity. The capacity of RM systems to act as selfish, mobile genetic elements may underlie the structure and function of RM enzymes. PMID:11557807

Behavior of restriction-modification systems as selfish mobile elements and their impact on genome evolution.

PubMed

Kobayashi, I

2001-09-15

Restriction-modification (RM) systems are composed of genes that encode a restriction enzyme and a modification methylase. RM systems sometimes behave as discrete units of life, like viruses and transposons. RM complexes attack invading DNA that has not been properly modified and thus may serve as a tool of defense for bacterial cells. However, any threat to their maintenance, such as a challenge by a competing genetic element (an incompatible plasmid or an allelic homologous stretch of DNA, for example) can lead to cell death through restriction breakage in the genome. This post-segregational or post-disturbance cell killing may provide the RM complexes (and any DNA linked with them) with a competitive advantage. There is evidence that they have undergone extensive horizontal transfer between genomes, as inferred from their sequence homology, codon usage bias and GC content difference. They are often linked with mobile genetic elements such as plasmids, viruses, transposons and integrons. The comparison of closely related bacterial genomes also suggests that, at times, RM genes themselves behave as mobile elements and cause genome rearrangements. Indeed some bacterial genomes that survived post-disturbance attack by an RM gene complex in the laboratory have experienced genome rearrangements. The avoidance of some restriction sites by bacterial genomes may result from selection by past restriction attacks. Both bacteriophages and bacteria also appear to use homologous recombination to cope with the selfish behavior of RM systems. RM systems compete with each other in several ways. One is competition for recognition sequences in post-segregational killing. Another is super-infection exclusion, that is, the killing of the cell carrying an RM system when it is infected with another RM system of the same regulatory specificity but of a different sequence specificity. The capacity of RM systems to act as selfish, mobile genetic elements may underlie the structure and function of RM enzymes.

[Ph-Sensor Properties of a Fluorescent Protein from Dendronephthya sp].

PubMed

Pakhomov, A A; Chertkova, R V; Martynov, V I

2015-01-01

Genetically encoded biosensors based on fluorescent proteins are now widely applicable for monitoring pH changes in live cells. Here, we have shown that a fluorescent protein from Dendronephthya sp. (DendFP) exhibits a pronounced pH-sensitivity. Unlike most of known genetically encoded pH-sensors, fluorescence of the protein is not quenched upon medium acidification, but is shifting from the red to green spectral range. Therefore, quantitative measurements of intracellular pH are feasible by ratiometric comparison of emission intensities in the red and green spectral ranges, which makes DendFP advantageous compared with other genetically encoded pH-sensors.

DNA methylase activity as a marker for the presence of a family of phage-like elements conferring efflux-mediated macrolide resistance in streptococci.

PubMed

Figueiredo, T A; Aguiar, S I; Melo-Cristino, J; Ramirez, M

2006-11-01

Recently, two related chimeric genetic elements (Tn1207.3 and Phi10394.4) were shown to carry the macrolide efflux gene mef in Streptococcus pyogenes (group A streptococci [GAS]). The dissemination of elements belonging to the Tn1207.3/Phi10394.4 family in recent isolates of GAS, Streptococcus dysgalactiae subsp. equisimilis, Streptococcus pneumoniae, and Streptococcus agalactiae recovered in Portugal was surveyed. In total, 149 GAS, 18 S. pneumoniae, 4 S. dysgalactiae subsp. equisimilis, and 5 S. agalactiae isolates from infections, presenting the M phenotype of macrolide resistance and containing the mef gene, were screened for the presence of Tn1207.3/Phi10394.4 by PCR targeting open reading frames (ORFs) specific for these related elements. All the GAS isolates tested and one of the S. dysgalactiae subsp. equisimilis isolates carried Tn1207.3. However, neither of these elements was found in the isolates of the other streptococcal species. It was also noted that the DNAs of the isolates carrying Tn1207.3 were resistant to cleavage by the endonuclease SmaI. Cloning and expression of ORF12 of Tn1207.3 in Escherichia coli showed that it encoded a methyltransferase that rendered DNA refractory to cleavage by SmaI (M.Spy10394I). Using this characteristic as a marker for the presence of the Tn1207.3/Phi10394.4 family, we reviewed the literature and concluded that these genetic elements are widely distributed among tetracycline-susceptible GAS isolates presenting the M phenotype from diverse geographic origins and may have played an important role in the dissemination of macrolide resistance in this species.

Ribosomal frameshifting and transcriptional slippage: From genetic steganography and cryptography to adventitious use

PubMed Central

Atkins, John F.; Loughran, Gary; Bhatt, Pramod R.; Firth, Andrew E.; Baranov, Pavel V.

2016-01-01

Genetic decoding is not ‘frozen’ as was earlier thought, but dynamic. One facet of this is frameshifting that often results in synthesis of a C-terminal region encoded by a new frame. Ribosomal frameshifting is utilized for the synthesis of additional products, for regulatory purposes and for translational ‘correction’ of problem or ‘savior’ indels. Utilization for synthesis of additional products occurs prominently in the decoding of mobile chromosomal element and viral genomes. One class of regulatory frameshifting of stable chromosomal genes governs cellular polyamine levels from yeasts to humans. In many cases of productively utilized frameshifting, the proportion of ribosomes that frameshift at a shift-prone site is enhanced by specific nascent peptide or mRNA context features. Such mRNA signals, which can be 5′ or 3′ of the shift site or both, can act by pairing with ribosomal RNA or as stem loops or pseudoknots even with one component being 4 kb 3′ from the shift site. Transcriptional realignment at slippage-prone sequences also generates productively utilized products encoded trans-frame with respect to the genomic sequence. This too can be enhanced by nucleic acid structure. Together with dynamic codon redefinition, frameshifting is one of the forms of recoding that enriches gene expression. PMID:27436286

Staphylococcus aureus genomics and the impact of horizontal gene transfer.

PubMed

Lindsay, Jodi A

2014-03-01

Whole genome sequencing and microarrays have revealed the population structure of Staphylococcus aureus, and identified epidemiological shifts, transmission routes, and adaptation of major clones. S. aureus genomes are highly diverse. This is partly due to a population structure of conserved lineages, each with unique combinations of genes encoding surface proteins, regulators, immune evasion and virulence pathways. Even more variable are the mobile genetic elements (MGE), which encode key proteins for antibiotic resistance, virulence and host-adaptation. MGEs can transfer at high frequency between isolates of the same lineage by horizontal gene transfer (HGT). There is increasing evidence that HGT is key to bacterial adaptation and success. Recent studies have shed light on new mechanisms of DNA transfer such as transformation, the identification of receptors for transduction, on integration of DNA pathways, mechanisms blocking transfer including CRISPR and new restriction systems, strategies for evasion of restriction barriers, as well as factors influencing MGE selection and stability. These studies have also lead to new tools enabling construction of genetically modified clinical S. aureus isolates. This review will focus on HGT mechanisms and their importance in shaping the evolution of new clones adapted to antibiotic resistance, healthcare, communities and livestock. Copyright © 2013 Elsevier GmbH. All rights reserved.

Quantitative imaging for discovery and assembly of the metabo-regulome

PubMed Central

Okumoto, Sakiko; Takanaga, Hitomi; Frommer, Wolf B.

2009-01-01

Summary Little is known about regulatory networks that control metabolic flux in plant cells. Detailed understanding of regulation is crucial for synthetic biology. The difficulty of measuring metabolites with cellular and subcellular precision is a major roadblock. New tools have been developed for monitoring extracellular, cytosolic, organellar and vacuolar ion and metabolite concentrations with a time resolution of milliseconds to hours. Genetically encoded sensors allow quantitative measurement of steady-state concentrations of ions, signaling molecules and metabolites and their respective changes over time. Fluorescence resonance energy transfer (FRET) sensors exploit conformational changes in polypeptides as a proxy for analyte concentrations. Subtle effects of analyte binding on the conformation of the recognition element are translated into a FRET change between two fused green fluorescent protein (GFP) variants, enabling simple monitoring of analyte concentrations using fluorimetry or fluorescence microscopy. Fluorimetry provides information averaged over cell populations, while microscopy detects differences between cells or populations of cells. The genetically encoded sensors can be targeted to subcellular compartments or the cell surface. Confocal microscopy ultimately permits observation of gradients or local differences within a compartment. The FRET assays can be adapted to high-throughput analysis to screen mutant populations in order to systematically identify signaling networks that control individual steps in metabolic flux. PMID:19138219

Diversity, virulence, and antimicrobial resistance of the KPC-producing Klebsiella pneumoniae ST307 clone.

PubMed

Villa, Laura; Feudi, Claudia; Fortini, Daniela; Brisse, Sylvain; Passet, Virginie; Bonura, Celestino; Endimiani, Andrea; Mammina, Caterina; Ocampo, Ana Maria; Jimenez, Judy Natalia; Doumith, Michel; Woodford, Neil; Hopkins, Katie; Carattoli, Alessandra

2017-04-01

The global spread of Klebsiella pneumoniae producing Klebsiella pneumoniae carbapenemase (KPC) has been mainly associated with the dissemination of high-risk clones. In the last decade, hospital outbreaks involving KPC-producing K. pneumoniae have been predominantly attributed to isolates belonging to clonal group (CG) 258. However, results of recent epidemiological analysis indicate that KPC-producing sequence type (ST) 307, is emerging in different parts of the world and is a candidate to become a prevalent high-risk clone in the near future. Here we show that the ST307 genome encodes genetic features that may provide an advantage in adaptation to the hospital environment and the human host. Sequence analysis revealed novel plasmid-located virulence factors, including a cluster for glycogen synthesis. Glycogen production is considered to be one of the possible adaptive responses to long-term survival and growth in environments outside the host. Chromosomally-encoded virulence traits in the clone comprised fimbriae, an integrative conjugative element carrying the yersiniabactin siderophore, and two different capsular loci. Compared with the ST258 clone, capsulated ST307 isolates showed higher resistance to complement-mediated killing. The acquired genetic features identified in the genome of this new emerging clone may contribute to increased persistence of ST307 in the hospital environment and shed light on its potential epidemiological success.

Recurrent emergence of structural variants of LTR retrotransposon CsRn1 evolving novel expression strategy and their selective expansion in a carcinogenic liver fluke, Clonorchis sinensis.

PubMed

Kim, Seon-Hee; Kong, Yoon; Bae, Young-An

2017-06-01

Autonomous retrotransposons, in which replication and transcription are coupled, encode the essential gag and pol genes as a fusion or separate overlapping form(s) that are expressed in single transcripts regulated by a common upstream promoter. The element-specific expression strategies have driven development of relevant translational recoding mechanisms including ribosomal frameshifting to satisfy the protein stoichiometry critical for the assembly of infectious virus-like particles. Retrotransposons with different recoding strategies exhibit a mosaic distribution pattern across the diverse families of reverse transcribing elements, even though their respective distributions are substantially skewed towards certain family groups. However, only a few investigations to date have focused on the emergence of retrotransposons evolving novel expression strategy and causal genetic drivers of the structural variants. In this study, the bulk of genomic and transcribed sequences of a Ty3/gypsy-like CsRn1 retrotransposon in Clonorchis sinensis were analyzed for the comprehensive examination of its expression strategy. Our results demonstrated that structural variants with single open reading frame (ORF) have recurrently emerged from precedential CsRn1 copies encoding overlapping gag-pol ORFs by a single-nucleotide insertion in an upstream region of gag stop codon. In the parasite genome, some of the newly evolved variants appeared to undergo proliferative burst as active master lineages together with their ancestral copies. The genetic event was similarly observed in Opisthorchis viverrini, the closest neighbor of C. sinensis, whereas the resulting structural variants might have failed to overcome purifying selection and comprised minor remnant copies in the Opisthorchis genome. Copyright © 2017 Elsevier B.V. All rights reserved.

Genome sequence and comparative microarray analysis of serotype M18 group A Streptococcus strains associated with acute rheumatic fever outbreaks.

PubMed

Smoot, James C; Barbian, Kent D; Van Gompel, Jamie J; Smoot, Laura M; Chaussee, Michael S; Sylva, Gail L; Sturdevant, Daniel E; Ricklefs, Stacy M; Porcella, Stephen F; Parkins, Larye D; Beres, Stephen B; Campbell, David S; Smith, Todd M; Zhang, Qing; Kapur, Vivek; Daly, Judy A; Veasy, L George; Musser, James M

2002-04-02

Acute rheumatic fever (ARF), a sequelae of group A Streptococcus (GAS) infection, is the most common cause of preventable childhood heart disease worldwide. The molecular basis of ARF and the subsequent rheumatic heart disease are poorly understood. Serotype M18 GAS strains have been associated for decades with ARF outbreaks in the U.S. As a first step toward gaining new insight into ARF pathogenesis, we sequenced the genome of strain MGAS8232, a serotype M18 organism isolated from a patient with ARF. The genome is a circular chromosome of 1,895,017 bp, and it shares 1.7 Mb of closely related genetic material with strain SF370 (a sequenced serotype M1 strain). Strain MGAS8232 has 178 ORFs absent in SF370. Phages, phage-like elements, and insertion sequences are the major sources of variation between the genomes. The genomes of strain MGAS8232 and SF370 encode many of the same proven or putative virulence factors. Importantly, strain MGAS8232 has genes encoding many additional secreted proteins involved in human-GAS interactions, including streptococcal pyrogenic exotoxin A (scarlet fever toxin) and two uncharacterized pyrogenic exotoxin homologues, all phage-associated. DNA microarray analysis of 36 serotype M18 strains from diverse localities showed that most regions of variation were phages or phage-like elements. Two epidemics of ARF occurring 12 years apart in Salt Lake City, UT, were caused by serotype M18 strains that were genetically identical, or nearly so. Our analysis provides a critical foundation for accelerated research into ARF pathogenesis and a molecular framework to study the plasticity of GAS genomes.

Antibiotic Resistance Genes in the Bacteriophage DNA Fraction of Environmental Samples

PubMed Central

Colomer-Lluch, Marta; Jofre, Juan; Muniesa, Maite

2011-01-01

Antibiotic resistance is an increasing global problem resulting from the pressure of antibiotic usage, greater mobility of the population, and industrialization. Many antibiotic resistance genes are believed to have originated in microorganisms in the environment, and to have been transferred to other bacteria through mobile genetic elements. Among others, β-lactam antibiotics show clinical efficacy and low toxicity, and they are thus widely used as antimicrobials. Resistance to β-lactam antibiotics is conferred by β-lactamase genes and penicillin-binding proteins, which are chromosomal- or plasmid-encoded, although there is little information available on the contribution of other mobile genetic elements, such as phages. This study is focused on three genes that confer resistance to β-lactam antibiotics, namely two β-lactamase genes (blaTEM and blaCTX-M9) and one encoding a penicillin-binding protein (mecA) in bacteriophage DNA isolated from environmental water samples. The three genes were quantified in the DNA isolated from bacteriophages collected from 30 urban sewage and river water samples, using quantitative PCR amplification. All three genes were detected in the DNA of phages from all the samples tested, in some cases reaching 104 gene copies (GC) of blaTEM or 102 GC of blaCTX-M and mecA. These values are consistent with the amount of fecal pollution in the sample, except for mecA, which showed a higher number of copies in river water samples than in urban sewage. The bla genes from phage DNA were transferred by electroporation to sensitive host bacteria, which became resistant to ampicillin. blaTEM and blaCTX were detected in the DNA of the resistant clones after transfection. This study indicates that phages are reservoirs of resistance genes in the environment. PMID:21390233

Distribution of the ACME-arcA gene among methicillin-resistant Staphylococcus aureus from England and Wales.

PubMed

Ellington, Matthew J; Yearwood, Lianne; Ganner, Mark; East, Claire; Kearns, Angela M

2008-01-01

The ST8-SCCmecIVa (USA300) methicillin-resistant Staphylococcus aureus (MRSA) clone can harbour the arginine catabolic mobile element (ACME). The arc gene cluster within the ACME may function as a virulence or strain survival factor. We determined the distribution of the ACME-associated arcA gene among genetically diverse MRSA from around England and Wales. MRSA isolates (n = 203) of diverse genetic types, referred to the England and Wales Staphylococcus reference laboratory, were tested for the presence of the ACME-arcA gene. ACME-arcA-positive isolates were characterized by toxin gene profiling, PFGE and spa sequence typing. MICs of a range of antimicrobials were also determined. The ACME-arcA gene was detected in 17 isolates. Twelve were related to known ST8-MRSA-SCCmecIVa isolates of the USA300 lineage by pulsotype and were resistant to oxacillin, with variable ciprofloxacin and erythromycin resistance. Outside the USA300 lineage, four of the remaining five ACME-arcA isolates were closely related ST97-MRSA-SCCmecV, Panton-Valentine leucocidin (PVL)-negative, resistant to oxacillin and variously resistant to erythromycin, ciprofloxacin, clindamycin, gentamicin, tetracycline and fusidic acid. The remaining isolate was ST1, PVL-positive and resistant to fusidic acid as well as oxacillin. Thirteen out of the 17 isolates were associated with skin and soft tissue infections. The detection of ACME-arcA in diverse MRSA types highlights the mobility of the elements encoding ACME-arcA genes. The diversity of strain types and resistance profiles among ACME-arcA-encoding MRSA is a cause for public-health concern and demands continued surveillance and close monitoring.

Sequencing and diversity analyses reveal extensive similarities between some epsilon-toxin-encoding plasmids and the pCPF5603 Clostridium perfringens enterotoxin plasmid.

PubMed

Miyamoto, Kazuaki; Li, Jihong; Sayeed, Sameera; Akimoto, Shigeru; McClane, Bruce A

2008-11-01

Clostridium perfringens type B and D isolates produce epsilon-toxin, the third most potent clostridial toxin. The epsilon-toxin gene (etx) is plasmid borne in type D isolates, but etx genetics have been poorly studied in type B isolates. This study reports the first sequencing of any etx plasmid, i.e., pCP8533etx, from type B strain NCTC8533. This etx plasmid is 64.7 kb, carries tcp conjugative transfer genes, and encodes additional potential virulence factors including beta2-toxin, sortase, and collagen adhesin but not beta-toxin. Interestingly, nearly 80% of pCP8533etx open reading frames (ORFs) are also present on pCPF5603, an enterotoxin-encoding plasmid from type A isolate F5603. Pulsed-field gel electrophoresis and overlapping PCR indicated that a pCP8533etx-like etx plasmid is also present in most, if not all, other type B isolates and some beta2-toxin-positive, cpe-negative type D isolates, while other type D isolates carry different etx plasmids. Sequences upstream of the etx gene vary between type B isolates and some type D isolates that do not carry a pCP8533etx-like etx plasmid. However, nearly all type B and D isolates have an etx locus with an upstream IS1151, and those etx loci typically reside near a dcm ORF. These results suggest that pCPF5603 and pCP8533etx evolved from insertion of mobile genetic elements carrying enterotoxin or etx genes, respectively, onto a common progenitor plasmid.

A User's Guide to the Encyclopedia of DNA Elements (ENCODE)

PubMed Central

2011-01-01

The mission of the Encyclopedia of DNA Elements (ENCODE) Project is to enable the scientific and medical communities to interpret the human genome sequence and apply it to understand human biology and improve health. The ENCODE Consortium is integrating multiple technologies and approaches in a collective effort to discover and define the functional elements encoded in the human genome, including genes, transcripts, and transcriptional regulatory regions, together with their attendant chromatin states and DNA methylation patterns. In the process, standards to ensure high-quality data have been implemented, and novel algorithms have been developed to facilitate analysis. Data and derived results are made available through a freely accessible database. Here we provide an overview of the project and the resources it is generating and illustrate the application of ENCODE data to interpret the human genome. PMID:21526222

Characterization of "cis"-regulatory elements ("c"RE) associated with mammary gland function

USDA-ARS?s Scientific Manuscript database

The Bos taurus genome assembly has propelled dairy science into a new era; still, most of the information encoded in the genome has not yet been decoded. The human Encyclopedia of DNA Elements (ENCODE) project has spearheaded the identification and annotation of functional genomic elements in the hu...

A globally distributed mobile genetic element inhibits natural transformation of Vibrio cholerae

PubMed Central

Dalia, Ankur B.; Seed, Kimberley D.; Calderwood, Stephen B.; Camilli, Andrew

2015-01-01

Natural transformation is one mechanism of horizontal gene transfer (HGT) in Vibrio cholerae, the causative agent of cholera. Recently, it was found that V. cholerae isolates from the Haiti outbreak were poorly transformed by this mechanism. Here, we show that an integrating conjugative element (ICE)-encoded DNase, which we name IdeA, is necessary and sufficient for inhibiting natural transformation of Haiti outbreak strains. We demonstrate that IdeA inhibits this mechanism of HGT in cis via DNA endonuclease activity that is localized to the periplasm. Furthermore, we show that natural transformation between cholera strains in a relevant environmental context is inhibited by IdeA. The ICE encoding IdeA is globally distributed. Therefore, we analyzed the prevalence and role for this ICE in limiting natural transformation of isolates from Bangladesh collected between 2001 and 2011. We found that IdeA+ ICEs were nearly ubiquitous in isolates from 2001 to 2005; however, their prevalence decreased to ∼40% from 2006 to 2011. Thus, IdeA+ ICEs may have limited the role of natural transformation in V. cholerae. However, the rise in prevalence of strains lacking IdeA may now increase the role of this conserved mechanism of HGT in the evolution of this pathogen. PMID:26240317

Fine-Scale Map of Encyclopedia of DNA Elements Regions in the Korean Population

PubMed Central

Yoo, Yeon-Kyeong; Ke, Xiayi; Hong, Sungwoo; Jang, Hye-Yoon; Park, Kyunghee; Kim, Sook; Ahn, TaeJin; Lee, Yeun-Du; Song, Okryeol; Rho, Na-Young; Lee, Moon Sue; Lee, Yeon-Su; Kim, Jaeheup; Kim, Young J.; Yang, Jun-Mo; Song, Kyuyoung; Kimm, Kyuchan; Weir, Bruce; Cardon, Lon R.; Lee, Jong-Eun; Hwang, Jung-Joo

2006-01-01

The International HapMap Project aims to generate detailed human genome variation maps by densely genotyping single-nucleotide polymorphisms (SNPs) in CEPH, Chinese, Japanese, and Yoruba samples. This will undoubtedly become an important facility for genetic studies of diseases and complex traits in the four populations. To address how the genetic information contained in such variation maps is transferable to other populations, the Korean government, industries, and academics have launched the Korean HapMap project to genotype high-density Encyclopedia of DNA Elements (ENCODE) regions in 90 Korean individuals. Here we show that the LD pattern, block structure, haplotype diversity, and recombination rate are highly concordant between Korean and the two HapMap Asian samples, particularly Japanese. The availability of information from both Chinese and Japanese samples helps to predict more accurately the possible performance of HapMap markers in Korean disease-gene studies. Tagging SNPs selected from the two HapMap Asian maps, especially the Japanese map, were shown to be very effective for Korean samples. These results demonstrate that the HapMap variation maps are robust in related populations and will serve as an important resource for the studies of the Korean population in particular. PMID:16702437

Primer-Independent DNA Synthesis by a Family B DNA Polymerase from Self-Replicating Mobile Genetic Elements.

PubMed

Redrejo-Rodríguez, Modesto; Ordóñez, Carlos D; Berjón-Otero, Mónica; Moreno-González, Juan; Aparicio-Maldonado, Cristian; Forterre, Patrick; Salas, Margarita; Krupovic, Mart

2017-11-07

Family B DNA polymerases (PolBs) play a central role during replication of viral and cellular chromosomes. Here, we report the discovery of a third major group of PolBs, which we denote primer-independent PolB (piPolB), that might be a link between the previously known protein-primed and RNA/DNA-primed PolBs. PiPolBs are encoded by highly diverse mobile genetic elements, pipolins, integrated in the genomes of diverse bacteria and also present as circular plasmids in mitochondria. Biochemical characterization showed that piPolB displays efficient DNA polymerization activity that can use undamaged and damaged templates and is endowed with proofreading and strand displacement capacities. Remarkably, the protein is also capable of template-dependent de novo DNA synthesis, i.e., DNA-priming activity, thereby breaking the long-standing dogma that replicative DNA polymerases require a pre-existing primer for DNA synthesis. We suggest that piPolBs are involved in self-replication of pipolins and may also contribute to bacterial DNA damage tolerance. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

VRprofile: gene-cluster-detection-based profiling of virulence and antibiotic resistance traits encoded within genome sequences of pathogenic bacteria.

PubMed

Li, Jun; Tai, Cui; Deng, Zixin; Zhong, Weihong; He, Yongqun; Ou, Hong-Yu

2017-01-10

VRprofile is a Web server that facilitates rapid investigation of virulence and antibiotic resistance genes, as well as extends these trait transfer-related genetic contexts, in newly sequenced pathogenic bacterial genomes. The used backend database MobilomeDB was firstly built on sets of known gene cluster loci of bacterial type III/IV/VI/VII secretion systems and mobile genetic elements, including integrative and conjugative elements, prophages, class I integrons, IS elements and pathogenicity/antibiotic resistance islands. VRprofile is thus able to co-localize the homologs of these conserved gene clusters using HMMer or BLASTp searches. With the integration of the homologous gene cluster search module with a sequence composition module, VRprofile has exhibited better performance for island-like region predictions than the other widely used methods. In addition, VRprofile also provides an integrated Web interface for aligning and visualizing identified gene clusters with MobilomeDB-archived gene clusters, or a variety set of bacterial genomes. VRprofile might contribute to meet the increasing demands of re-annotations of bacterial variable regions, and aid in the real-time definitions of disease-relevant gene clusters in pathogenic bacteria of interest. VRprofile is freely available at http://bioinfo-mml.sjtu.edu.cn/VRprofile. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

HiView: an integrative genome browser to leverage Hi-C results for the interpretation of GWAS variants.

PubMed

Xu, Zheng; Zhang, Guosheng; Duan, Qing; Chai, Shengjie; Zhang, Baqun; Wu, Cong; Jin, Fulai; Yue, Feng; Li, Yun; Hu, Ming

2016-03-11

Genome-wide association studies (GWAS) have identified thousands of genetic variants associated with complex traits and diseases. However, most of them are located in the non-protein coding regions, and therefore it is challenging to hypothesize the functions of these non-coding GWAS variants. Recent large efforts such as the ENCODE and Roadmap Epigenomics projects have predicted a large number of regulatory elements. However, the target genes of these regulatory elements remain largely unknown. Chromatin conformation capture based technologies such as Hi-C can directly measure the chromatin interactions and have generated an increasingly comprehensive catalog of the interactome between the distal regulatory elements and their potential target genes. Leveraging such information revealed by Hi-C holds the promise of elucidating the functions of genetic variants in human diseases. In this work, we present HiView, the first integrative genome browser to leverage Hi-C results for the interpretation of GWAS variants. HiView is able to display Hi-C data and statistical evidence for chromatin interactions in genomic regions surrounding any given GWAS variant, enabling straightforward visualization and interpretation. We believe that as the first GWAS variants-centered Hi-C genome browser, HiView is a useful tool guiding post-GWAS functional genomics studies. HiView is freely accessible at: http://www.unc.edu/~yunmli/HiView .

pTC Plasmids from Sulfolobus Species in the Geothermal Area of Tengchong, China: Genomic Conservation and Naturally-Occurring Variations as a Result of Transposition by Mobile Genetic Elements

PubMed Central

Xiang, Xiaoyu; Huang, Xiaoxing; Wang, Haina; Huang, Li

2015-01-01

Plasmids occur frequently in Archaea. A novel plasmid (denoted pTC1) containing typical conjugation functions has been isolated from Sulfolobus tengchongensis RT8-4, a strain obtained from a hot spring in Tengchong, China, and characterized. The plasmid is a circular double-stranded DNA molecule of 20,417 bp. Among a total of 26 predicted pTC1 ORFs, 23 have homologues in other known Sulfolobus conjugative plasmids (CPs). pTC1 resembles other Sulfolobus CPs in genome architecture, and is most highly conserved in the genomic region encoding conjugation functions. However, attempts to demonstrate experimentally the capacity of the plasmid for conjugational transfer were unsuccessful. A survey revealed that pTC1 and its closely related plasmid variants were widespread in the geothermal area of Tengchong. Variations of the plasmids at the target sites for transposition by an insertion sequence (IS) and a miniature inverted-repeat transposable element (MITE) were readily detected. The IS was efficiently inserted into the pTC1 genome, and the inserted sequence was inactivated and degraded more frequently in an imprecise manner than in a precise manner. These results suggest that the host organism has evolved a strategy to maintain a balance between the insertion and elimination of mobile genetic elements to permit genomic plasticity while inhibiting their fast spreading. PMID:25686154

pTC Plasmids from Sulfolobus Species in the Geothermal Area of Tengchong, China: Genomic Conservation and Naturally-Occurring Variations as a Result of Transposition by Mobile Genetic Elements.

PubMed

Xiang, Xiaoyu; Huang, Xiaoxing; Wang, Haina; Huang, Li

2015-02-12

Plasmids occur frequently in Archaea. A novel plasmid (denoted pTC1) containing typical conjugation functions has been isolated from Sulfolobus tengchongensis RT8-4, a strain obtained from a hot spring in Tengchong, China, and characterized. The plasmid is a circular double-stranded DNA molecule of 20,417 bp. Among a total of 26 predicted pTC1 ORFs, 23 have homologues in other known Sulfolobus conjugative plasmids (CPs). pTC1 resembles other Sulfolobus CPs in genome architecture, and is most highly conserved in the genomic region encoding conjugation functions. However, attempts to demonstrate experimentally the capacity of the plasmid for conjugational transfer were unsuccessful. A survey revealed that pTC1 and its closely related plasmid variants were widespread in the geothermal area of Tengchong. Variations of the plasmids at the target sites for transposition by an insertion sequence (IS) and a miniature inverted-repeat transposable element (MITE) were readily detected. The IS was efficiently inserted into the pTC1 genome, and the inserted sequence was inactivated and degraded more frequently in an imprecise manner than in a precise manner. These results suggest that the host organism has evolved a strategy to maintain a balance between the insertion and elimination of mobile genetic elements to permit genomic plasticity while inhibiting their fast spreading.

Genomic epidemiology of global Klebsiella pneumoniae carbapenemase (KPC)-producing Escherichia coli.

PubMed

Stoesser, N; Sheppard, A E; Peirano, G; Anson, L W; Pankhurst, L; Sebra, R; Phan, H T T; Kasarskis, A; Mathers, A J; Peto, T E A; Bradford, P; Motyl, M R; Walker, A S; Crook, D W; Pitout, J D

2017-07-19

The dissemination of carbapenem resistance in Escherichia coli has major implications for the management of common infections. bla KPC , encoding a transmissible carbapenemase (KPC), has historically largely been associated with Klebsiella pneumoniae, a predominant plasmid (pKpQIL), and a specific transposable element (Tn4401, ~10 kb). Here we characterize the genetic features of bla KPC emergence in global E. coli, 2008-2013, using both long- and short-read whole-genome sequencing. Amongst 43/45 successfully sequenced bla KPC -E. coli strains, we identified substantial strain diversity (n = 21 sequence types, 18% of annotated genes in the core genome); substantial plasmid diversity (≥9 replicon types); and substantial bla KPC -associated, mobile genetic element (MGE) diversity (50% not within complete Tn4401 elements). We also found evidence of inter-species, regional and international plasmid spread. In several cases bla KPC was found on high copy number, small Col-like plasmids, previously associated with horizontal transmission of resistance genes in the absence of antimicrobial selection pressures. E. coli is a common human pathogen, but also a commensal in multiple environmental and animal reservoirs, and easily transmissible. The association of bla KPC with a range of MGEs previously linked to the successful spread of widely endemic resistance mechanisms (e.g. bla TEM , bla CTX-M ) suggests that it may become similarly prevalent.

The Persistent Contributions of RNA to Eukaryotic Gen(om)e Architecture and Cellular Function

PubMed Central

Brosius, Jürgen

2014-01-01

Currently, the best scenario for earliest forms of life is based on RNA molecules as they have the proven ability to catalyze enzymatic reactions and harbor genetic information. Evolutionary principles valid today become apparent in such models already. Furthermore, many features of eukaryotic genome architecture might have their origins in an RNA or RNA/protein (RNP) world, including the onset of a further transition, when DNA replaced RNA as the genetic bookkeeper of the cell. Chromosome maintenance, splicing, and regulatory function via RNA may be deeply rooted in the RNA/RNP worlds. Mostly in eukaryotes, conversion from RNA to DNA is still ongoing, which greatly impacts the plasticity of extant genomes. Raw material for novel genes encoding protein or RNA, or parts of genes including regulatory elements that selection can act on, continues to enter the evolutionary lottery. PMID:25081515

Imaging dynamic redox processes with genetically encoded probes.

PubMed

Ezeriņa, Daria; Morgan, Bruce; Dick, Tobias P

2014-08-01

Redox signalling plays an important role in many aspects of physiology, including that of the cardiovascular system. Perturbed redox regulation has been associated with numerous pathological conditions; nevertheless, the causal relationships between redox changes and pathology often remain unclear. Redox signalling involves the production of specific redox species at specific times in specific locations. However, until recently, the study of these processes has been impeded by a lack of appropriate tools and methodologies that afford the necessary redox species specificity and spatiotemporal resolution. Recently developed genetically encoded fluorescent redox probes now allow dynamic real-time measurements, of defined redox species, with subcellular compartment resolution, in intact living cells. Here we discuss the available genetically encoded redox probes in terms of their sensitivity and specificity and highlight where uncertainties or controversies currently exist. Furthermore, we outline major goals for future probe development and describe how progress in imaging methodologies will improve our ability to employ genetically encoded redox probes in a wide range of situations. This article is part of a special issue entitled "Redox Signalling in the Cardiovascular System." Copyright © 2014 Elsevier Ltd. All rights reserved.

Aerodynamic Shape Optimization Using A Real-Number-Encoded Genetic Algorithm

NASA Technical Reports Server (NTRS)

Holst, Terry L.; Pulliam, Thomas H.

2001-01-01

A new method for aerodynamic shape optimization using a genetic algorithm with real number encoding is presented. The algorithm is used to optimize three different problems, a simple hill climbing problem, a quasi-one-dimensional nozzle problem using an Euler equation solver and a three-dimensional transonic wing problem using a nonlinear potential solver. Results indicate that the genetic algorithm is easy to implement and extremely reliable, being relatively insensitive to design space noise.

Design and construction of a double inversion recombination switch for heritable sequential genetic memory.

PubMed

Ham, Timothy S; Lee, Sung K; Keasling, Jay D; Arkin, Adam P

2008-07-30

Inversion recombination elements present unique opportunities for computing and information encoding in biological systems. They provide distinct binary states that are encoded into the DNA sequence itself, allowing us to overcome limitations posed by other biological memory or logic gate systems. Further, it is in theory possible to create complex sequential logics by careful positioning of recombinase recognition sites in the sequence. In this work, we describe the design and synthesis of an inversion switch using the fim and hin inversion recombination systems to create a heritable sequential memory switch. We have integrated the two inversion systems in an overlapping manner, creating a switch that can have multiple states. The switch is capable of transitioning from state to state in a manner analogous to a finite state machine, while encoding the state information into DNA. This switch does not require protein expression to maintain its state, and "remembers" its state even upon cell death. We were able to demonstrate transition into three out of the five possible states showing the feasibility of such a switch. We demonstrate that a heritable memory system that encodes its state into DNA is possible, and that inversion recombination system could be a starting point for more complex memory circuits. Although the circuit did not fully behave as expected, we showed that a multi-state, temporal memory is achievable.

Design and Construction of a Double Inversion Recombination Switch for Heritable Sequential Genetic Memory

PubMed Central

Ham, Timothy S.; Lee, Sung K.; Keasling, Jay D.; Arkin, Adam P.

2008-01-01

Background Inversion recombination elements present unique opportunities for computing and information encoding in biological systems. They provide distinct binary states that are encoded into the DNA sequence itself, allowing us to overcome limitations posed by other biological memory or logic gate systems. Further, it is in theory possible to create complex sequential logics by careful positioning of recombinase recognition sites in the sequence. Methodology/Principal Findings In this work, we describe the design and synthesis of an inversion switch using the fim and hin inversion recombination systems to create a heritable sequential memory switch. We have integrated the two inversion systems in an overlapping manner, creating a switch that can have multiple states. The switch is capable of transitioning from state to state in a manner analogous to a finite state machine, while encoding the state information into DNA. This switch does not require protein expression to maintain its state, and “remembers” its state even upon cell death. We were able to demonstrate transition into three out of the five possible states showing the feasibility of such a switch. Conclusions/Significance We demonstrate that a heritable memory system that encodes its state into DNA is possible, and that inversion recombination system could be a starting point for more complex memory circuits. Although the circuit did not fully behave as expected, we showed that a multi-state, temporal memory is achievable. PMID:18665232

Genetic Control of L-a and L-(Bc) Dsrna Copy Number in Killer Systems of SACCHAROMYCES CEREVISIAE

PubMed Central

Ball, Steven G.; Tirtiaux, Catherine; Wickner, Reed B.

1984-01-01

M dsRNA in yeast encodes a toxin precursor and immunity protein, whereas L-A dsRNA encodes the 81,000-dalton major protein of the intracellular particles in which both L-A and M are found. L-(BC) dsRNA(s) are found in particles with different coat proteins. We find that M dsRNA lowers the copy number of L-A, but not L-(BC). The SKI gene products lower the copy number of L-(BC), L-A, M1 and M2. This is the first known interaction of L-(BC) with any element of the killer systems. The MAK3, MAK10 and PET18 gene products are necessary for L-A maintenance and replication, but mutations in these genes do not affect L-(BC) copy number. Mutations in MAK1, MAK4, MAK7, MAK17 and MAK24 do not detectably affect copy number of L-(BC) or L-A. PMID:17246214

Towards an understanding of the role of Clostridium perfringens toxins in human and animal disease

PubMed Central

Uzal, Francisco A; Freedman, John C; Shrestha, Archana; Theoret, James R; Garcia, Jorge; Awad, Milena M; Adams, Vicki; Moore, Robert J; Rood, Julian I; McClane, Bruce A

2014-01-01

Clostridium perfringens uses its arsenal of >16 toxins to cause histotoxic and intestinal infections in humans and animals. It has been unclear why this bacterium produces so many different toxins, especially since many target the plasma membrane of host cells. However, it is now established that C. perfringens uses chromosomally encoded alpha toxin (a phospholipase C) and perfringolysin O (a pore-forming toxin) during histotoxic infections. In contrast, this bacterium causes intestinal disease by employing toxins encoded by mobile genetic elements, including C. perfringens enterotoxin, necrotic enteritis toxin B-like, epsilon toxin and beta toxin. Like perfringolysin O, the toxins with established roles in intestinal disease form membrane pores. However, the intestinal disease-associated toxins vary in their target specificity, when they are produced (sporulation vs vegetative growth), and in their sensitivity to intestinal proteases. Producing many toxins with diverse characteristics likely imparts virulence flexibility to C. perfringens so it can cause an array of diseases. PMID:24762309

Evidence of a bigenomic regulation of mitochondrial gene expression by thyroid hormone during rat brain development.

PubMed

Sinha, Rohit Anthony; Pathak, Amrita; Mohan, Vishwa; Babu, Satish; Pal, Amit; Khare, Drirh; Godbole, Madan M

2010-07-02

Hypothyroidism during early mammalian brain development is associated with decreased expression of various mitochondrial encoded genes along with evidence for mitochondrial dysfunction. However, in-spite of the similarities between neurological disorders caused by perinatal hypothyroidism and those caused by various genetic mitochondrial defects we still do not know as to how thyroid hormone (TH) regulates mitochondrial transcription during development and whether this regulation by TH is nuclear mediated or through mitochondrial TH receptors? We here in rat cerebellum show that hypothyroidism causes reduction in expression of nuclear encoded genes controlling mitochondrial biogenesis like PGC-1alpha, NRF-1alpha and Tfam. Also, we for the first time demonstrate a mitochondrial localization of thyroid hormone receptor (mTR) isoform in developing brain capable of binding a TH response element (DR2) present in D-loop region of mitochondrial DNA. These results thus indicate an integrated nuclear-mitochondrial cross talk in regulation of mitochondrial transcription by TH during brain development. Copyright 2010 Elsevier Inc. All rights reserved.

viking: identification and characterization of a second type IV collagen in Drosophila.

PubMed

Yasothornsrikul, S; Davis, W J; Cramer, G; Kimbrell, D A; Dearolf, C R

1997-10-01

We have taken an enhancer trap approach to identify genes that are expressed in hematopoietic cells and tissues of Drosophila. We conducted a molecular analysis of two P-element insertion strains that have reporter gene expression in embryonic hemocytes, strain 197 and vikingICO. This analysis has determined that viking encodes a collagen type IV gene, alpha2(IV). The viking locus is located adjacent to the previously described DCg1, which encodes collagen alpha1(IV), and in the opposite orientation. The alpha2(IV) and alpha1(IV) collagens are structurally very similar to one another, and to vertebrate type IV collagens. In early development, viking and DCg1 are transcribed in the same tissue-specific pattern, primarily in the hemocytes and fat body cells. Our results suggest that both the alpha1 and alpha2 collagen IV chains may contribute to basement membranes in Drosophila. This work also provides the foundation for a more complete genetic dissection of collagen type IV molecules and their developmental function in Drosophila.

Divergently overlapping cis-encoded antisense RNA regulating toxin-antitoxin systems from E. coli: hok/sok, ldr/rdl, symE/symR.

PubMed

Kawano, Mitsuoki

2012-12-01

Toxin-antitoxin (TA) systems are categorized into three classes based on the type of antitoxin. In type I TA systems, the antitoxin is a small antisense RNA that inhibits translation of small toxic proteins by binding to the corresponding mRNAs. Those type I TA systems were originally identified as plasmid stabilization modules rendering a post-segregational killing (PSK) effect on the host cells. The type I TA loci also exist on the Escherichia coli chromosome but their biological functions are less clear. Genetic organization and regulatory elements of hok/sok and ldr/rdl families are very similar and the toxins are predicted to contain a transmembrane domain, but otherwise share no detectable sequence similarity. This review will give an overview of the type I TA modules of E. coli K-12, especially hok/sok, ldr/rdl and SOS-inducible symE/symR systems, which are regulated by divergently overlapping cis-encoded antisense RNAs.

Evidence of a bigenomic regulation of mitochondrial gene expression by thyroid hormone during rat brain development

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

Sinha, Rohit Anthony; Pathak, Amrita; Mohan, Vishwa

Hypothyroidism during early mammalian brain development is associated with decreased expression of various mitochondrial encoded genes along with evidence for mitochondrial dysfunction. However, in-spite of the similarities between neurological disorders caused by perinatal hypothyroidism and those caused by various genetic mitochondrial defects we still do not know as to how thyroid hormone (TH) regulates mitochondrial transcription during development and whether this regulation by TH is nuclear mediated or through mitochondrial TH receptors? We here in rat cerebellum show that hypothyroidism causes reduction in expression of nuclear encoded genes controlling mitochondrial biogenesis like PGC-1{alpha}, NRF-1{alpha} and Tfam. Also, we for themore » first time demonstrate a mitochondrial localization of thyroid hormone receptor (mTR) isoform in developing brain capable of binding a TH response element (DR2) present in D-loop region of mitochondrial DNA. These results thus indicate an integrated nuclear-mitochondrial cross talk in regulation of mitochondrial transcription by TH during brain development.« less

Toxins, Targets, and Triggers: An Overview of Toxin-Antitoxin Biology.

PubMed

Harms, Alexander; Brodersen, Ditlev Egeskov; Mitarai, Namiko; Gerdes, Kenn

2018-06-07

Bacterial toxin-antitoxin (TA) modules are abundant genetic elements that encode a toxin protein capable of inhibiting cell growth and an antitoxin that counteracts the toxin. The majority of toxins are enzymes that interfere with translation or DNA replication, but a wide variety of molecular activities and cellular targets have been described. Antitoxins are proteins or RNAs that often control their cognate toxins through direct interactions and, in conjunction with other signaling elements, through transcriptional and translational regulation of TA module expression. Three major biological functions of TA modules have been discovered, post-segregational killing ("plasmid addiction"), abortive infection (bacteriophage immunity through altruistic suicide), and persister formation (antibiotic tolerance through dormancy). In this review, we summarize the current state of the field and highlight how multiple levels of regulation shape the conditions of toxin activation to achieve the different biological functions of TA modules. Copyright © 2018 Elsevier Inc. All rights reserved.

The Microprocessor controls the activity of mammalian retrotransposons

PubMed Central

Heras, Sara R.; Macias, Sara; Plass, Mireya; Fernandez, Noemí; Cano, David; Eyras, Eduardo; Garcia-Perez, José L.; Cáceres, Javier F.

2013-01-01

More than half of the human genome is made of Transposable Elements. Their ongoing mobilization is a driving force in genetic diversity; however, little is known about how the host regulates their activity. Here, we show that the Microprocessor (Drosha-DGCR8), which is required for microRNA biogenesis, also recognizes and binds RNAs derived from human LINE-1 (Long INterspersed Element 1), Alu and SVA retrotransposons. Expression analyses demonstrate that cells lacking a functional Microprocessor accumulate LINE-1 mRNA and encoded proteins. Furthermore, we show that structured regions of the LINE-1 mRNA can be cleaved in vitro by Drosha. Additionally, we used a cell culture-based assay to show that the Microprocessor negatively regulates LINE-1 and Alu retrotransposition in vivo. Altogether, these data reveal a new role for the Microprocessor as a post-transcriptional repressor of mammalian retrotransposons acting as a defender of human genome integrity. PMID:23995758

The Microprocessor controls the activity of mammalian retrotransposons.

PubMed

Heras, Sara R; Macias, Sara; Plass, Mireya; Fernandez, Noemí; Cano, David; Eyras, Eduardo; Garcia-Perez, José L; Cáceres, Javier F

2013-10-01

More than half of the human genome is made of transposable elements whose ongoing mobilization is a driving force in genetic diversity; however, little is known about how the host regulates their activity. Here, we show that the Microprocessor (Drosha-DGCR8), which is required for microRNA biogenesis, also recognizes and binds RNAs derived from human long interspersed element 1 (LINE-1), Alu and SVA retrotransposons. Expression analyses demonstrate that cells lacking a functional Microprocessor accumulate LINE-1 mRNA and encoded proteins. Furthermore, we show that structured regions of the LINE-1 mRNA can be cleaved in vitro by Drosha. Additionally, we used a cell culture-based assay to show that the Microprocessor negatively regulates LINE-1 and Alu retrotransposition in vivo. Altogether, these data reveal a new role for the Microprocessor as a post-transcriptional repressor of mammalian retrotransposons and a defender of human genome integrity.

Stochastic Predator-Prey Dynamics of Transposons in the Human Genome

NASA Astrophysics Data System (ADS)

Xue, Chi; Goldenfeld, Nigel

2016-11-01

Transposable elements, or transposons, are DNA sequences that can jump from site to site in the genome during the life cycle of a cell, usually encoding the very enzymes which perform their excision. However, some transposons are parasitic, relying on the enzymes produced by the regular transposons. In this case, we show that a stochastic model, which takes into account the small copy numbers of the active transposons in a cell, predicts noise-induced predator-prey oscillations with a characteristic time scale that is much longer than the cell replication time, indicating that the state of the predator-prey oscillator is stored in the genome and transmitted to successive generations. Our work demonstrates the important role of the number fluctuations in the expression of mobile genetic elements, and shows explicitly how ecological concepts can be applied to the dynamics and fluctuations of living genomes.

Resveratrol stimulates c-Fos gene transcription via activation of ERK1/2 involving multiple genetic elements.

PubMed

Thiel, Gerald; Rössler, Oliver G

2018-06-05

The polyphenol resveratrol is found in many plant and fruits and is a constituent of our diet. Resveratrol has been proposed to have chemopreventive and anti-inflammatory activities. On the cellular level, resveratrol activates stimulus-regulated transcription factors. To identify resveratrol-responsive elements within a natural gene promoter, the molecular pathway leading to c-Fos gene expression by resveratrol was dissected. The c-Fos gene encodes a basic region leucine zipper transcription factor and is a prototype of an immediate-early gene that is regulated by a wide range of signaling molecules. We analyzed chromatin-integrated c-Fos promoter-luciferase reporter genes where transcription factor binding sites were destroyed by point mutations or deletion mutagenesis. The results show that mutation of the binding sites for serum response factor (SRF), activator protein-1 (AP-1) and cAMP response element binding protein (CREB) significantly reduced reporter gene transcription following stimulation of the cells with resveratrol. Inactivation of the binding sites for signal transducer and activator of transcription (STAT) or ternary complex factors did not influence resveratrol-regulated c-Fos promoter activity. Thus, the c-Fos promoter contains three resveratrol-responsive elements, the cAMP response element (CRE), and the binding sites for SRF and AP-1. Moreover, we show that the transcriptional activation potential of the c-Fos protein is increased in resveratrol-stimulated cells, indicating that the biological activity of c-Fos is elevated by resveratrol stimulation. Pharmacological and genetic experiments revealed that the protein kinase ERK1/2 is the signal transducer that connects resveratrol treatment with the c-Fos gene. Copyright © 2018 Elsevier B.V. All rights reserved.

Minimal genomes of mycoplasma-related endobacteria are plastic and contain host-derived genes for sustained life within Glomeromycota.

PubMed

Naito, Mizue; Morton, Joseph B; Pawlowska, Teresa E

2015-06-23

Arbuscular mycorrhizal fungi (AMF, Glomeromycota) colonize roots of the majority of terrestrial plants. They provide essential minerals to their plant hosts and receive photosynthates in return. All major lineages of AMF harbor endobacteria classified as Mollicutes, and known as mycoplasma-related endobacteria (MRE). Except for their substantial intrahost genetic diversity and ability to transmit vertically, virtually nothing is known about the life history of these endobacteria. To understand MRE biology, we sequenced metagenomes of three MRE populations, each associated with divergent AMF hosts. We found that each AMF species harbored a genetically distinct group of MRE. Despite vertical transmission, all MRE populations showed extensive chromosomal rearrangements, which we attributed to genetic recombination, activity of mobile elements, and a history of plectroviral invasion. The MRE genomes are characterized by a highly reduced gene content, indicating metabolic dependence on the fungal host, with the mechanism of energy production remaining unclear. Several MRE genes encode proteins with domains involved in protein-protein interactions with eukaryotic hosts. In addition, the MRE genomes harbor genes horizontally acquired from AMF. Some of these genes encode small ubiquitin-like modifier (SUMO) proteases specific to the SUMOylation systems of eukaryotes, which MRE likely use to manipulate their fungal host. The extent of MRE genome plasticity and reduction, along with the large number of horizontally acquired host genes, suggests a high degree of adaptation to the fungal host. These features, together with the ubiquity of the MRE-Glomeromycota associations, emphasize the significance of MRE in the biology of Glomeromycota.

Chemical fingerprints encode mother–offspring similarity, colony membership, relatedness, and genetic quality in fur seals

PubMed Central

Stoffel, Martin A.; Caspers, Barbara A.; Forcada, Jaume; Giannakara, Athina; Baier, Markus; Eberhart-Phillips, Luke; Müller, Caroline; Hoffman, Joseph I.

2015-01-01

Chemical communication underpins virtually all aspects of vertebrate social life, yet remains poorly understood because of its highly complex mechanistic basis. We therefore used chemical fingerprinting of skin swabs and genetic analysis to explore the chemical cues that may underlie mother–offspring recognition in colonially breeding Antarctic fur seals. By sampling mother–offspring pairs from two different colonies, using a variety of statistical approaches and genotyping a large panel of microsatellite loci, we show that colony membership, mother–offspring similarity, heterozygosity, and genetic relatedness are all chemically encoded. Moreover, chemical similarity between mothers and offspring reflects a combination of genetic and environmental influences, the former partly encoded by substances resembling known pheromones. Our findings reveal the diversity of information contained within chemical fingerprints and have implications for understanding mother–offspring communication, kin recognition, and mate choice. PMID:26261311

Incidence and Characterization of Integrons, Genetic Elements Mediating Multiple-Drug Resistance, in Avian Escherichia coli

PubMed Central

Bass, Lydia; Liebert, Cynthia A.; Lee, Margie D.; Summers, Anne O.; White, David G.; Thayer, Stephan G.; Maurer, John J.

1999-01-01

Antibiotic resistance among avian bacterial isolates is common and is of great concern to the poultry industry. Approximately 36% (n = 100) of avian, pathogenic Escherichia coli isolates obtained from diseased poultry exhibited multiple-antibiotic resistance to tetracycline, oxytetracycline, streptomycin, sulfonamides, and gentamicin. Clinical avian E. coli isolates were further screened for the presence of markers for class 1 integrons, the integron recombinase intI1 and the quaternary ammonium resistance gene qacEΔ1, in order to determine the contribution of integrons to the observed multiple-antibiotic resistance phenotypes. Sixty-three percent of the clinical isolates were positive for the class 1 integron markers intI1 and qacEΔ1. PCR analysis with the conserved class 1 integron primers yielded amplicons of approximately 1 kb from E. coli isolates positive for intI1 and qacEΔ1. These PCR amplicons contained the spectinomycin-streptomycin resistance gene aadA1. Further characterization of the identified integrons revealed that many were part of the transposon Tn21, a genetic element that encodes both antibiotic resistance and heavy-metal resistance to mercuric compounds. Fifty percent of the clinical isolates positive for the integron marker gene intI1 as well as for the qacEΔ1 and aadA1 cassettes also contained the mercury reductase gene merA. The correlation between the presence of the merA gene with that of the integrase and antibiotic resistance genes suggests that these integrons are located in Tn21. The presence of these elements among avian E. coli isolates of diverse genetic makeup as well as in Salmonella suggests the mobility of Tn21 among pathogens in humans as well as poultry. PMID:10582884

Genetically encoded Ca2+ indicators: using genetics and molecular design to understand complex physiology

PubMed Central

Kotlikoff, Michael I

2007-01-01

This article reviews genetically encoded Ca2+ indicators (GECIs), with a focus on the use of these novel molecules in the context of understanding complex cell signalling in mammals, in vivo. The review focuses on the advantages and limitations of specific GECI design strategies and the results of experiments in which these molecules have been expressed in transgenic mice, concentrating particularly on recent experiments from our laboratory in which physiological signalling could be monitored in vivo. Finally, newer strategies for effective genetic specification of GECIs are briefly reviewed. PMID:17038427

A Eukaryotic-Type Serine/Threonine Protein Kinase Is Required for Biofilm Formation, Genetic Competence, and Acid Resistance in Streptococcus mutans

PubMed Central

Hussain, Haitham; Branny, Pavel; Allan, Elaine

2006-01-01

We report an operon encoding a eukaryotic-type serine/threonine protein kinase (STPK) and its cognate phosphatase (STPP) in Streptococcus mutans. Mutation of the gene encoding the STPK produced defects in biofilm formation, genetic competence, and acid resistance, determinants important in caries pathogenesis. PMID:16452447

Genetic encoding of a bicyclo[6.1.0]nonyne-charged amino acid enables fast cellular protein imaging by metal-free ligation.

PubMed

Borrmann, Annika; Milles, Sigrid; Plass, Tilman; Dommerholt, Jan; Verkade, Jorge M M; Wiessler, Manfred; Schultz, Carsten; van Hest, Jan C M; van Delft, Floris L; Lemke, Edward A

2012-09-24

Visualizing biomolecules by fluorescent tagging is a powerful method for studying their behaviour and function inside cells. We prepared and genetically encoded an unnatural amino acid (UAA) that features a bicyclononyne moiety. This UAA offered exceptional reactivity in strain-promoted azide-alkyne cycloadditions. Kinetic measurements revealed that the UAA reacted also remarkably fast in the inverse-electron-demand Diels-Alder cycloaddition with tetrazine-conjugated dyes. Genetic encoding of the new UAA inside mammalian cells and its subsequent selective labeling at low dye concentrations demonstrate the usefulness of the new amino acid for future imaging studies. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Developmental control of transcriptional and proliferative potency during the evolutionary emergence of animals

PubMed Central

Arenas-Mena, Cesar; Coffman, James A.

2016-01-01

Summary It is proposed that the evolution of complex animals required repressive genetic mechanisms for controlling the transcriptional and proliferative potency of cells. Unicellular organisms are transcriptionally potent, able to express their full genetic complement as the need arises through their life cycle, whereas differentiated cells of multicellular organisms can only express a fraction of their genomic potential. Likewise, whereas cell proliferation in unicellular organisms is primarily limited by nutrient availability, cell proliferation in multicellular organisms is developmentally regulated. Repressive genetic controls limiting the potency of cells at the end of ontogeny would have stabilized the gene expression states of differentiated cells and prevented disruptive proliferation, allowing the emergence of diverse cell types and functional shapes. We propose that distal cis-regulatory elements represent the primary innovations that set the stage for the evolution of developmental gene regulatory networks and the repressive control of key multipotency and cell-cycle control genes. The testable prediction of this model is that the genomes of extant animals, unlike those of our unicellular relatives, encode gene regulatory circuits dedicated to the developmental control of transcriptional and proliferative potency. PMID:26173445

Minimal Increase Network Coding for Dynamic Networks.

PubMed

Zhang, Guoyin; Fan, Xu; Wu, Yanxia

2016-01-01

Because of the mobility, computing power and changeable topology of dynamic networks, it is difficult for random linear network coding (RLNC) in static networks to satisfy the requirements of dynamic networks. To alleviate this problem, a minimal increase network coding (MINC) algorithm is proposed. By identifying the nonzero elements of an encoding vector, it selects blocks to be encoded on the basis of relationship between the nonzero elements that the controls changes in the degrees of the blocks; then, the encoding time is shortened in a dynamic network. The results of simulations show that, compared with existing encoding algorithms, the MINC algorithm provides reduced computational complexity of encoding and an increased probability of delivery.

Minimal Increase Network Coding for Dynamic Networks

PubMed Central

Wu, Yanxia

2016-01-01

Because of the mobility, computing power and changeable topology of dynamic networks, it is difficult for random linear network coding (RLNC) in static networks to satisfy the requirements of dynamic networks. To alleviate this problem, a minimal increase network coding (MINC) algorithm is proposed. By identifying the nonzero elements of an encoding vector, it selects blocks to be encoded on the basis of relationship between the nonzero elements that the controls changes in the degrees of the blocks; then, the encoding time is shortened in a dynamic network. The results of simulations show that, compared with existing encoding algorithms, the MINC algorithm provides reduced computational complexity of encoding and an increased probability of delivery. PMID:26867211

The Xylella fastidosa RTX operons: evidence for the evolution of protein mosaics through novel genetic exchanges.

PubMed

Gambetta, Gregory A; Matthews, Mark A; Syvanen, Michael

2018-05-04

Xylella fastidiosa (Xf) is a gram negative bacterium inhabiting the plant vascular system. In most species this bacterium lives as a benign symbiote, but in several agriculturally important plants (e.g. coffee, citrus, grapevine) Xf is pathogenic. Xf has four loci encoding homologues to hemolysin RTX proteins, virulence factors involved in a wide range of plant pathogen interactions. We show that all four genes are expressed during pathogenesis in grapevine. The sequences from these four genes have a complex repetitive structure. At the C-termini, sequence diversity between strains is what would be expected from orthologous genes. However, within strains there is no N-terminal homology, indicating these loci encode RTXs of different functions and/or specificities. More striking is that many of the orthologous loci between strains share this extreme variation at the N-termini. Thus these RTX orthologues are most easily visualized as fusions between the orthologous C-termini and different N-termini. Further, the four genes are found in operons having a peculiar structure with an extensively duplicated module encoding a small protein with homology to the N-terminal region of the full length RTX. Surprisingly, some of these small peptides are most similar not to their corresponding full length RTX, but to the N-termini of RTXs from other Xf strains, and even other remotely related species. These results demonstrate that these genes are expressed in planta during pathogenesis. Their structure suggests extensive evolutionary restructuring through horizontal gene transfers and heterologous recombination mechanisms. The sum of the evidence suggests these repetitive modules are a novel kind of mobile genetic element.

The mouse genome displays highly dynamic populations of KRAB-zinc finger protein genes and related genetic units

PubMed Central

Kauzlaric, Annamaria; Ecco, Gabriela; Cassano, Marco; Duc, Julien; Imbeault, Michael; Trono, Didier

2017-01-01

KRAB-containing poly-zinc finger proteins (KZFPs) constitute the largest family of transcription factors encoded by mammalian genomes, and growing evidence indicates that they fulfill functions critical to both embryonic development and maintenance of adult homeostasis. KZFP genes underwent broad and independent waves of expansion in many higher vertebrates lineages, yet comprehensive studies of members harbored by a given species are scarce. Here we present a thorough analysis of KZFP genes and related units in the murine genome. We first identified about twice as many elements than previously annotated as either KZFP genes or pseudogenes, notably by assigning to this family an entity formerly considered as a large group of Satellite repeats. We then could delineate an organization in clusters distributed throughout the genome, with signs of recombination, translocation, duplication and seeding of new sites by retrotransposition of KZFP genes and related genetic units (KZFP/rGUs). Moreover, we harvested evidence indicating that closely related paralogs had evolved through both drifting and shifting of sequences encoding for zinc finger arrays. Finally, we could demonstrate that the KAP1-SETDB1 repressor complex tames the expression of KZFP/rGUs within clusters, yet that the primary targets of this regulation are not the KZFP/rGUs themselves but enhancers contained in neighboring endogenous retroelements and that, underneath, KZFPs conserve highly individualized patterns of expression. PMID:28334004

Ribosomal frameshifting and transcriptional slippage: From genetic steganography and cryptography to adventitious use.

PubMed

Atkins, John F; Loughran, Gary; Bhatt, Pramod R; Firth, Andrew E; Baranov, Pavel V

2016-09-06

Genetic decoding is not 'frozen' as was earlier thought, but dynamic. One facet of this is frameshifting that often results in synthesis of a C-terminal region encoded by a new frame. Ribosomal frameshifting is utilized for the synthesis of additional products, for regulatory purposes and for translational 'correction' of problem or 'savior' indels. Utilization for synthesis of additional products occurs prominently in the decoding of mobile chromosomal element and viral genomes. One class of regulatory frameshifting of stable chromosomal genes governs cellular polyamine levels from yeasts to humans. In many cases of productively utilized frameshifting, the proportion of ribosomes that frameshift at a shift-prone site is enhanced by specific nascent peptide or mRNA context features. Such mRNA signals, which can be 5' or 3' of the shift site or both, can act by pairing with ribosomal RNA or as stem loops or pseudoknots even with one component being 4 kb 3' from the shift site. Transcriptional realignment at slippage-prone sequences also generates productively utilized products encoded trans-frame with respect to the genomic sequence. This too can be enhanced by nucleic acid structure. Together with dynamic codon redefinition, frameshifting is one of the forms of recoding that enriches gene expression. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

The mouse genome displays highly dynamic populations of KRAB-zinc finger protein genes and related genetic units.

PubMed

Kauzlaric, Annamaria; Ecco, Gabriela; Cassano, Marco; Duc, Julien; Imbeault, Michael; Trono, Didier

2017-01-01

KRAB-containing poly-zinc finger proteins (KZFPs) constitute the largest family of transcription factors encoded by mammalian genomes, and growing evidence indicates that they fulfill functions critical to both embryonic development and maintenance of adult homeostasis. KZFP genes underwent broad and independent waves of expansion in many higher vertebrates lineages, yet comprehensive studies of members harbored by a given species are scarce. Here we present a thorough analysis of KZFP genes and related units in the murine genome. We first identified about twice as many elements than previously annotated as either KZFP genes or pseudogenes, notably by assigning to this family an entity formerly considered as a large group of Satellite repeats. We then could delineate an organization in clusters distributed throughout the genome, with signs of recombination, translocation, duplication and seeding of new sites by retrotransposition of KZFP genes and related genetic units (KZFP/rGUs). Moreover, we harvested evidence indicating that closely related paralogs had evolved through both drifting and shifting of sequences encoding for zinc finger arrays. Finally, we could demonstrate that the KAP1-SETDB1 repressor complex tames the expression of KZFP/rGUs within clusters, yet that the primary targets of this regulation are not the KZFP/rGUs themselves but enhancers contained in neighboring endogenous retroelements and that, underneath, KZFPs conserve highly individualized patterns of expression.

Diversity, virulence, and antimicrobial resistance of the KPC-producing Klebsiella pneumoniae ST307 clone

PubMed Central

Villa, Laura; Feudi, Claudia; Fortini, Daniela; Brisse, Sylvain; Passet, Virginie; Bonura, Celestino; Endimiani, Andrea; Mammina, Caterina; Ocampo, Ana Maria; Jimenez, Judy Natalia; Doumith, Michel; Woodford, Neil; Hopkins, Katie

2017-01-01

The global spread of Klebsiella pneumoniae producing Klebsiella pneumoniae carbapenemase (KPC) has been mainly associated with the dissemination of high-risk clones. In the last decade, hospital outbreaks involving KPC-producing K. pneumoniae have been predominantly attributed to isolates belonging to clonal group (CG) 258. However, results of recent epidemiological analysis indicate that KPC-producing sequence type (ST) 307, is emerging in different parts of the world and is a candidate to become a prevalent high-risk clone in the near future. Here we show that the ST307 genome encodes genetic features that may provide an advantage in adaptation to the hospital environment and the human host. Sequence analysis revealed novel plasmid-located virulence factors, including a cluster for glycogen synthesis. Glycogen production is considered to be one of the possible adaptive responses to long-term survival and growth in environments outside the host. Chromosomally-encoded virulence traits in the clone comprised fimbriae, an integrative conjugative element carrying the yersiniabactin siderophore, and two different capsular loci. Compared with the ST258 clone, capsulated ST307 isolates showed higher resistance to complement-mediated killing. The acquired genetic features identified in the genome of this new emerging clone may contribute to increased persistence of ST307 in the hospital environment and shed light on its potential epidemiological success. PMID:28785421

Annotation and sequence diversity of transposable elements in common bean (Phaseolus vulgaris).

PubMed

Gao, Dongying; Abernathy, Brian; Rohksar, Daniel; Schmutz, Jeremy; Jackson, Scott A

2014-01-01

Common bean (Phaseolus vulgaris) is an important legume crop grown and consumed worldwide. With the availability of the common bean genome sequence, the next challenge is to annotate the genome and characterize functional DNA elements. Transposable elements (TEs) are the most abundant component of plant genomes and can dramatically affect genome evolution and genetic variation. Thus, it is pivotal to identify TEs in the common bean genome. In this study, we performed a genome-wide transposon annotation in common bean using a combination of homology and sequence structure-based methods. We developed a 2.12-Mb transposon database which includes 791 representative transposon sequences and is available upon request or from www.phytozome.org. Of note, nearly all transposons in the database are previously unrecognized TEs. More than 5,000 transposon-related expressed sequence tags (ESTs) were detected which indicates that some transposons may be transcriptionally active. Two Ty1-copia retrotransposon families were found to encode the envelope-like protein which has rarely been identified in plant genomes. Also, we identified an extra open reading frame (ORF) termed ORF2 from 15 Ty3-gypsy families that was located between the ORF encoding the retrotransposase and the 3'LTR. The ORF2 was in opposite transcriptional orientation to retrotransposase. Sequence homology searches and phylogenetic analysis suggested that the ORF2 may have an ancient origin, but its function is not clear. These transposon data provide a useful resource for understanding the genome organization and evolution and may be used to identify active TEs for developing transposon-tagging system in common bean and other related genomes.

CRISPR-based screening of genomic island excision events in bacteria.

PubMed

Selle, Kurt; Klaenhammer, Todd R; Barrangou, Rodolphe

2015-06-30

Genomic analysis of Streptococcus thermophilus revealed that mobile genetic elements (MGEs) likely contributed to gene acquisition and loss during evolutionary adaptation to milk. Clustered regularly interspaced short palindromic repeats-CRISPR-associated genes (CRISPR-Cas), the adaptive immune system in bacteria, limits genetic diversity by targeting MGEs including bacteriophages, transposons, and plasmids. CRISPR-Cas systems are widespread in streptococci, suggesting that the interplay between CRISPR-Cas systems and MGEs is one of the driving forces governing genome homeostasis in this genus. To investigate the genetic outcomes resulting from CRISPR-Cas targeting of integrated MGEs, in silico prediction revealed four genomic islands without essential genes in lengths from 8 to 102 kbp, totaling 7% of the genome. In this study, the endogenous CRISPR3 type II system was programmed to target the four islands independently through plasmid-based expression of engineered CRISPR arrays. Targeting lacZ within the largest 102-kbp genomic island was lethal to wild-type cells and resulted in a reduction of up to 2.5-log in the surviving population. Genotyping of Lac(-) survivors revealed variable deletion events between the flanking insertion-sequence elements, all resulting in elimination of the Lac-encoding island. Chimeric insertion sequence footprints were observed at the deletion junctions after targeting all of the four genomic islands, suggesting a common mechanism of deletion via recombination between flanking insertion sequences. These results established that self-targeting CRISPR-Cas systems may direct significant evolution of bacterial genomes on a population level, influencing genome homeostasis and remodeling.

Genetically Encoded Voltage Indicators: Opportunities and Challenges

PubMed Central

Yang, Helen H.

2016-01-01

A longstanding goal in neuroscience is to understand how spatiotemporal patterns of neuronal electrical activity underlie brain function, from sensory representations to decision making. An emerging technology for monitoring electrical dynamics, voltage imaging using genetically encoded voltage indicators (GEVIs), couples the power of genetics with the advantages of light. Here, we review the properties that determine indicator performance and applicability, discussing both recent progress and technical limitations. We then consider GEVI applications, highlighting studies that have already deployed GEVIs for biological discovery. We also examine which classes of biological questions GEVIs are primed to address and which ones are beyond their current capabilities. As GEVIs are further developed, we anticipate that they will become more broadly used by the neuroscience community to eavesdrop on brain activity with unprecedented spatiotemporal resolution. SIGNIFICANCE STATEMENT Genetically encoded voltage indicators are engineered light-emitting protein sensors that typically report neuronal voltage dynamics as changes in brightness. In this review, we systematically discuss the current state of this emerging method, considering both its advantages and limitations for imaging neural activity. We also present recent applications of this technology and discuss what is feasible now and what we anticipate will become possible with future indicator development. This review will inform neuroscientists of recent progress in the field and help potential users critically evaluate the suitability of genetically encoded voltage indicator imaging to answer their specific biological questions. PMID:27683896

Utilisation of ISA Reverse Genetics and Large-Scale Random Codon Re-Encoding to Produce Attenuated Strains of Tick-Borne Encephalitis Virus within Days.

PubMed

de Fabritus, Lauriane; Nougairède, Antoine; Aubry, Fabien; Gould, Ernest A; de Lamballerie, Xavier

2016-01-01

Large-scale codon re-encoding is a new method of attenuating RNA viruses. However, the use of infectious clones to generate attenuated viruses has inherent technical problems. We previously developed a bacterium-free reverse genetics protocol, designated ISA, and now combined it with large-scale random codon-re-encoding method to produce attenuated tick-borne encephalitis virus (TBEV), a pathogenic flavivirus which causes febrile illness and encephalitis in humans. We produced wild-type (WT) and two re-encoded TBEVs, containing 273 or 273+284 synonymous mutations in the NS5 and NS5+NS3 coding regions respectively. Both re-encoded viruses were attenuated when compared with WT virus using a laboratory mouse model and the relative level of attenuation increased with the degree of re-encoding. Moreover, all infected animals produced neutralizing antibodies. This novel, rapid and efficient approach to engineering attenuated viruses could potentially expedite the development of safe and effective new-generation live attenuated vaccines.

Polintons: a hotbed of eukaryotic virus, transposon and plasmid evolution

PubMed Central

Krupovic, Mart; Koonin, Eugene V.

2018-01-01

Polintons (also known as Mavericks) are large DNA transposons that are widespread in the genomes of eukaryotes. We have recently shown that Polintons encode virus capsid proteins, which suggests that these transposons might form virions, at least under some conditions. In this Opinion article, we delineate the evolutionary relationships among bacterial tectiviruses, Polintons, adenoviruses, virophages, large and giant DNA viruses of eukaryotes of the proposed order ‘Megavirales’, and linear mitochondrial and cytoplasmic plasmids. We hypothesize that Polintons were the first group of eukaryotic double-stranded DNA viruses to evolve from bacteriophages and that they gave rise to most large DNA viruses of eukaryotes and various other selfish genetic elements. PMID:25534808

Transcriptional analysis of the bglP gene from Streptococcus mutans.

PubMed

Cote, Christopher K; Honeyman, Allen L

2006-04-21

An open reading frame encoding a putative antiterminator protein, LicT, was identified in the genomic sequence of Streptococcus mutans. A potential ribonucleic antitermination (RAT) site to which the LicT protein would potentially bind has been identified immediately adjacent to this open reading frame. The licT gene and RAT site are both located 5' to a beta-glucoside PTS regulon previously described in S. mutans that is responsible for esculin utilization in the presence of glucose. It was hypothesized that antitermination is the regulatory mechanism that is responsible for the control of the bglP gene expression, which encodes an esculin-specific PTS enzyme II. To localize the promoter activity associated with the bglP locus, a series of transcriptional lacZ gene fusions was formed on a reporter shuttle vector using various DNA fragments from the bglP promoter region. Subsequent beta-galactosidase assays in S. mutans localized the bglP promoter region and identified putative -35 and -10 promoter elements. Primer extension analysis identified the bglP transcriptional start site. In addition, a terminated bglP transcript formed by transcriptional termination was identified via transcript mapping experiments. The physical location of these genetic elements, the RAT site and the promoter regions, and the identification of a short terminated mRNA support the hypothesis that antitermination regulates the bglP transcript.

Transcriptional analysis of the bglP gene from Streptococcus mutans

PubMed Central

Cote, Christopher K; Honeyman, Allen L

2006-01-01

Background An open reading frame encoding a putative antiterminator protein, LicT, was identified in the genomic sequence of Streptococcus mutans. A potential ribonucleic antitermination (RAT) site to which the LicT protein would potentially bind has been identified immediately adjacent to this open reading frame. The licT gene and RAT site are both located 5' to a beta-glucoside PTS regulon previously described in S. mutans that is responsible for esculin utilization in the presence of glucose. It was hypothesized that antitermination is the regulatory mechanism that is responsible for the control of the bglP gene expression, which encodes an esculin-specific PTS enzyme II. Results To localize the promoter activity associated with the bglP locus, a series of transcriptional lacZ gene fusions was formed on a reporter shuttle vector using various DNA fragments from the bglP promoter region. Subsequent beta-galactosidase assays in S. mutans localized the bglP promoter region and identified putative -35 and -10 promoter elements. Primer extension analysis identified the bglP transcriptional start site. In addition, a terminated bglP transcript formed by transcriptional termination was identified via transcript mapping experiments. Conclusion The physical location of these genetic elements, the RAT site and the promoter regions, and the identification of a short terminated mRNA support the hypothesis that antitermination regulates the bglP transcript. PMID:16630357

Evolutionary, ecological and biotechnological perspectives on plasmids resident in the human gut mobile metagenome

PubMed Central

Ogilvie, Lesley A.; Firouzmand, Sepinoud; Jones, Brian V.

2012-01-01

Numerous mobile genetic elements (MGE) are associated with the human gut microbiota and collectively referred to as the gut mobile metagenome. The role of this flexible gene pool in development and functioning of the gut microbial community remains largely unexplored, yet recent evidence suggests that at least some MGE comprising this fraction of the gut microbiome reflect the co-evolution of host and microbe in the gastro-intestinal tract. In conjunction, the high level of novel gene content typical of MGE coupled with their predicted high diversity, suggests that the mobile metagenome constitutes an immense and largely unexplored gene-space likely to encode many novel activities with potential biotechnological or pharmaceutical value, as well as being important to the development and functioning of the gut microbiota. Of the various types of MGE that comprise the gut mobile metagenome, plasmids are of particular importance since these elements are often capable of autonomous transfer between disparate bacterial species, and are known to encode accessory functions that increase bacterial fitness in a given environment facilitating bacterial adaptation. In this article current knowledge regarding plasmids resident in the human gut mobile metagenome is reviewed, and available strategies to access and characterize this portion of the gut microbiome are described. The relative merits of these methods and their present as well as prospective impact on our understanding of the human gut microbiota is discussed. PMID:22126801

Chicken genome analysis reveals novel genes encoding biotin-binding proteins related to avidin family

PubMed Central

Niskanen, Einari A; Hytönen, Vesa P; Grapputo, Alessandro; Nordlund, Henri R; Kulomaa, Markku S; Laitinen, Olli H

2005-01-01

Background A chicken egg contains several biotin-binding proteins (BBPs), whose complete DNA and amino acid sequences are not known. In order to identify and characterise these genes and proteins we studied chicken cDNAs and genes available in the NCBI database and chicken genome database using the reported N-terminal amino acid sequences of chicken egg-yolk BBPs as search strings. Results Two separate hits showing significant homology for these N-terminal sequences were discovered. For one of these hits, the chromosomal location in the immediate proximity of the avidin gene family was found. Both of these hits encode proteins having high sequence similarity with avidin suggesting that chicken BBPs are paralogous to avidin family. In particular, almost all residues corresponding to biotin binding in avidin are conserved in these putative BBP proteins. One of the found DNA sequences, however, seems to encode a carboxy-terminal extension not present in avidin. Conclusion We describe here the predicted properties of the putative BBP genes and proteins. Our present observations link BBP genes together with avidin gene family and shed more light on the genetic arrangement and variability of this family. In addition, comparative modelling revealed the potential structural elements important for the functional and structural properties of the putative BBP proteins. PMID:15777476

A super-family of transcriptional activators regulates bacteriophage packaging and lysis in Gram-positive bacteria

PubMed Central

Quiles-Puchalt, Nuria; Tormo-Más, María Ángeles; Campoy, Susana; Toledo-Arana, Alejandro; Monedero, Vicente; Lasa, Íñigo; Novick, Richard P.; Christie, Gail E.; Penadés, José R.

2013-01-01

The propagation of bacteriophages and other mobile genetic elements requires exploitation of the phage mechanisms involved in virion assembly and DNA packaging. Here, we identified and characterized four different families of phage-encoded proteins that function as activators required for transcription of the late operons (morphogenetic and lysis genes) in a large group of phages infecting Gram-positive bacteria. These regulators constitute a super-family of proteins, here named late transcriptional regulators (Ltr), which share common structural, biochemical and functional characteristics and are unique to this group of phages. They are all small basic proteins, encoded by genes present at the end of the early gene cluster in their respective phage genomes and expressed under cI repressor control. To control expression of the late operon, the Ltr proteins bind to a DNA repeat region situated upstream of the terS gene, activating its transcription. This involves the C-terminal part of the Ltr proteins, which control specificity for the DNA repeat region. Finally, we show that the Ltr proteins are the only phage-encoded proteins required for the activation of the packaging and lysis modules. In summary, we provide evidence that phage packaging and lysis is a conserved mechanism in Siphoviridae infecting a wide variety of Gram-positive bacteria. PMID:23771138

Refactored M13 Bacteriophage as a Platform for Tumor Cell Imaging and Drug Delivery

PubMed Central

MOSER, FELIX; ENDY, DREW; BELCHER, ANGELA M.

2014-01-01

M13 bacteriophage is a well-characterized platform for peptide display. The utility of the M13 display platform is derived from the ability to encode phage protein fusions with display peptides at the genomic level. However, the genome of the phage is complicated by overlaps of key genetic elements. These overlaps directly couple the coding sequence of one gene to the coding or regulatory sequence of another, making it difficult to alter one gene without disrupting the other. Specifically, overlap of the end of gene VII and the beginning of gene IX has prevented the functional genomic modification of the N-terminus of p9. By redesigning the M13 genome to physically separate these overlapping genetic elements, a process known as “refactoring,” we enabled independent manipulation of gene VII and gene IX and the construction of the first N-terminal genomic modification of p9 for peptide display. We demonstrate the utility of this refactored genome by developing an M13 bacteriophage-based platform for targeted imaging of and drug delivery to prostate cancer cells in vitro. This successful use of refactoring principles to reengineer a natural biological system strengthens the suggestion that natural genomes can be rationally designed for a number of applications. PMID:23656279

Refactored M13 bacteriophage as a platform for tumor cell imaging and drug delivery.

PubMed

Ghosh, Debadyuti; Kohli, Aditya G; Moser, Felix; Endy, Drew; Belcher, Angela M

2012-12-21

M13 bacteriophage is a well-characterized platform for peptide display. The utility of the M13 display platform is derived from the ability to encode phage protein fusions with display peptides at the genomic level. However, the genome of the phage is complicated by overlaps of key genetic elements. These overlaps directly couple the coding sequence of one gene to the coding or regulatory sequence of another, making it difficult to alter one gene without disrupting the other. Specifically, overlap of the end of gene VII and the beginning of gene IX has prevented the functional genomic modification of the N-terminus of p9. By redesigning the M13 genome to physically separate these overlapping genetic elements, a process known as "refactoring," we enabled independent manipulation of gene VII and gene IX and the construction of the first N-terminal genomic modification of p9 for peptide display. We demonstrate the utility of this refactored genome by developing an M13 bacteriophage-based platform for targeted imaging of and drug delivery to prostate cancer cells in vitro. This successful use of refactoring principles to re-engineer a natural biological system strengthens the suggestion that natural genomes can be rationally designed for a number of applications.

The ENCODE Project at UC Santa Cruz.

PubMed

Thomas, Daryl J; Rosenbloom, Kate R; Clawson, Hiram; Hinrichs, Angie S; Trumbower, Heather; Raney, Brian J; Karolchik, Donna; Barber, Galt P; Harte, Rachel A; Hillman-Jackson, Jennifer; Kuhn, Robert M; Rhead, Brooke L; Smith, Kayla E; Thakkapallayil, Archana; Zweig, Ann S; Haussler, David; Kent, W James

2007-01-01

The goal of the Encyclopedia Of DNA Elements (ENCODE) Project is to identify all functional elements in the human genome. The pilot phase is for comparison of existing methods and for the development of new methods to rigorously analyze a defined 1% of the human genome sequence. Experimental datasets are focused on the origin of replication, DNase I hypersensitivity, chromatin immunoprecipitation, promoter function, gene structure, pseudogenes, non-protein-coding RNAs, transcribed RNAs, multiple sequence alignment and evolutionarily constrained elements. The ENCODE project at UCSC website (http://genome.ucsc.edu/ENCODE) is the primary portal for the sequence-based data produced as part of the ENCODE project. In the pilot phase of the project, over 30 labs provided experimental results for a total of 56 browser tracks supported by 385 database tables. The site provides researchers with a number of tools that allow them to visualize and analyze the data as well as download data for local analyses. This paper describes the portal to the data, highlights the data that has been made available, and presents the tools that have been developed within the ENCODE project. Access to the data and types of interactive analysis that are possible are illustrated through supplemental examples.

Genetics Home Reference: atelosteogenesis type 3

MedlinePlus

... in the gene encoding filamin B disrupt vertebral segmentation, joint formation and skeletogenesis. Nat Genet. 2004 Apr; ... Celebrates Its 15th Anniversary Genetic Information Non-Discrimination Act (GINA) Turns 10 All Bulletins Features What is ...

Genetics Home Reference: boomerang dysplasia

MedlinePlus

... in the gene encoding filamin B disrupt vertebral segmentation, joint formation and skeletogenesis. Nat Genet. 2004 Apr; ... Celebrates Its 15th Anniversary Genetic Information Non-Discrimination Act (GINA) Turns 10 All Bulletins Features What is ...

Genetics Home Reference: atelosteogenesis type 1

MedlinePlus

... in the gene encoding filamin B disrupt vertebral segmentation, joint formation and skeletogenesis. Nat Genet. 2004 Apr; ... Celebrates Its 15th Anniversary Genetic Information Non-Discrimination Act (GINA) Turns 10 All Bulletins Features What is ...

Is junk DNA bunk? A critique of ENCODE.

PubMed

Doolittle, W Ford

2013-04-02

Do data from the Encyclopedia Of DNA Elements (ENCODE) project render the notion of junk DNA obsolete? Here, I review older arguments for junk grounded in the C-value paradox and propose a thought experiment to challenge ENCODE's ontology. Specifically, what would we expect for the number of functional elements (as ENCODE defines them) in genomes much larger than our own genome? If the number were to stay more or less constant, it would seem sensible to consider the rest of the DNA of larger genomes to be junk or, at least, assign it a different sort of role (structural rather than informational). If, however, the number of functional elements were to rise significantly with C-value then, (i) organisms with genomes larger than our genome are more complex phenotypically than we are, (ii) ENCODE's definition of functional element identifies many sites that would not be considered functional or phenotype-determining by standard uses in biology, or (iii) the same phenotypic functions are often determined in a more diffuse fashion in larger-genomed organisms. Good cases can be made for propositions ii and iii. A larger theoretical framework, embracing informational and structural roles for DNA, neutral as well as adaptive causes of complexity, and selection as a multilevel phenomenon, is needed.

Draft genome sequence of a KPC-2-producing Klebsiella pneumoniae ST340 carrying blaCTX-M-15 and blaCTX-M-59 genes: a rich genome of mobile genetic elements and genes encoding antibiotic resistance.

PubMed

Casella, Tiago; de Morais, Andressa Batista Zequini; de Paula Barcelos, Diego Diniz; Tolentino, Fernanda Modesto; Cerdeira, Louise Teixeira; Bueno, Maria Fernanda Campagnari; Francisco, Gabriela Rodrigues; de Andrade, Leonardo Neves; da Costa Darini, Ana Lucia; de Oliveira Garcia, Doroti; Lincopan, Nilton; Nogueira, Mara Corrêa Lelles

2018-06-01

Klebsiella pneumoniae is considered an opportunistic pathogen and an important agent of nosocomial and community infections. It presents the ability to capture and harbour several antimicrobial resistance genes and, in this context, the extensive use of carbapenems to treat serious infections has been responsible for the selection of several resistance genes. This study reports the draft genome sequence of a KPC-2-producing K. pneumoniae strain (Kp10) simultaneously harbouring bla CTX-M-15 and bla CTX-M-59 genes isolated from urine culture of a patient with Parkinson's disease. Classical microbiological methods were applied to isolate and identify the strain, and PCR and sequencing were used to identify and characterise the genes and the genetic environment. Whole-genome sequencing (WGS) was performed using a Nextera XT DNA library and a NextSeq platform. WGS analysis revealed the presence of 5915 coding genes, 46 RNA-encoding genes and 255 pseudogenes. Kp10 belonged to sequence type 340 (ST340) of clonal complex 258 (CC258) and carried 20 transferable genes associated with antimicrobial resistance, comprising seven drug classes. Although the simultaneous presence of different bla CTX-M genes in the same strain is rarely reported, the bla KPC-2 , bla CTX-M-15 and bla CTX-M-59 genes were not associated with the same genetic mobile structure in Kp10. These results confirm the capacity of K. pneumoniae to harbour several antimicrobial resistance genes. Thus, this draft genome could help in future epidemiological studies regarding the dissemination of clinically relevant resistance genes. Copyright © 2018 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

The Hmr and Lhr Hybrid Incompatibility Genes Suppress a Broad Range of Heterochromatic Repeats

PubMed Central

Satyaki, P. R. V.; Cuykendall, Tawny N.; Wei, Kevin H-C.; Brideau, Nicholas J.; Kwak, Hojoong; Aruna, S.; Ferree, Patrick M.; Ji, Shuqing; Barbash, Daniel A.

2014-01-01

Hybrid incompatibilities (HIs) cause reproductive isolation between species and thus contribute to speciation. Several HI genes encode adaptively evolving proteins that localize to or interact with heterochromatin, suggesting that HIs may result from co-evolution with rapidly evolving heterochromatic DNA. Little is known, however, about the intraspecific function of these HI genes, the specific sequences they interact with, or the evolutionary forces that drive their divergence. The genes Hmr and Lhr genetically interact to cause hybrid lethality between Drosophila melanogaster and D. simulans, yet mutations in both genes are viable. Here, we report that Hmr and Lhr encode proteins that form a heterochromatic complex with Heterochromatin Protein 1 (HP1a). Using RNA-Seq analyses we discovered that Hmr and Lhr are required to repress transcripts from satellite DNAs and many families of transposable elements (TEs). By comparing Hmr and Lhr function between D. melanogaster and D. simulans we identify several satellite DNAs and TEs that are differentially regulated between the species. Hmr and Lhr mutations also cause massive overexpression of telomeric TEs and significant telomere lengthening. Hmr and Lhr therefore regulate three types of heterochromatic sequences that are responsible for the significant differences in genome size and structure between D. melanogaster and D. simulans and have high potential to cause genetic conflicts with host fitness. We further find that many TEs are overexpressed in hybrids but that those specifically mis-expressed in lethal hybrids do not closely correlate with Hmr function. Our results therefore argue that adaptive divergence of heterochromatin proteins in response to repetitive DNAs is an important underlying force driving the evolution of hybrid incompatibility genes, but that hybrid lethality likely results from novel epistatic genetic interactions that are distinct to the hybrid background. PMID:24651406

Negative emotional content disrupts the coherence of episodic memories.

PubMed

Bisby, James A; Horner, Aidan J; Bush, Daniel; Burgess, Neil

2018-02-01

Events are thought to be stored in episodic memory as coherent representations, in which the constituent elements are bound together so that a cue can trigger reexperience of all elements via pattern completion. Negative emotional content can strongly influence memory, but opposing theories predict strengthening or weakening of memory coherence. Across a series of experiments, participants imagined a number of person-location-object events with half of the events including a negative element (e.g., an injured person), and memory was tested across all within event associations. We show that the presence of a negative element reduces memory for associations between event elements, including between neutral elements encoded after a negative element. The presence of a negative element reduces the coherence with which a multimodal event is remembered. Our results, supported by a computational model, suggest that coherent retrieval from neutral events is supported by pattern completion, but that negative content weakens associative encoding which impairs this process. Our findings have important implications for understanding the way traumatic events are encoded and support therapeutic strategies aimed at restoring associations between negative content and its surrounding context. (PsycINFO Database Record (c) 2018 APA, all rights reserved).

Dual genetically encoded phage-displayed ligands.

PubMed

Mohan, Kritika; Weiss, Gregory A

2014-05-15

M13 bacteriophage display presents polypeptides as fusions to phage coat proteins. Such phage-displayed ligands offer useful reagents for biosensors. Here, we report a modified phage propagation protocol for the consistent and robust display of two different genetically encoded ligands on the major coat protein, P8. The results demonstrate that the phage surface reaches a saturation point for maximum peptide display. Copyright © 2014 Elsevier Inc. All rights reserved.

Protein conformation by EPR spectroscopy using gadolinium tags clicked to genetically encoded p-azido-L-phenylalanine.

PubMed

Abdelkader, E H; Feintuch, A; Yao, X; Adams, L A; Aurelio, L; Graham, B; Goldfarb, D; Otting, G

2015-11-14

Quantitative cysteine-independent ligation of a Gd(3+) tag to genetically encoded p-azido-L-phenylalanine via Cu(I)-catalyzed click chemistry is shown to deliver an exceptionally powerful tool for Gd(3+)-Gd(3+) distance measurements by double electron-electron resonance (DEER) experiments, as the position of the Gd(3+) ion relative to the protein can be predicted with high accuracy.

Regulation of ecmF gene expression and genetic hierarchy among STATa, CudA, and MybC on several prestalk A-specific gene expressions in Dictyostelium.

PubMed

Saga, Yukika; Inamura, Tomoka; Shimada, Nao; Kawata, Takefumi

2016-05-01

STATa, a Dictyostelium homologue of metazoan signal transducer and activator of transcription, is important for the organizer function in the tip region of the migrating Dictyostelium slug. We previously showed that ecmF gene expression depends on STATa in prestalk A (pstA) cells, where STATa is activated. Deletion and site-directed mutagenesis analysis of the ecmF/lacZ fusion gene in wild-type and STATa null strains identified an imperfect inverted repeat sequence, ACAAATANTATTTGT, as a STATa-responsive element. An upstream sequence element was required for efficient expression in the rear region of pstA zone; an element downstream of the inverted repeat was necessary for sufficient prestalk expression during culmination. Band shift analyses using purified STATa protein detected no sequence-specific binding to those ecmF elements. The only verified upregulated target gene of STATa is cudA gene; CudA directly activates expL7 gene expression in prestalk cells. However, ecmF gene expression was almost unaffected in a cudA null mutant. Several previously reported putative STATa target genes were also expressed in cudA null mutant but were downregulated in STATa null mutant. Moreover, mybC, which encodes another transcription factor, belonged to this category, and ecmF expression was downregulated in a mybC null mutant. These findings demonstrate the existence of a genetic hierarchy for pstA-specific genes, which can be classified into two distinct STATa downstream pathways, CudA dependent and independent. The ecmF expression is indirectly upregulated by STATa in a CudA-independent activation manner but dependent on MybC, whose expression is positively regulated by STATa. © 2016 Japanese Society of Developmental Biologists.

Metagenomic Analysis Revealing Antibiotic Resistance Genes (ARGs) and Their Genetic Compartments in the Tibetan Environment.

PubMed

Chen, Baowei; Yuan, Ke; Chen, Xin; Yang, Ying; Zhang, Tong; Wang, Yawei; Luan, Tiangang; Zou, Shichun; Li, Xiangdong

2016-07-05

Comprehensive profiles of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in a minimally impacted environment are essential to understanding the evolution and dissemination of modern antibiotic resistance. Chemical analyses of the samples collected from Tibet demonstrated that the region under investigation was almost devoid of anthropogenic antibiotics. The soils, animal wastes, and sediments were different from each other in terms of bacterial community structures, and in the typical profiles of ARGs and MGEs. Diverse ARGs that encoded resistance to common antibiotics (e.g., beta-lactams, fluoroquinolones, etc.) were found mainly via an efflux mechanism completely distinct from modern antibiotic resistome. In addition, a very small fraction of ARGs in the Tibetan environment were carried by MGEs, indicating the low potential of these ARGs to be transferred among bacteria. In comparison to the ARG profiles in relatively pristine Tibet, contemporary ARGs and MGEs in human-impacted environments have evolved substantially since the broad use of anthropogenic antibiotics.

Complete Sequencing of pNDM-HK Encoding NDM-1 Carbapenemase from a Multidrug-Resistant Escherichia coli Strain Isolated in Hong Kong

PubMed Central

Ho, Pak Leung; Lo, Wai U.; Yeung, Man Kiu; Lin, Chi Ho; Chow, Kin Hung; Ang, Irene; Tong, Amy Hin Yan; Bao, Jessie Yun-Juan; Lok, Si; Lo, Janice Yee Chi

2011-01-01

Background The emergence of plasmid-mediated carbapenemases, such as NDM-1 in Enterobacteriaceae is a major public health issue. Since they mediate resistance to virtually all β-lactam antibiotics and there is often co-resistance to other antibiotic classes, the therapeutic options for infections caused by these organisms are very limited. Methodology We characterized the first NDM-1 producing E. coli isolate recovered in Hong Kong. The plasmid encoding the metallo-β-lactamase gene was sequenced. Principal Findings The plasmid, pNDM-HK readily transferred to E. coli J53 at high frequencies. It belongs to the broad host range IncL/M incompatibility group and is 88803 bp in size. Sequence alignment showed that pNDM-HK has a 55 kb backbone which shared 97% homology with pEL60 originating from the plant pathogen, Erwina amylovora in Lebanon and a 28.9 kb variable region. The plasmid backbone includes the mucAB genes mediating ultraviolet light resistance. The 28.9 kb region has a composite transposon-like structure which includes intact or truncated genes associated with resistance to β-lactams (bla TEM-1, bla NDM-1, Δbla DHA-1), aminoglycosides (aacC2, armA), sulphonamides (sul1) and macrolides (mel, mph2). It also harbors the following mobile elements: IS26, ISCR1, tnpU, tnpAcp2, tnpD, ΔtnpATn1 and insL. Certain blocks within the 28.9 kb variable region had homology with the corresponding sequences in the widely disseminated plasmids, pCTX-M3, pMUR050 and pKP048 originating from bacteria in Poland in 1996, in Spain in 2002 and in China in 2006, respectively. Significance The genetic support of NDM-1 gene suggests that it has evolved through complex pathways. The association with broad host range plasmid and multiple mobile genetic elements explain its observed horizontal mobility in multiple bacterial taxa. PMID:21445317

Genetically Encoded Voltage Indicators: Opportunities and Challenges.

PubMed

Yang, Helen H; St-Pierre, François

2016-09-28

A longstanding goal in neuroscience is to understand how spatiotemporal patterns of neuronal electrical activity underlie brain function, from sensory representations to decision making. An emerging technology for monitoring electrical dynamics, voltage imaging using genetically encoded voltage indicators (GEVIs), couples the power of genetics with the advantages of light. Here, we review the properties that determine indicator performance and applicability, discussing both recent progress and technical limitations. We then consider GEVI applications, highlighting studies that have already deployed GEVIs for biological discovery. We also examine which classes of biological questions GEVIs are primed to address and which ones are beyond their current capabilities. As GEVIs are further developed, we anticipate that they will become more broadly used by the neuroscience community to eavesdrop on brain activity with unprecedented spatiotemporal resolution. Genetically encoded voltage indicators are engineered light-emitting protein sensors that typically report neuronal voltage dynamics as changes in brightness. In this review, we systematically discuss the current state of this emerging method, considering both its advantages and limitations for imaging neural activity. We also present recent applications of this technology and discuss what is feasible now and what we anticipate will become possible with future indicator development. This review will inform neuroscientists of recent progress in the field and help potential users critically evaluate the suitability of genetically encoded voltage indicator imaging to answer their specific biological questions. Copyright © 2016 the authors 0270-6474/16/369977-13$15.00/0.

The effect of Mycobacterium tuberculosis CRISPR-associated Cas2 (Rv2816c) on stress response genes expression, morphology and macrophage survival of Mycobacterium smegmatis.

PubMed

Huang, Qinqin; Luo, Hongping; Liu, Minqiang; Zeng, Jie; Abdalla, Abualgasim Elgaili; Duan, Xiangke; Li, Qiming; Xie, Jianping

2016-06-01

Clustered regularly interspaced short palindromic repeats (CRISPR) are present in the genome of 40% bacteria and 90% archaea. CRISPR and accompanying Cas proteins constitute an adaptive immune system against disruptive mobile genetic elements. Two CRISPRs and 9 genes encoding CRISPR-associated proteins have been found in the genome of Mycobacterium tuberculosis. The CRISPR-associated Cas2 is an endoribonuclease required for the acquisition of new spacers. In this study, Cas2 encoded by Rv2816c was expressed in Mycobacterium smegmatis lacking CRISPR-Cas system and its role in stress responses of M. smegmatis in vitro and within macrophages was studied. We found that Cas2 mediated M. smegmatis stress response changes were associated with the altered expression of sigma factors which involved in mycobacterial stress response and virulence. We also found that Cas2 decreased the survival of M. smegmatis within macrophages. This study provides new insights on the role of Cas2. Copyright © 2015 Elsevier B.V. All rights reserved.

The Tcp conjugation system of Clostridium perfringens.

PubMed

Wisniewski, Jessica A; Rood, Julian I

2017-05-01

The Gram-positive pathogen Clostridium perfringens possesses a family of large conjugative plasmids that is typified by the tetracycline resistance plasmid pCW3. Since these plasmids may carry antibiotic resistance genes or genes encoding extracellular or sporulation-associated toxins, the conjugative transfer of these plasmids appears to be important for the epidemiology of C. perfringens-mediated diseases. Sequence analysis of members of this plasmid family identified a highly conserved 35kb region that encodes proteins with various functions, including plasmid replication and partitioning. The tcp conjugation locus also was identified in this region, initially based on low-level amino acid sequence identity to conjugation proteins from the integrative conjugative element Tn916. Genetic studies confirmed that the tcp locus is required for conjugative transfer and combined with biochemical and structural analyses have led to the development of a functional model of the Tcp conjugation apparatus. This review summarises our current understanding of the Tcp conjugation system, which is now one of the best-characterized conjugation systems in Gram-positive bacteria. Copyright © 2017 Elsevier Inc. All rights reserved.

Constitutional Epi/Genetic Conditions: Genetic, Epigenetic, and Environmental Factors

PubMed Central

Schenkel, Laila C.; Rodenhiser, David; Siu, Victoria; McCready, Elizabeth; Ainsworth, Peter; Sadikovic, Bekim

2016-01-01

There are more than 4,000 phenotypes for which the molecular basis is at least partly known. Though defects in primary DNA structure constitute a major cause of these disorders, epigenetic disruption is emerging as an important alternative mechanism in the etiology of a broad range of congenital and developmental conditions. These include epigenetic defects caused by either localized (in cis) genetic alterations or more distant (in trans) genetic events but can also include environmental effects. Emerging evidence suggests interplay between genetic and environmental factors in the epigenetic etiology of several constitutional “epi/genetic” conditions. This review summarizes our broadening understanding of how epigenetics contributes to pediatric disease by exploring different classes of epigenomic disorders. It further challenges the simplistic dogma of “DNA encodes RNA encodes protein” to best understand the spectrum of factors that can influence genetic traits in a pediatric population. PMID:28180025

Live-cell Imaging with Genetically Encoded Protein Kinase Activity Reporters.

PubMed

Maryu, Gembu; Miura, Haruko; Uda, Youichi; Komatsubara, Akira T; Matsuda, Michiyuki; Aoki, Kazuhiro

2018-04-25

Protein kinases play pivotal roles in intracellular signal transduction, and dysregulation of kinases leads to pathological results such as malignant tumors. Kinase activity has hitherto been measured by biochemical methods such as in vitro phosphorylation assay and western blotting. However, these methods are less useful to explore spatial and temporal changes in kinase activity and its cell-to-cell variation. Recent advances in fluorescent proteins and live-cell imaging techniques enable us to visualize kinase activity in living cells with high spatial and temporal resolutions. Several genetically encoded kinase activity reporters, which are based on the modes of action of kinase activation and phosphorylation, are currently available. These reporters are classified into single-fluorophore kinase activity reporters and Förster (or fluorescence) resonance energy transfer (FRET)-based kinase activity reporters. Here, we introduce the principles of genetically encoded kinase activity reporters, and discuss the advantages and disadvantages of these reporters.Key words: kinase, FRET, phosphorylation, KTR.

New tools for redox biology: From imaging to manipulation.

PubMed

Bilan, Dmitry S; Belousov, Vsevolod V

2017-08-01

Redox reactions play a key role in maintaining essential biological processes. Deviations in redox pathways result in the development of various pathologies at cellular and organismal levels. Until recently, studies on transformations in the intracellular redox state have been significantly hampered in living systems. The genetically encoded indicators, based on fluorescent proteins, have provided new opportunities in biomedical research. The existing indicators already enable monitoring of cellular redox parameters in different processes including embryogenesis, aging, inflammation, tissue regeneration, and pathogenesis of various diseases. In this review, we summarize information about all genetically encoded redox indicators developed to date. We provide the description of each indicator and discuss its advantages and limitations, as well as points that need to be considered when choosing an indicator for a particular experiment. One chapter is devoted to the important discoveries that have been made by using genetically encoded redox indicators. Copyright © 2016 Elsevier Inc. All rights reserved.

A genetically-encoded chloride and pH sensor for dissociating ion dynamics in the nervous system

PubMed Central

Raimondo, Joseph V.; Joyce, Bradley; Kay, Louise; Schlagheck, Theresa; Newey, Sarah E.; Srinivas, Shankar; Akerman, Colin J.

2013-01-01

Within the nervous system, intracellular Cl− and pH regulate fundamental processes including cell proliferation, metabolism, synaptic transmission, and network excitability. Cl− and pH are often co-regulated, and network activity results in the movement of both Cl− and H+. Tools to accurately measure these ions are crucial for understanding their role under physiological and pathological conditions. Although genetically-encoded Cl− and pH sensors have been described previously, these either lack ion specificity or are unsuitable for neuronal use. Here we present ClopHensorN—a new genetically-encoded ratiometric Cl− and pH sensor that is optimized for the nervous system. We demonstrate the ability of ClopHensorN to dissociate and simultaneously quantify Cl− and H+ concentrations under a variety of conditions. In addition, we establish the sensor's utility by characterizing activity-dependent ion dynamics in hippocampal neurons. PMID:24312004

A genetically-encoded chloride and pH sensor for dissociating ion dynamics in the nervous system.

PubMed

Raimondo, Joseph V; Joyce, Bradley; Kay, Louise; Schlagheck, Theresa; Newey, Sarah E; Srinivas, Shankar; Akerman, Colin J

2013-01-01

Within the nervous system, intracellular Cl(-) and pH regulate fundamental processes including cell proliferation, metabolism, synaptic transmission, and network excitability. Cl(-) and pH are often co-regulated, and network activity results in the movement of both Cl(-) and H(+). Tools to accurately measure these ions are crucial for understanding their role under physiological and pathological conditions. Although genetically-encoded Cl(-) and pH sensors have been described previously, these either lack ion specificity or are unsuitable for neuronal use. Here we present ClopHensorN-a new genetically-encoded ratiometric Cl(-) and pH sensor that is optimized for the nervous system. We demonstrate the ability of ClopHensorN to dissociate and simultaneously quantify Cl(-) and H(+) concentrations under a variety of conditions. In addition, we establish the sensor's utility by characterizing activity-dependent ion dynamics in hippocampal neurons.

Genetically Encoded Biosensors in Plants: Pathways to Discovery.

PubMed

Walia, Ankit; Waadt, Rainer; Jones, Alexander M

2018-04-29

Genetically encoded biosensors that directly interact with a molecule of interest were first introduced more than 20 years ago with fusion proteins that served as fluorescent indicators for calcium ions. Since then, the technology has matured into a diverse array of biosensors that have been deployed to improve our spatiotemporal understanding of molecules whose dynamics have profound influence on plant physiology and development. In this review, we address several types of biosensors with a focus on genetically encoded calcium indicators, which are now the most diverse and advanced group of biosensors. We then consider the discoveries in plant biology made by using biosensors for calcium, pH, reactive oxygen species, redox conditions, primary metabolites, phytohormones, and nutrients. These discoveries were dependent on the engineering, characterization, and optimization required to develop a successful biosensor; they were also dependent on the methodological developments required to express, detect, and analyze the readout of such biosensors.

Silencing of the PiAvr3a effector-encoding gene from Phytophthora infestans by transcriptional fusion to a short interspersed element.

PubMed

Vetukuri, Ramesh R; Tian, Zhendong; Avrova, Anna O; Savenkov, Eugene I; Dixelius, Christina; Whisson, Stephen C

2011-12-01

Phytophthora infestans is the notorious oomycete causing late blight of potato and tomato. A large proportion of the P. infestans genome is composed of transposable elements, the activity of which may be controlled by RNA silencing. Accumulation of small RNAs is one of the hallmarks of RNA silencing. Here we demonstrate the presence of small RNAs corresponding to the sequence of a short interspersed retrotransposable element (SINE) suggesting that small RNAs might be involved in silencing of SINEs in P. infestans. This notion was exploited to develop novel tools for gene silencing in P. infestans by engineering transcriptional fusions of the PiAvr3a gene, encoding an RXLR avirulence effector, to the infSINEm retroelement. Transgenic P. infestans lines expressing either 5'-infSINEm::PiAvr3a-3' or 5'-PiAvr3a::SINEm-3' chimeric transcripts initially exhibited partial silencing of PiAvr3a. Over time, PiAvr3a either recovered wild type transcript levels in some lines, or became fully silenced in others. Introduction of an inverted repeat construct was also successful in yielding P. infestans transgenic lines silenced for PiAvr3a. In contrast, constructs expressing antisense or aberrant RNA transcripts failed to initiate silencing of PiAvr3a. Lines exhibiting the most effective silencing of PiAvr3a were either weakly or non-pathogenic on susceptible potato cv. Bintje. This study expands the repertoire of reverse genetics tools available for P. infestans research, and provides insights into a possible mode of variation in effector expression through spread of silencing from adjacent retroelements. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

A large gene family in fission yeast encodes spore killers that subvert Mendel’s law

PubMed Central

Hu, Wen; Jiang, Zhao-Di; Suo, Fang; Zheng, Jin-Xin; He, Wan-Zhong; Du, Li-Lin

2017-01-01

Spore killers in fungi are selfish genetic elements that distort Mendelian segregation in their favor. It remains unclear how many species harbor them and how diverse their mechanisms are. Here, we discover two spore killers from a natural isolate of the fission yeast Schizosaccharomyces pombe. Both killers belong to the previously uncharacterized wtf gene family with 25 members in the reference genome. These two killers act in strain-background-independent and genome-location-independent manners to perturb the maturation of spores not inheriting them. Spores carrying one killer are protected from its killing effect but not that of the other killer. The killing and protecting activities can be uncoupled by mutation. The numbers and sequences of wtf genes vary considerably between S. pombe isolates, indicating rapid divergence. We propose that wtf genes contribute to the extensive intraspecific reproductive isolation in S. pombe, and represent ideal models for understanding how segregation-distorting elements act and evolve. DOI: http://dx.doi.org/10.7554/eLife.26057.001 PMID:28631610

SnoVault and encodeD: A novel object-based storage system and applications to ENCODE metadata.

PubMed

Hitz, Benjamin C; Rowe, Laurence D; Podduturi, Nikhil R; Glick, David I; Baymuradov, Ulugbek K; Malladi, Venkat S; Chan, Esther T; Davidson, Jean M; Gabdank, Idan; Narayana, Aditi K; Onate, Kathrina C; Hilton, Jason; Ho, Marcus C; Lee, Brian T; Miyasato, Stuart R; Dreszer, Timothy R; Sloan, Cricket A; Strattan, J Seth; Tanaka, Forrest Y; Hong, Eurie L; Cherry, J Michael

2017-01-01

The Encyclopedia of DNA elements (ENCODE) project is an ongoing collaborative effort to create a comprehensive catalog of functional elements initiated shortly after the completion of the Human Genome Project. The current database exceeds 6500 experiments across more than 450 cell lines and tissues using a wide array of experimental techniques to study the chromatin structure, regulatory and transcriptional landscape of the H. sapiens and M. musculus genomes. All ENCODE experimental data, metadata, and associated computational analyses are submitted to the ENCODE Data Coordination Center (DCC) for validation, tracking, storage, unified processing, and distribution to community resources and the scientific community. As the volume of data increases, the identification and organization of experimental details becomes increasingly intricate and demands careful curation. The ENCODE DCC has created a general purpose software system, known as SnoVault, that supports metadata and file submission, a database used for metadata storage, web pages for displaying the metadata and a robust API for querying the metadata. The software is fully open-source, code and installation instructions can be found at: http://github.com/ENCODE-DCC/snovault/ (for the generic database) and http://github.com/ENCODE-DCC/encoded/ to store genomic data in the manner of ENCODE. The core database engine, SnoVault (which is completely independent of ENCODE, genomic data, or bioinformatic data) has been released as a separate Python package.

SnoVault and encodeD: A novel object-based storage system and applications to ENCODE metadata

PubMed Central

Podduturi, Nikhil R.; Glick, David I.; Baymuradov, Ulugbek K.; Malladi, Venkat S.; Chan, Esther T.; Davidson, Jean M.; Gabdank, Idan; Narayana, Aditi K.; Onate, Kathrina C.; Hilton, Jason; Ho, Marcus C.; Lee, Brian T.; Miyasato, Stuart R.; Dreszer, Timothy R.; Sloan, Cricket A.; Strattan, J. Seth; Tanaka, Forrest Y.; Hong, Eurie L.; Cherry, J. Michael

2017-01-01

The Encyclopedia of DNA elements (ENCODE) project is an ongoing collaborative effort to create a comprehensive catalog of functional elements initiated shortly after the completion of the Human Genome Project. The current database exceeds 6500 experiments across more than 450 cell lines and tissues using a wide array of experimental techniques to study the chromatin structure, regulatory and transcriptional landscape of the H. sapiens and M. musculus genomes. All ENCODE experimental data, metadata, and associated computational analyses are submitted to the ENCODE Data Coordination Center (DCC) for validation, tracking, storage, unified processing, and distribution to community resources and the scientific community. As the volume of data increases, the identification and organization of experimental details becomes increasingly intricate and demands careful curation. The ENCODE DCC has created a general purpose software system, known as SnoVault, that supports metadata and file submission, a database used for metadata storage, web pages for displaying the metadata and a robust API for querying the metadata. The software is fully open-source, code and installation instructions can be found at: http://github.com/ENCODE-DCC/snovault/ (for the generic database) and http://github.com/ENCODE-DCC/encoded/ to store genomic data in the manner of ENCODE. The core database engine, SnoVault (which is completely independent of ENCODE, genomic data, or bioinformatic data) has been released as a separate Python package. PMID:28403240

Comparative genomics of pathogenic lineages of Vibrio nigripulchritudo identifies virulence-associated traits

PubMed Central

Goudenège, David; Labreuche, Yannick; Krin, Evelyne; Ansquer, Dominique; Mangenot, Sophie; Calteau, Alexandra; Médigue, Claudine; Mazel, Didier; Polz, Martin F; Le Roux, Frédérique

2013-01-01

Vibrio nigripulchritudo is an emerging pathogen of farmed shrimp in New Caledonia and other regions in the Indo-Pacific. The molecular determinants of V. nigripulchritudo pathogenicity are unknown; however, molecular epidemiological studies have suggested that pathogenicity is linked to particular lineages. Here, we performed high-throughput sequencing-based comparative genome analysis of 16 V. nigripulchritudo strains to explore the genomic diversity and evolutionary history of pathogen-containing lineages and to identify pathogen-specific genetic elements. Our phylogenetic analysis revealed three pathogen-containing V. nigripulchritudo clades, including two clades previously identified from New Caledonia and one novel clade comprising putatively pathogenic isolates from septicemic shrimp in Madagascar. The similar genetic distance between the three clades indicates that they have diverged from an ancestral population roughly at the same time and recombination analysis indicates that these genomes have, in the past, shared a common gene pool and exchanged genes. As each contemporary lineage is comprised of nearly identical strains, comparative genomics allowed differentiation of genetic elements specific to shrimp pathogenesis of varying severity. Notably, only a large plasmid present in all highly pathogenic (HP) strains encodes a toxin. Although less/non-pathogenic strains contain related plasmids, these are differentiated by a putative toxin locus. Expression of this gene by a non-pathogenic V. nigripulchritudo strain resulted in production of toxic culture supernatant, normally an exclusive feature of HP strains. Thus, this protein, here termed ‘nigritoxin', is implicated to an extent that remains to be precisely determined in the toxicity of V. nigripulchritudo. PMID:23739050

Engineered cell-cell communication via DNA messaging

PubMed Central

2012-01-01

Background Evolution has selected for organisms that benefit from genetically encoded cell-cell communication. Engineers have begun to repurpose elements of natural communication systems to realize programmed pattern formation and coordinate other population-level behaviors. However, existing engineered systems rely on system-specific small molecules to send molecular messages among cells. Thus, the information transmission capacity of current engineered biological communication systems is physically limited by specific biomolecules that are capable of sending only a single message, typically “regulate transcription.” Results We have engineered a cell-cell communication platform using bacteriophage M13 gene products to autonomously package and deliver heterologous DNA messages of varying lengths and encoded functions. We demonstrate the decoupling of messages from a common communication channel via the autonomous transmission of various arbitrary genetic messages. Further, we increase the range of engineered DNA messaging across semisolid media by linking message transmission or receipt to active cellular chemotaxis. Conclusions We demonstrate decoupling of a communication channel from message transmission within engineered biological systems via the autonomous targeted transduction of user-specified heterologous DNA messages. We also demonstrate that bacteriophage M13 particle production and message transduction occurs among chemotactic bacteria. We use chemotaxis to improve the range of DNA messaging, increasing both transmission distance and communication bit rates relative to existing small molecule-based communication systems. We postulate that integration of different engineered cell-cell communication platforms will allow for more complex spatial programming of dynamic cellular consortia. PMID:22958599

Disruption of Boundary Encoding During Sensorimotor Sequence Learning: An MEG Study.

PubMed

Michail, Georgios; Nikulin, Vadim V; Curio, Gabriel; Maess, Burkhard; Herrojo Ruiz, María

2018-01-01

Music performance relies on the ability to learn and execute actions and their associated sounds. The process of learning these auditory-motor contingencies depends on the proper encoding of the serial order of the actions and sounds. Among the different serial positions of a behavioral sequence, the first and last (boundary) elements are particularly relevant. Animal and patient studies have demonstrated a specific neural representation for boundary elements in prefrontal cortical regions and in the basal ganglia, highlighting the relevance of their proper encoding. The neural mechanisms underlying the encoding of sequence boundaries in the general human population remain, however, largely unknown. In this study, we examined how alterations of auditory feedback, introduced at different ordinal positions (boundary or within-sequence element), affect the neural and behavioral responses during sensorimotor sequence learning. Analysing the neuromagnetic signals from 20 participants while they performed short piano sequences under the occasional effect of altered feedback (AF), we found that at around 150-200 ms post-keystroke, the neural activities in the dorsolateral prefrontal cortex (DLPFC) and supplementary motor area (SMA) were dissociated for boundary and within-sequence elements. Furthermore, the behavioral data demonstrated that feedback alterations on boundaries led to greater performance costs, such as more errors in the subsequent keystrokes. These findings jointly support the idea that the proper encoding of boundaries is critical in acquiring sensorimotor sequences. They also provide evidence for the involvement of a distinct neural circuitry in humans including prefrontal and higher-order motor areas during the encoding of the different classes of serial order.

Staphylococcal SCCmec elements encode an active MCM-like helicase and thus may be replicative

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

Mir-Sanchis, Ignacio; Roman, Christina A.; Misiura, Agnieszka

2016-08-29

Methicillin-resistant Staphylococcus aureus (MRSA) is a public-health threat worldwide. Although the mobile genomic island responsible for this phenotype, staphylococcal cassette chromosome (SCC), has been thought to be nonreplicative, we predicted DNA-replication-related functions for some of the conserved proteins encoded by SCC. We show that one of these, Cch, is homologous to the self-loading initiator helicases of an unrelated family of genomic islands, that it is an active 3'-to-5' helicase and that the adjacent ORF encodes a single-stranded DNA–binding protein. Our 2.9-Å crystal structure of intact Cch shows that it forms a hexameric ring. Cch, like the archaeal and eukaryotic MCM-familymore » replicative helicases, belongs to the pre–sensor II insert clade of AAA+ ATPases. Additionally, we found that SCC elements are part of a broader family of mobile elements, all of which encode a replication initiator upstream of their recombinases. Replication after excision would enhance the efficiency of horizontal gene transfer.« less

Constructs and methods for genome editing and genetic engineering of fungi and protists

DOEpatents

Hittinger, Christopher Todd; Alexander, William Gerald

2018-01-30

Provided herein are constructs for genome editing or genetic engineering in fungi or protists, methods of using the constructs and media for use in selecting cells. The construct include a polynucleotide encoding a thymidine kinase operably connected to a promoter, suitably a constitutive promoter; a polynucleotide encoding an endonuclease operably connected to an inducible promoter; and a recognition site for the endonuclease. The constructs may also include selectable markers for use in selecting recombinations.

Live-cell imaging of cell signaling using genetically encoded fluorescent reporters.

PubMed

Ni, Qiang; Mehta, Sohum; Zhang, Jin

2018-01-01

Synergistic advances in fluorescent protein engineering and live-cell imaging techniques in recent years have fueled the concurrent development and application of genetically encoded fluorescent reporters that are tailored for tracking signaling dynamics in living systems over multiple length and time scales. These biosensors are uniquely suited for this challenging task, owing to their specificity, sensitivity, and versatility, as well as to the noninvasive and nondestructive nature of fluorescence and the power of genetic encoding. Over the past 10 years, a growing number of fluorescent reporters have been developed for tracking a wide range of biological signals in living cells and animals, including second messenger and metabolite dynamics, enzyme activation and activity, and cell cycle progression and neuronal activity. Many of these biosensors are gaining wide use and are proving to be indispensable for unraveling the complex biological functions of individual signaling molecules in their native environment, the living cell, shedding new light on the structural and molecular underpinnings of cell signaling. In this review, we highlight recent advances in protein engineering that are likely to help expand and improve the design and application of these valuable tools. We then turn our focus to specific examples of live-cell imaging using genetically encoded fluorescent reporters as an important platform for advancing our understanding of G protein-coupled receptor signaling and neuronal activity. © 2017 Federation of European Biochemical Societies.

Cryogenic Optical Position Encoders for Mechanisms in the JWST Optical Telescope Element Simulator (OSIM)

NASA Technical Reports Server (NTRS)

Leviton, Douglas B.; Anderjaska, Thomas; Badger, James (Inventor); Capon, Tom; Davis, CLinton; Dicks, Brent (Inventor); Eichhorn, William; Garza, Mario; Guishard, Corina; Haghani, Shadan;

DNA transposon-based gene vehicles - scenes from an evolutionary drive

PubMed Central

2013-01-01

DNA transposons are primitive genetic elements which have colonized living organisms from plants to bacteria and mammals. Through evolution such parasitic elements have shaped their host genomes by replicating and relocating between chromosomal loci in processes catalyzed by the transposase proteins encoded by the elements themselves. DNA transposable elements are constantly adapting to life in the genome, and self-suppressive regulation as well as defensive host mechanisms may assist in buffering ‘cut-and-paste’ DNA mobilization until accumulating mutations will eventually restrict events of transposition. With the reconstructed Sleeping Beauty DNA transposon as a powerful engine, a growing list of transposable elements with activity in human cells have moved into biomedical experimentation and preclinical therapy as versatile vehicles for delivery and genomic insertion of transgenes. In this review, we aim to link the mechanisms that drive transposon evolution with the realities and potential challenges we are facing when adapting DNA transposons for gene transfer. We argue that DNA transposon-derived vectors may carry inherent, and potentially limiting, traits of their mother elements. By understanding in detail the evolutionary journey of transposons, from host colonization to element multiplication and inactivation, we may better exploit the potential of distinct transposable elements. Hence, parallel efforts to investigate and develop distinct, but potent, transposon-based vector systems will benefit the broad applications of gene transfer. Insight and clever optimization have shaped new DNA transposon vectors, which recently debuted in the first DNA transposon-based clinical trial. Learning from an evolutionary drive may help us create gene vehicles that are safer, more efficient, and less prone for suppression and inactivation. PMID:24320156

[The ENCODE project and functional genomics studies].

PubMed

Ding, Nan; Qu, Hongzhu; Fang, Xiangdong

2014-03-01

Upon the completion of the Human Genome Project, scientists have been trying to interpret the underlying genomic code for human biology. Since 2003, National Human Genome Research Institute (NHGRI) has invested nearly $0.3 billion and gathered over 440 scientists from more than 32 institutions in the United States, China, United Kingdom, Japan, Spain and Singapore to initiate the Encyclopedia of DNA Elements (ENCODE) project, aiming to identify and analyze all regulatory elements in the human genome. Taking advantage of the development of next-generation sequencing technologies and continuous improvement of experimental methods, ENCODE had made remarkable achievements: identified methylation and histone modification of DNA sequences and their regulatory effects on gene expression through altering chromatin structures, categorized binding sites of various transcription factors and constructed their regulatory networks, further revised and updated database for pseudogenes and non-coding RNA, and identified SNPs in regulatory sequences associated with diseases. These findings help to comprehensively understand information embedded in gene and genome sequences, the function of regulatory elements as well as the molecular mechanism underlying the transcriptional regulation by noncoding regions, and provide extensive data resource for life sciences, particularly for translational medicine. We re-viewed the contributions of high-throughput sequencing platform development and bioinformatical technology improve-ment to the ENCODE project, the association between epigenetics studies and the ENCODE project, and the major achievement of the ENCODE project. We also provided our prospective on the role of the ENCODE project in promoting the development of basic and clinical medicine.

The missing link: Bordetella petrii is endowed with both the metabolic versatility of environmental bacteria and virulence traits of pathogenic Bordetellae.

PubMed

Gross, Roy; Guzman, Carlos A; Sebaihia, Mohammed; dos Santos, Vítor A P Martins; Pieper, Dietmar H; Koebnik, Ralf; Lechner, Melanie; Bartels, Daniela; Buhrmester, Jens; Choudhuri, Jomuna V; Ebensen, Thomas; Gaigalat, Lars; Herrmann, Stefanie; Khachane, Amit N; Larisch, Christof; Link, Stefanie; Linke, Burkhard; Meyer, Folker; Mormann, Sascha; Nakunst, Diana; Rückert, Christian; Schneiker-Bekel, Susanne; Schulze, Kai; Vorhölter, Frank-Jörg; Yevsa, Tetyana; Engle, Jacquelyn T; Goldman, William E; Pühler, Alfred; Göbel, Ulf B; Goesmann, Alexander; Blöcker, Helmut; Kaiser, Olaf; Martinez-Arias, Rosa

2008-09-30

Bordetella petrii is the only environmental species hitherto found among the otherwise host-restricted and pathogenic members of the genus Bordetella. Phylogenetically, it connects the pathogenic Bordetellae and environmental bacteria of the genera Achromobacter and Alcaligenes, which are opportunistic pathogens. B. petrii strains have been isolated from very different environmental niches, including river sediment, polluted soil, marine sponges and a grass root. Recently, clinical isolates associated with bone degenerative disease or cystic fibrosis have also been described. In this manuscript we present the results of the analysis of the completely annotated genome sequence of the B. petrii strain DSMZ12804. B. petrii has a mosaic genome of 5,287,950 bp harboring numerous mobile genetic elements, including seven large genomic islands. Four of them are highly related to the clc element of Pseudomonas knackmussii B13, which encodes genes involved in the degradation of aromatics. Though being an environmental isolate, the sequenced B. petrii strain also encodes proteins related to virulence factors of the pathogenic Bordetellae, including the filamentous hemagglutinin, which is a major colonization factor of B. pertussis, and the master virulence regulator BvgAS. However, it lacks all known toxins of the pathogenic Bordetellae. The genomic analysis suggests that B. petrii represents an evolutionary link between free-living environmental bacteria and the host-restricted obligate pathogenic Bordetellae. Its remarkable metabolic versatility may enable B. petrii to thrive in very different ecological niches.

The ATP-sensitive potassium (KATP) channel-encoded dSUR gene is required for Drosophila heart function and is regulated by tinman

PubMed Central

Akasaka, Takeshi; Klinedinst, Susan; Ocorr, Karen; Bustamante, Erika L.; Kim, Seung K.; Bodmer, Rolf

2006-01-01

The homeobox transcription factor Tinman plays an important role in the initiation of heart development. Later functions of Tinman, including the target genes involved in cardiac physiology, are less well studied. We focused on the dSUR gene, which encodes an ATP-binding cassette transmembrane protein that is expressed in the heart. Mammalian SUR genes are associated with KATP (ATP-sensitive potassium) channels, which are involved in metabolic homeostasis. We provide experimental evidence that Tinman directly regulates dSUR expression in the developing heart. We identified a cis-regulatory element in the first intron of dSUR, which contains Tinman consensus binding sites and is sufficient for faithful dSUR expression in the fly’s myocardium. Site-directed mutagenesis of this element shows that these Tinman sites are critical to dSUR expression, and further genetic manipulations suggest that the GATA transcription factor Pannier is synergistically involved in cardiac-restricted dSUR expression in vivo. Physiological analysis of dSUR knock-down flies supports the idea that dSUR plays a protective role against hypoxic stress and pacing-induced heart failure. Because dSUR expression dramatically decreases with age, it is likely to be a factor involved in the cardiac aging phenotype of Drosophila. dSUR provides a model for addressing how embryonic regulators of myocardial cell commitment can contribute to the establishment and maintenance of cardiac performance. PMID:16882722

Characterization of Integrons and Sulfonamide Resistance Genes among Bacteria from Drinking Water Distribution Systems in Southwestern Nigeria.

PubMed

Adesoji, Ayodele T; Ogunjobi, Adeniyi A; Olatoye, Isaac O

2017-01-01

The emergence of antibiotic resistance among pathogenic bacteria in clinical and environmental settings is a global problem. Many antibiotic resistance genes are located on mobile genetic elements such as plasmids and integrons, enabling their transfer among a variety of bacterial species. Water distribution systems may be reservoirs for the spread of antibiotic resistance. Bacteria isolated from raw, treated, and municipal tap water samples from selected water distribution systems in south-western Nigeria were investigated using the point inoculation method with seeded antibiotics, PCR amplification, and sequencing for the determination of bacterial resistance profiles and class 1/2 integrase genes and gene cassettes, respectively. sul1,sul2, and sul3 were detected in 21.6, 27.8, and 0% of the isolates, respectively (n = 162). Class 1 and class 2 integrons were detected in 21.42 and 3.6% of the isolates, respectively (n = 168). Genes encoding resistance to aminoglycosides (aadA2, aadA1, and aadB), trimethoprim (dfrA15, dfr7, and dfrA1), and sulfonamide (sul1) were detected among bacteria with class 1 integrons, while genes that encodes resistance to strepthothricin (sat2) and trimethoprim (dfrA15) were detected among bacteria with class 2 integrons. Bacteria from these water samples are a potential reservoir of multidrug-resistant traits including sul genes and mobile resistance elements, i.e. the integrase gene. © 2016 S. Karger AG, Basel.

The missing link: Bordetella petrii is endowed with both the metabolic versatility of environmental bacteria and virulence traits of pathogenic Bordetellae

PubMed Central

Gross, Roy; Guzman, Carlos A; Sebaihia, Mohammed; Martins dos Santos, Vítor AP; Pieper, Dietmar H; Koebnik, Ralf; Lechner, Melanie; Bartels, Daniela; Buhrmester, Jens; Choudhuri, Jomuna V; Ebensen, Thomas; Gaigalat, Lars; Herrmann, Stefanie; Khachane, Amit N; Larisch, Christof; Link, Stefanie; Linke, Burkhard; Meyer, Folker; Mormann, Sascha; Nakunst, Diana; Rückert, Christian; Schneiker-Bekel, Susanne; Schulze, Kai; Vorhölter, Frank-Jörg; Yevsa, Tetyana; Engle, Jacquelyn T; Goldman, William E; Pühler, Alfred; Göbel, Ulf B; Goesmann, Alexander; Blöcker, Helmut; Kaiser, Olaf; Martinez-Arias, Rosa

2008-01-01

Background Bordetella petrii is the only environmental species hitherto found among the otherwise host-restricted and pathogenic members of the genus Bordetella. Phylogenetically, it connects the pathogenic Bordetellae and environmental bacteria of the genera Achromobacter and Alcaligenes, which are opportunistic pathogens. B. petrii strains have been isolated from very different environmental niches, including river sediment, polluted soil, marine sponges and a grass root. Recently, clinical isolates associated with bone degenerative disease or cystic fibrosis have also been described. Results In this manuscript we present the results of the analysis of the completely annotated genome sequence of the B. petrii strain DSMZ12804. B. petrii has a mosaic genome of 5,287,950 bp harboring numerous mobile genetic elements, including seven large genomic islands. Four of them are highly related to the clc element of Pseudomonas knackmussii B13, which encodes genes involved in the degradation of aromatics. Though being an environmental isolate, the sequenced B. petrii strain also encodes proteins related to virulence factors of the pathogenic Bordetellae, including the filamentous hemagglutinin, which is a major colonization factor of B. pertussis, and the master virulence regulator BvgAS. However, it lacks all known toxins of the pathogenic Bordetellae. Conclusion The genomic analysis suggests that B. petrii represents an evolutionary link between free-living environmental bacteria and the host-restricted obligate pathogenic Bordetellae. Its remarkable metabolic versatility may enable B. petrii to thrive in very different ecological niches. PMID:18826580

Genetic analysis reveals the identity of the photoreceptor for phototaxis in hormogonium filaments of Nostoc punctiforme.

PubMed

Campbell, Elsie L; Hagen, Kari D; Chen, Rui; Risser, Douglas D; Ferreira, Daniela P; Meeks, John C

2015-02-15

In cyanobacterial Nostoc species, substratum-dependent gliding motility is confined to specialized nongrowing filaments called hormogonia, which differentiate from vegetative filaments as part of a conditional life cycle and function as dispersal units. Here we confirm that Nostoc punctiforme hormogonia are positively phototactic to white light over a wide range of intensities. N. punctiforme contains two gene clusters (clusters 2 and 2i), each of which encodes modular cyanobacteriochrome-methyl-accepting chemotaxis proteins (MCPs) and other proteins that putatively constitute a basic chemotaxis-like signal transduction complex. Transcriptional analysis established that all genes in clusters 2 and 2i, plus two additional clusters (clusters 1 and 3) with genes encoding MCPs lacking cyanobacteriochrome sensory domains, are upregulated during the differentiation of hormogonia. Mutational analysis determined that only genes in cluster 2i are essential for positive phototaxis in N. punctiforme hormogonia; here these genes are designated ptx (for phototaxis) genes. The cluster is unusual in containing complete or partial duplicates of genes encoding proteins homologous to the well-described chemotaxis elements CheY, CheW, MCP, and CheA. The cyanobacteriochrome-MCP gene (ptxD) lacks transmembrane domains and has 7 potential binding sites for bilins. The transcriptional start site of the ptx genes does not resemble a sigma 70 consensus recognition sequence; moreover, it is upstream of two genes encoding gas vesicle proteins (gvpA and gvpC), which also are expressed only in the hormogonium filaments of N. punctiforme. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Association of Antibiotic Resistance in Agricultural Escherichia coli Isolates with Attachment to Quartz▿

PubMed Central

Liu, Ping; Soupir, Michelle L.; Zwonitzer, Martha; Huss, Bridgette; Jarboe, Laura R.

2011-01-01

Surface water can be contaminated by bacteria from various sources, including manure from agricultural facilities. Attachment of these bacteria to soil and organic particles contributes to their transport through the environment, though the mechanism of attachment is unknown. As bacterial attachment to human tissues is known to be correlated with antibiotic resistance, we have investigated here the relationship between bacterial attachment to environmental particles and antibiotic resistance in agricultural isolates. We evaluated 203 Escherichia coli isolates collected from swine facilities for attachment to quartz, resistance to 13 antibiotics, and the presence of genes encoding 13 attachment factors. The genes encoding type I, EcpA, P pili, and Ag43 were detected, though none was significantly related to attachment. Quartz attachment was positively and significantly (P < 0.0038) related to combined resistance to amoxicillin/streptomycin/tetracycline/sulfamethazine/tylosin/chlortetracycline and negatively and significantly (P < 0.0038) related to combined resistance to nalidixic acid/kanamycin/neomycin. These results provide clear evidence for a link between antibiotic resistance and attachment to quartz in agricultural isolates. We propose that this may be due to encoding by the responsible genes on a mobile genetic element. Further exploration of the relationship between antibiotic resistance and attachment to environmental particles will improve the understanding and modeling of environmental transport processes, with the goal of preventing human exposure to antibiotic-resistant or virulent microorganisms. PMID:21821756

Association of antibiotic resistance in agricultural Escherichia coli isolates with attachment to quartz.

PubMed

Liu, Ping; Soupir, Michelle L; Zwonitzer, Martha; Huss, Bridgette; Jarboe, Laura R

2011-10-01

Surface water can be contaminated by bacteria from various sources, including manure from agricultural facilities. Attachment of these bacteria to soil and organic particles contributes to their transport through the environment, though the mechanism of attachment is unknown. As bacterial attachment to human tissues is known to be correlated with antibiotic resistance, we have investigated here the relationship between bacterial attachment to environmental particles and antibiotic resistance in agricultural isolates. We evaluated 203 Escherichia coli isolates collected from swine facilities for attachment to quartz, resistance to 13 antibiotics, and the presence of genes encoding 13 attachment factors. The genes encoding type I, EcpA, P pili, and Ag43 were detected, though none was significantly related to attachment. Quartz attachment was positively and significantly (P < 0.0038) related to combined resistance to amoxicillin/streptomycin/tetracycline/sulfamethazine/tylosin/chlortetracycline and negatively and significantly (P < 0.0038) related to combined resistance to nalidixic acid/kanamycin/neomycin. These results provide clear evidence for a link between antibiotic resistance and attachment to quartz in agricultural isolates. We propose that this may be due to encoding by the responsible genes on a mobile genetic element. Further exploration of the relationship between antibiotic resistance and attachment to environmental particles will improve the understanding and modeling of environmental transport processes, with the goal of preventing human exposure to antibiotic-resistant or virulent microorganisms.

Gene Expression in Class 2 Integrons Is SOS-Independent and Involves Two Pc Promoters.

PubMed

Jové, Thomas; Da Re, Sandra; Tabesse, Aurore; Gassama-Sow, Amy; Ploy, Marie-Cécile

2017-01-01

Integrons are powerful bacterial genetic elements that permit the expression and dissemination of antibiotic-resistance gene cassettes. They contain a promoter Pc that allows the expression of gene cassettes captured through site-specific recombination catalyzed by IntI, the integron-encoded integrase. Class 1 and 2 integrons are found in both clinical and environmental settings. The regulation of intI and of Pc promoters has been extensively studied in class 1 integrons and the regulatory role of the SOS response on intI expression has been shown. Here we investigated class 2 integrons. We characterized the P intI2 promoter and showed that intI2 expression is not regulated via the SOS response. We also showed that, unlike class 1 integrons, class 2 integrons possess not one but two active Pc promoters that are located within the attI2 region that seem to contribute equally to gene cassette expression. Class 2 integrons mostly encode an inactive truncated integrase, but the rare class 2 integrons that encode an active integrase are associated with less efficient Pc2 promoter variants. We propose an evolutionary model for class 2 integrons in which the absence of repression of the integrase gene expression led to mutations resulting in either inactive integrase or Pc variants of weaker activity, thereby reducing the potential fitness cost of these integrons.

Absolute angular encoder based on optical diffraction

NASA Astrophysics Data System (ADS)

Wu, Jian; Zhou, Tingting; Yuan, Bo; Wang, Liqiang

2015-08-01

A new encoding method for absolute angular encoder based on optical diffraction was proposed in the present study. In this method, an encoder disc is specially designed that a series of elements are uniformly spaced in one circle and each element is consisted of four diffraction gratings, which are tilted in the directions of 30°, 60°, -60° and -30°, respectively. The disc is illuminated by a coherent light and the diffractive signals are received. The positions of diffractive spots are used for absolute encoding and their intensities are for subdivision, which is different from the traditional optical encoder based on transparent/opaque binary principle. Since the track's width in the disc is not limited in the diffraction pattern, it provides a new way to solve the contradiction between the size and resolution, which is good for minimization of encoder. According to the proposed principle, the diffraction pattern disc with a diameter of 40 mm was made by lithography in the glass substrate. A prototype of absolute angular encoder with a resolution of 20" was built up. Its maximum error was tested as 78" by comparing with a small angle measuring system based on laser beam deflection.

Characteristic and intermingled neocortical circuits encode different visual object discriminations.

PubMed

Zhang, Guo-Rong; Zhao, Hua; Cook, Nathan; Svestka, Michael; Choi, Eui M; Jan, Mary; Cook, Robert G; Geller, Alfred I

2017-07-28

Synaptic plasticity and neural network theories hypothesize that the essential information for advanced cognitive tasks is encoded in specific circuits and neurons within distributed neocortical networks. However, these circuits are incompletely characterized, and we do not know if a specific discrimination is encoded in characteristic circuits among multiple animals. Here, we determined the spatial distribution of active neurons for a circuit that encodes some of the essential information for a cognitive task. We genetically activated protein kinase C pathways in several hundred spatially-grouped glutamatergic and GABAergic neurons in rat postrhinal cortex, a multimodal associative area that is part of a distributed circuit that encodes visual object discriminations. We previously established that this intervention enhances accuracy for specific discriminations. Moreover, the genetically-modified, local circuit in POR cortex encodes some of the essential information, and this local circuit is preferentially activated during performance, as shown by activity-dependent gene imaging. Here, we mapped the positions of the active neurons, which revealed that two image sets are encoded in characteristic and different circuits. While characteristic circuits are known to process sensory information, in sensory areas, this is the first demonstration that characteristic circuits encode specific discriminations, in a multimodal associative area. Further, the circuits encoding the two image sets are intermingled, and likely overlapping, enabling efficient encoding. Consistent with reconsolidation theories, intermingled and overlapping encoding could facilitate formation of associations between related discriminations, including visually similar discriminations or discriminations learned at the same time or place. Copyright © 2017 Elsevier B.V. All rights reserved.

Chromatin Accessibility Mapping Identifies Mediators of Basal Transcription and Retinoid-Induced Repression of OTX2 in Medulloblastoma

PubMed Central

Zhang, Monica; Song, Lingyun; Lee, Bum-Kyu; Iyer, Vishwanath R.; Furey, Terrence S.; Crawford, Gregory E.; Yan, Hai; He, Yiping

2014-01-01

Despite an emerging understanding of the genetic alterations giving rise to various tumors, the mechanisms whereby most oncogenes are overexpressed remain unclear. Here we have utilized an integrated approach of genomewide regulatory element mapping via DNase-seq followed by conventional reporter assays and transcription factor binding site discovery to characterize the transcriptional regulation of the medulloblastoma oncogene Orthodenticle Homeobox 2 (OTX2). Through these studies we have revealed that OTX2 is differentially regulated in medulloblastoma at the level of chromatin accessibility, which is in part mediated by DNA methylation. In cell lines exhibiting chromatin accessibility of OTX2 regulatory regions, we found that autoregulation maintains OTX2 expression. Comparison of medulloblastoma regulatory elements with those of the developing brain reveals that these tumors engage a developmental regulatory program to drive OTX2 transcription. Finally, we have identified a transcriptional regulatory element mediating retinoid-induced OTX2 repression in these tumors. This work characterizes for the first time the mechanisms of OTX2 overexpression in medulloblastoma. Furthermore, this study establishes proof of principle for applying ENCODE datasets towards the characterization of upstream trans-acting factors mediating expression of individual genes. PMID:25198066

The Tc1/mariner transposable element family shapes genetic variation and gene expression in the protist Trichomonas vaginalis

PubMed Central

2014-01-01

Background Trichomonas vaginalis is the most prevalent non-viral sexually transmitted parasite. Although the protist is presumed to reproduce asexually, 60% of its haploid genome contains transposable elements (TEs), known contributors to genome variability. The availability of a draft genome sequence and our collection of >200 global isolates of T. vaginalis facilitate the study and analysis of TE population dynamics and their contribution to genomic variability in this protist. Results We present here a pilot study of a subset of class II Tc1/mariner TEs that belong to the T. vaginalis Tvmar1 family. We report the genetic structure of 19 Tvmar1 loci, their ability to encode a full-length transposase protein, and their insertion frequencies in 94 global isolates from seven regions of the world. While most of the Tvmar1 elements studied exhibited low insertion frequencies, two of the 19 loci (locus 1 and locus 9) show high insertion frequencies of 1.00 and 0.96, respectively. The genetic structuring of the global populations identified by principal component analysis (PCA) of the Tvmar1 loci is in general agreement with published data based on genotyping, showing that Tvmar1 polymorphisms are a robust indicator of T. vaginalis genetic history. Analysis of expression of 22 genes flanking 13 Tvmar1 loci indicated significantly altered expression of six of the genes next to five Tvmar1 insertions, suggesting that the insertions have functional implications for T. vaginalis gene expression. Conclusions Our study is the first in T. vaginalis to describe Tvmar1 population dynamics and its contribution to genetic variability of the parasite. We show that a majority of our studied Tvmar1 insertion loci exist at very low frequencies in the global population, and insertions are variable between geographical isolates. In addition, we observe that low frequency insertion is related to reduced or abolished expression of flanking genes. While low insertion frequencies might be expected, we identified two Tvmar1 insertion loci that are fixed across global populations. This observation indicates that Tvmar1 insertion may have differing impacts and fitness costs in the host genome and may play varying roles in the adaptive evolution of T. vaginalis. PMID:24834134

Role of penA polymorphisms for penicillin susceptibility in Neisseria lactamica and Neisseria meningitidis.

PubMed

Karch, André; Vogel, Ulrich; Claus, Heike

2015-10-01

In meningococci, reduced penicillin susceptibility is associated with five specific mutations in the transpeptidase region of penicillin binding protein 2 (PBP2). We showed that the same set of mutations was present in 64 of 123 Neisseria lactamica strains obtained from a carriage study (MIC range: 0.125-2.0mg/L). The PBP2 encoding penA alleles in these strains were genetically similar to those found in intermediate resistant meningococci suggesting frequent interspecies genetic exchange. Fifty-six N. lactamica isolates with mostly lower penicillin MICs (range: 0.064-0.38mg/L) exhibited only three of the five mutations. The corresponding penA alleles were unique to N. lactamica and formed a distinct genetic clade. PenA alleles with no mutations on the other hand were unique to meningococci. Under penicillin selective pressure, genetic transformation of N. lactamica penA alleles in meningococci was only possible for alleles encoding five mutations, but not for those encoding three mutations; the transfer resulted in MICs comparable to those of meningococci harboring penA alleles that encoded PBP2 with five mutations, but considerably lower than those of the corresponding N. lactamica donor strains. Due to a transformation barrier the complete N. lactamica penA could not be transformed into N. meningitidis. In summary, penicillin MICs in N. lactamica were associated with the number of mutations in the transpeptidase region of PBP2. Evidence for interspecific genetic transfer was only observed for penA alleles associated with higher MICs, suggesting that alleles encoding only three mutations in the transpeptidase region are biologically not effective in N. meningitidis. Factors other than PBP2 seem to be responsible for the high levels of penicillin resistance in N. lactamica. A reduction of penicillin susceptibility in N. meningitidis by horizontal gene transfer from N. lactamica is unlikely to happen. Copyright © 2015 Elsevier GmbH. All rights reserved.

Into the Wild: Dissemination of Antibiotic Resistance Determinants via a Species Recovery Program

PubMed Central

Power, Michelle L.; Emery, Samantha; Gillings, Michael R.

2013-01-01

Management strategies associated with captive breeding of endangered species can establish opportunities for transfer of pathogens and genetic elements between human and animal microbiomes. The class 1 integron is a mobile genetic element associated with clinical antibiotic resistance in gram-negative bacteria. We examined the gut microbiota of endangered brush-tail rock wallabies Petrogale penicillata to determine if they carried class 1 integrons. No integrons were detected in 65 animals from five wild populations. In contrast, class 1 integrons were detected in 48% of fecal samples from captive wallabies. The integrons contained diverse cassette arrays that encoded resistance to streptomycin, spectinomycin, and trimethoprim. Evidence suggested that captive wallabies had acquired typical class 1 integrons on a number of independent occasions, and had done so in the absence of strong selection afforded by antibiotic therapy. Sufficient numbers of bacteria containing diverse class 1 integrons must have been present in the general environment occupied by the wallabies to account for this acquisition. The captive wallabies have now been released, in an attempt to bolster wild populations of the species. Consequently, they can potentially spread resistance integrons into wild wallabies and into new environments. This finding highlights the potential for genes and pathogens from human sources to be acquired during captive breeding and to be unwittingly spread to other populations. PMID:23717399

Evolution of Sphingomonad Gene Clusters Related to Pesticide Catabolism Revealed by Genome Sequence and Mobilomics of Sphingobium herbicidovorans MH

PubMed Central

Nielsen, Tue Kjærgaard; Rasmussen, Morten; Demanèche, Sandrine; Cecillon, Sébastien; Vogel, Timothy M.

2017-01-01

Abstract Bacterial degraders of chlorophenoxy herbicides have been isolated from various ecosystems, including pristine environments. Among these degraders, the sphingomonads constitute a prominent group that displays versatile xenobiotic-degradation capabilities. Four separate sequencing strategies were required to provide the complete sequence of the complex and plastic genome of the canonical chlorophenoxy herbicide-degrading Sphingobium herbicidovorans MH. The genome has an intricate organization of the chlorophenoxy-herbicide catabolic genes sdpA, rdpA, and cadABCD that encode the (R)- and (S)-enantiomer-specific 2,4-dichlorophenoxypropionate dioxygenases and four subunits of a Rieske non-heme iron oxygenase involved in 2-methyl-chlorophenoxyacetic acid degradation, respectively. Several major genomic rearrangements are proposed to help understand the evolution and mobility of these important genes and their genetic context. Single-strain mobilomic sequence analysis uncovered plasmids and insertion sequence-associated circular intermediates in this environmentally important bacterium and enabled the description of evolutionary models for pesticide degradation in strain MH and related organisms. The mobilome presented a complex mosaic of mobile genetic elements including four plasmids and several circular intermediate DNA molecules of insertion-sequence elements and transposons that are central to the evolution of xenobiotics degradation. Furthermore, two individual chromosomally integrated prophages were shown to excise and form free circular DNA molecules. This approach holds great potential for improving the understanding of genome plasticity, evolution, and microbial ecology. PMID:28961970

Simplification of genotyping techniques of the ABO blood type experiment and exploration of population genetics.

PubMed

Hu, Jian; Zhou, Yi-ren; Ding, Jia-lin; Wang, Zhi-yuan; Liu, Ling; Wang, Ye-kai; Lou, Hui-ling; Qiao, Shou-yi; Wu, Yan-hua

2017-05-20

The ABO blood type is one of the most common and widely used genetic traits in humans. Three glycosyltransferase-encoding gene alleles, I A , I B and i, produce three red blood cell surface antigens, by which the ABO blood type is classified. By using the ABO blood type experiment as an ideal case for genetics teaching, we can easily introduce to the students several genetic concepts, including multiple alleles, gene interaction, single nucleotide polymorphism (SNP) and gene evolution. Herein we have innovated and integrated our ABO blood type genetics experiments. First, in the section of Molecular Genetics, a new method of ABO blood genotyping was established: specific primers based on SNP sites were designed to distinguish three alleles through quantitative real-time PCR. Next, the experimental teaching method of Gene Evolution was innovated in the Population Genetics section: a gene-evolution software was developed to simulate the evolutionary tendency of the ABO genotype encoding alleles under diverse conditions. Our reform aims to extend the contents of genetics experiments, to provide additional teaching approaches, and to improve the learning efficiency of our students eventually.

Silk Materials Functionalized via Genetic Engineering for Biomedical Applications.

PubMed

Deptuch, Tomasz; Dams-Kozlowska, Hanna

2017-12-12

The great mechanical properties, biocompatibility and biodegradability of silk-based materials make them applicable to the biomedical field. Genetic engineering enables the construction of synthetic equivalents of natural silks. Knowledge about the relationship between the structure and function of silk proteins enables the design of bioengineered silks that can serve as the foundation of new biomaterials. Furthermore, in order to better address the needs of modern biomedicine, genetic engineering can be used to obtain silk-based materials with new functionalities. Sequences encoding new peptides or domains can be added to the sequences encoding the silk proteins. The expression of one cDNA fragment indicates that each silk molecule is related to a functional fragment. This review summarizes the proposed genetic functionalization of silk-based materials that can be potentially useful for biomedical applications.

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.

Engineering of a genetically encodable fluorescent voltage sensor exploiting fast Ci-VSP voltage-sensing movements.

PubMed

Lundby, Alicia; Mutoh, Hiroki; Dimitrov, Dimitar; Akemann, Walther; Knöpfel, Thomas

2008-06-25

Ci-VSP contains a voltage-sensing domain (VSD) homologous to that of voltage-gated potassium channels. Using charge displacement ('gating' current) measurements we show that voltage-sensing movements of this VSD can occur within 1 ms in mammalian membranes. Our analysis lead to development of a genetically encodable fluorescent protein voltage sensor (VSFP) in which the fast, voltage-dependent conformational changes of the Ci-VSP voltage sensor are transduced to similarly fast fluorescence read-outs.

Caprine prion gene polymorphisms are associated with decreased incidence of classical scrapie in goat herds in the United Kingdom

PubMed Central

2011-01-01

The application of genetic breeding programmes to eradicate transmissible spongiform encephalopathies in goats is an important aim for reasons of animal welfare as well as human food safety and food security. Based on the positive impact of Prnp genetics on sheep scrapie in Europe in the past decade, we have established caprine Prnp gene variation in more than 1100 goats from the United Kingdom and studied the association of Prnp alleles with disease phenotypes in 150 scrapie-positive goats. This investigation confirms the association of the Met142 encoding Prnp allele with increased resistance to preclinical and clinical scrapie. It reveals a novel association of the Ser127 encoding allele with a reduced probability to develop clinical signs of scrapie in goats that are already positive for the accumulation of disease-specific prion protein in brain or periphery. A United Kingdom survey of Prnp genotypes in eight common breeds revealed eleven alleles in over thirty genotypes. The Met142 encoding allele had a high overall mean allele frequency of 22.6%, whereas the Ser127 encoding allele frequency was considerably lower with 6.4%. In contrast, a well known resistance associated allele encoding Lys222 was found to be rare (0.9%) in this survey. The analysis of Prnp genotypes in Mexican Criollas goats revealed nine alleles, including a novel Phe to Leu substitution in codon 201, confirming that high genetic variability of Prnp can be found in scrapie-free populations. Our study implies that it should be feasible to lower scrapie prevalence in goat herds in the United Kingdom by genetic selection. PMID:22040234

Genomic Evidence for the Evolution of Streptococcus equi: Host Restriction, Increased Virulence, and Genetic Exchange with Human Pathogens

PubMed Central

Paillot, Romain; Steward, Karen F.; Webb, Katy; Ainslie, Fern; Jourdan, Thibaud; Bason, Nathalie C.; Holroyd, Nancy E.; Mungall, Karen; Quail, Michael A.; Sanders, Mandy; Simmonds, Mark; Willey, David; Brooks, Karen; Aanensen, David M.; Spratt, Brian G.; Jolley, Keith A.; Maiden, Martin C. J.; Kehoe, Michael; Chanter, Neil; Bentley, Stephen D.; Robinson, Carl; Maskell, Duncan J.; Parkhill, Julian; Waller, Andrew S.

2009-01-01

The continued evolution of bacterial pathogens has major implications for both human and animal disease, but the exchange of genetic material between host-restricted pathogens is rarely considered. Streptococcus equi subspecies equi (S. equi) is a host-restricted pathogen of horses that has evolved from the zoonotic pathogen Streptococcus equi subspecies zooepidemicus (S. zooepidemicus). These pathogens share approximately 80% genome sequence identity with the important human pathogen Streptococcus pyogenes. We sequenced and compared the genomes of S. equi 4047 and S. zooepidemicus H70 and screened S. equi and S. zooepidemicus strains from around the world to uncover evidence of the genetic events that have shaped the evolution of the S. equi genome and led to its emergence as a host-restricted pathogen. Our analysis provides evidence of functional loss due to mutation and deletion, coupled with pathogenic specialization through the acquisition of bacteriophage encoding a phospholipase A2 toxin, and four superantigens, and an integrative conjugative element carrying a novel iron acquisition system with similarity to the high pathogenicity island of Yersinia pestis. We also highlight that S. equi, S. zooepidemicus, and S. pyogenes share a common phage pool that enhances cross-species pathogen evolution. We conclude that the complex interplay of functional loss, pathogenic specialization, and genetic exchange between S. equi, S. zooepidemicus, and S. pyogenes continues to influence the evolution of these important streptococci. PMID:19325880

Genetic Nature of Elemental Contents in Wheat Grains and Its Genomic Prediction: Toward the Effective Use of Wheat Landraces from Afghanistan

PubMed Central

Yamaoka, Shuhei; Yoshimura, Kazusa; Kondou, Youichi; Onogi, Akio; Matsui, Minami; Iwata, Hiroyoshi; Ban, Tomohiro

2017-01-01

Profiling elemental contents in wheat grains and clarifying the underlying genetic systems are important for the breeding of biofortified crops. Our objective was to evaluate the genetic potential of 269 Afghan wheat landraces for increasing elemental contents in wheat cultivars. The contents of three major (Mg, K, and P) and three minor (Mn, Fe, and Zn) elements in wheat grains were measured by energy dispersive X-ray fluorescence spectrometry. Large variations in elemental contents were observed among landraces. Marker-based heritability estimates were low to moderate, suggesting that the elemental contents are complex quantitative traits. Genetic correlations between two locations (Japan and Afghanistan) and among the six elements were estimated using a multi-response Bayesian linear mixed model. Low-to-moderate genetic correlations were observed among major elements and among minor elements respectively, but not between major and minor elements. A single-response genome-wide association study detected only one significant marker, which was associated with Zn, suggesting it will be difficult to increase the elemental contents of wheat by conventional marker-assisted selection. Genomic predictions for major elemental contents were moderately or highly accurate, whereas those for minor elements were mostly low or moderate. Our results indicate genomic selection may be useful for the genetic improvement of elemental contents in wheat. PMID:28072876

Amplifying genetic logic gates.

PubMed

Bonnet, Jerome; Yin, Peter; Ortiz, Monica E; Subsoontorn, Pakpoom; Endy, Drew

2013-05-03

Organisms must process information encoded via developmental and environmental signals to survive and reproduce. Researchers have also engineered synthetic genetic logic to realize simpler, independent control of biological processes. We developed a three-terminal device architecture, termed the transcriptor, that uses bacteriophage serine integrases to control the flow of RNA polymerase along DNA. Integrase-mediated inversion or deletion of DNA encoding transcription terminators or a promoter modulates transcription rates. We realized permanent amplifying AND, NAND, OR, XOR, NOR, and XNOR gates actuated across common control signal ranges and sequential logic supporting autonomous cell-cell communication of DNA encoding distinct logic-gate states. The single-layer digital logic architecture developed here enables engineering of amplifying logic gates to control transcription rates within and across diverse organisms.

[Genetic diversity of extraintestinal Escherichia coli strains producers of beta-lactamases TEM, SHV and CTX-M associated with healthcare].

PubMed

Varela, Yasmin; Millán, Beatriz; Araque, María

2017-06-01

There are few reports from Venezuela describing the genetic basis that sustains the pathogenic potential and phylogenetics of Escherichia coli extraintestinal strains isolated in health care units. To establish the genetic diversity of extraintestinal E. coli strains producers of betalactamases TEM, SHV and CTX-M associated with healthcare. We studied a collection of 12 strains of extraintestinal E. coli with diminished sensitivity to broad-spectrum cephalosporins. Antimicrobial susceptibility was determined by minimum inhibitory concentration. We determined the phylogenetic groups, virulence factors and genes encoding antimicrobial resistance using PCR, and clonal characterization by repetitive element palindromic-PCR rep-PCR. All strains showed resistance to cephalosporins and joint resistance to quinolones and aminoglycosides. The phylogenetic distribution showed that the A and B1 groups were the most frequent, followed by D and B2. We found all the virulence factors analyzed in the B2 group, and fimH gene was the most frequent among them. We found blaCTX-M in all strains,with a higher prevalence of blaCTX-M-8; two of these strains showed coproduction of blaCTX-M-9 and were genetically identified as blaCTXM-65 and blaCTX-M-147 by sequencing. The strains under study showed genetic diversity, hosting a variety of virulence genes, as well as antimicrobial resistance with no particular phylogroup prevalence. This is the first report of blaCTX-M alleles in Venezuela and in the world associated to non-genetically related strains isolated in health care units, a situation that deserves attention, as well as the rationalization of antimicrobials use.

Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

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

Thompson, David N.; Apel, William A.; Thompson, Vicki S.

A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extractsmore » are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.« less

Red fluorescent genetically encoded indicator for intracellular hydrogen peroxide

NASA Astrophysics Data System (ADS)

Ermakova, Yulia G.; Bilan, Dmitry S.; Matlashov, Mikhail E.; Mishina, Natalia M.; Markvicheva, Ksenia N.; Subach, Oksana M.; Subach, Fedor V.; Bogeski, Ivan; Hoth, Markus; Enikolopov, Grigori; Belousov, Vsevolod V.

2014-10-01

Reactive oxygen species (ROS) are conserved regulators of numerous cellular functions, and overproduction of ROS is a hallmark of various pathological processes. Genetically encoded fluorescent probes are unique tools to study ROS production in living systems of different scale and complexity. However, the currently available recombinant redox sensors have green emission, which overlaps with the spectra of many other probes. Expanding the spectral range of recombinant in vivo ROS probes would enable multiparametric in vivo ROS detection. Here we present the first genetically encoded red fluorescent sensor for hydrogen peroxide detection, HyPerRed. The performance of this sensor is similar to its green analogues. We demonstrate the utility of the sensor by tracing low concentrations of H2O2 produced in the cytoplasm of cultured cells upon growth factor stimulation. Moreover, using HyPerRed we detect local and transient H2O2 production in the mitochondrial matrix upon inhibition of the endoplasmic reticulum Ca2+ uptake.

Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

DOEpatents

Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Ward, Thomas E.

2016-03-22

A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.

Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

DOEpatents

Thompson, David N; Apel, William A; Thompson, Vicki S; Ward, Thomas E

2013-07-23

A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.

Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

DOEpatents

Thompson, David N; Apel, William A; Thompson, Vicki S; Ward, Thomas E

2014-04-08

A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.

Modular assembly of transposable element arrays by microsatellite targeting in the guayule and rice genomes.

PubMed

Valdes Franco, José A; Wang, Yi; Huo, Naxin; Ponciano, Grisel; Colvin, Howard A; McMahan, Colleen M; Gu, Yong Q; Belknap, William R

2018-04-19

Guayule (Parthenium argentatum A. Gray) is a rubber-producing desert shrub native to Mexico and the United States. Guayule represents an alternative to Hevea brasiliensis as a source for commercial natural rubber. The efficient application of modern molecular/genetic tools to guayule improvement requires characterization of its genome. The 1.6 Gb guayule genome was sequenced, assembled and annotated. The final 1.5 Gb assembly, while fragmented (N 50  = 22 kb), maps > 95% of the shotgun reads and is essentially complete. Approximately 40,000 transcribed, protein encoding genes were annotated on the assembly. Further characterization of this genome revealed 15 families of small, microsatellite-associated, transposable elements (TEs) with unexpected chromosomal distribution profiles. These SaTar (Satellite Targeted) elements, which are non-autonomous Mu-like elements (MULEs), were frequently observed in multimeric linear arrays of unrelated individual elements within which no individual element is interrupted by another. This uniformly non-nested TE multimer architecture has not been previously described in either eukaryotic or prokaryotic genomes. Five families of similarly distributed non-autonomous MULEs (microsatellite associated, modularly assembled) were characterized in the rice genome. Families of TEs with similar structures and distribution profiles were identified in sorghum and citrus. The sequencing and assembly of the guayule genome provides a foundation for application of current crop improvement technologies to this plant. In addition, characterization of this genome revealed SaTar elements with distribution profiles unique among TEs. Satar targeting appears based on an alternative MULE recombination mechanism with the potential to impact gene evolution.

Genetics Home Reference: Hartnup disease

MedlinePlus

... Hartnup disorder is caused by mutations in the gene encoding the neutral amino acid transporter SLC6A19. Nat Genet. 2004 ... are genome editing and CRISPR-Cas9? What is precision medicine? What ...

Recent developments of genetically encoded optical sensors for cell biology.

PubMed

Bolbat, Andrey; Schultz, Carsten

2017-01-01

Optical sensors are powerful tools for live cell research as they permit to follow the location, concentration changes or activities of key cellular players such as lipids, ions and enzymes. Most of the current sensor probes are based on fluorescence which provides great spatial and temporal precision provided that high-end microscopy is used and that the timescale of the event of interest fits the response time of the sensor. Many of the sensors developed in the past 20 years are genetically encoded. There is a diversity of designs leading to simple or sometimes complicated applications for the use in live cells. Genetically encoded sensors began to emerge after the discovery of fluorescent proteins, engineering of their improved optical properties and the manipulation of their structure through application of circular permutation. In this review, we will describe a variety of genetically encoded biosensor concepts, including those for intensiometric and ratiometric sensors based on single fluorescent proteins, Forster resonance energy transfer-based sensors, sensors utilising bioluminescence, sensors using self-labelling SNAP- and CLIP-tags, and finally tetracysteine-based sensors. We focus on the newer developments and discuss the current approaches and techniques for design and application. This will demonstrate the power of using optical sensors in cell biology and will help opening the field to more systematic applications in the future. © 2016 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

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.

Silk Materials Functionalized via Genetic Engineering for Biomedical Applications

PubMed Central

Deptuch, Tomasz

2017-01-01

The great mechanical properties, biocompatibility and biodegradability of silk-based materials make them applicable to the biomedical field. Genetic engineering enables the construction of synthetic equivalents of natural silks. Knowledge about the relationship between the structure and function of silk proteins enables the design of bioengineered silks that can serve as the foundation of new biomaterials. Furthermore, in order to better address the needs of modern biomedicine, genetic engineering can be used to obtain silk-based materials with new functionalities. Sequences encoding new peptides or domains can be added to the sequences encoding the silk proteins. The expression of one cDNA fragment indicates that each silk molecule is related to a functional fragment. This review summarizes the proposed genetic functionalization of silk-based materials that can be potentially useful for biomedical applications. PMID:29231863

Thirty years of Alzheimer's disease genetics: the implications of systematic meta-analyses.

PubMed

Bertram, Lars; Tanzi, Rudolph E

2008-10-01

The genetic underpinnings of Alzheimer's disease (AD) remain largely elusive despite early successes in identifying three genes that cause early-onset familial AD (those that encode amyloid precursor protein (APP) and the presenilins (PSEN1 and PSEN2)), and one genetic risk factor for late-onset AD (the gene that encodes apolipoprotein E (APOE)). A large number of studies that aimed to help uncover the remaining disease-related loci have been published in recent decades, collectively proposing or refuting the involvement of over 500 different gene candidates. Systematic meta-analyses of these studies currently highlight more than 20 loci that have modest but significant effects on AD risk. This Review discusses the putative pathogenetic roles and common biochemical pathways of some of the most genetically and biologically compelling of these potential AD risk factors.

Natural autoantibodies: from 'horror autotoxicus' to 'gnothi seauton'.

PubMed

Avrameas, S

1991-05-01

The immune system of normal unimmunized animals is characterized by the presence of B cells synthesizing and secreting mainly polyreactive, but also monoreactive, IgM and IgG natural antibodies that can react with a variety of self constituents. These antibodies, like the autoantibodies appearing in several immunopathological states, use the same genetic elements as the antibodies directed against environmental antigens, and seem to be encoded by unmutated germ-line genes. Accumulating evidence indicates that these natural auto-antibodies exert various biological roles, both related and unrelated to the immune system. In this article, Stratis Avrameas proposes that natural auto-antibodies, by interacting with the large number of self constituents present in an organism, establish an extensive dynamic network that contributes to the general homeostasis of the organism.

A novel de novo POGZ mutation in a patient with intellectual disability.

PubMed

Tan, Bo; Zou, Yongyi; Zhang, Yue; Zhang, Rui; Ou, Jianjun; Shen, Yidong; Zhao, Jingping; Luo, Xiaomei; Guo, Jing; Zeng, Lanlan; Hu, Yiqiao; Zheng, Yu; Pan, Qian; Liang, Desheng; Wu, Lingqian

2016-04-01

POGZ, the gene encoding pogo transposable element-derived protein with zinc-finger domain, has been implicated in autism spectrum disorder and it is widely expressed in the human tissues, including the brain. Intellectual disability (ID) is highly heterogeneous neurodevelopment disorder and affects ~2-3% of the general population. Here we report the identification of a novel frameshift mutation in the coding region of the POGZ gene (c.1277_1278insC), which occurred de novo in a Chinese patient with ID. In silico analysis and western blotting revealed this frameshift mutation generating truncated protein in peripheral blood lymphocytes, and this may disrupt several important domains of POGZ gene. Our finding broadens the spectrum of POGZ mutations and may help to understand the molecular basis of ID and aid genetic counseling.

Learning Intelligent Genetic Algorithms Using Japanese Nonograms

ERIC Educational Resources Information Center

Tsai, Jinn-Tsong; Chou, Ping-Yi; Fang, Jia-Cen

2012-01-01

An intelligent genetic algorithm (IGA) is proposed to solve Japanese nonograms and is used as a method in a university course to learn evolutionary algorithms. The IGA combines the global exploration capabilities of a canonical genetic algorithm (CGA) with effective condensed encoding, improved fitness function, and modified crossover and…

A deep auto-encoder model for gene expression prediction.

PubMed

Xie, Rui; Wen, Jia; Quitadamo, Andrew; Cheng, Jianlin; Shi, Xinghua

2017-11-17

Gene expression is a key intermediate level that genotypes lead to a particular trait. Gene expression is affected by various factors including genotypes of genetic variants. With an aim of delineating the genetic impact on gene expression, we build a deep auto-encoder model to assess how good genetic variants will contribute to gene expression changes. This new deep learning model is a regression-based predictive model based on the MultiLayer Perceptron and Stacked Denoising Auto-encoder (MLP-SAE). The model is trained using a stacked denoising auto-encoder for feature selection and a multilayer perceptron framework for backpropagation. We further improve the model by introducing dropout to prevent overfitting and improve performance. To demonstrate the usage of this model, we apply MLP-SAE to a real genomic datasets with genotypes and gene expression profiles measured in yeast. Our results show that the MLP-SAE model with dropout outperforms other models including Lasso, Random Forests and the MLP-SAE model without dropout. Using the MLP-SAE model with dropout, we show that gene expression quantifications predicted by the model solely based on genotypes, align well with true gene expression patterns. We provide a deep auto-encoder model for predicting gene expression from SNP genotypes. This study demonstrates that deep learning is appropriate for tackling another genomic problem, i.e., building predictive models to understand genotypes' contribution to gene expression. With the emerging availability of richer genomic data, we anticipate that deep learning models play a bigger role in modeling and interpreting genomics.

Genetic Dissection of Sexual Reproduction in a Primary Homothallic Basidiomycete

PubMed Central

Sampaio, José Paulo; Gonçalves, Paula

2016-01-01

In fungi belonging to the phylum Basidiomycota, sexual compatibility is usually determined by two genetically unlinked MAT loci, one of which encodes one or more pheromone receptors (P/R) and pheromone precursors, and the other comprehends at least one pair of divergently transcribed genes encoding homeodomain (HD) transcription factors. Most species are heterothallic, meaning that sexual reproduction requires mating between two sexually compatible individuals harboring different alleles at both MAT loci. However, some species are known to be homothallic, one individual being capable of completing the sexual cycle without mating with a genetically distinct partner. While the molecular underpinnings of the heterothallic life cycles of several basidiomycete model species have been dissected in great detail, much less is known concerning the molecular basis for homothallism. Following the discovery in available draft genomes of the homothallic basidiomycetous yeast Phaffia rhodozyma of P/R and HD genes, we employed available genetic tools to determine their role in sexual development. Two P/R clusters, each harboring one pheromone receptor and one pheromone precursor gene were found in close vicinity of each other and were shown to form two redundant P/R pairs, each receptor being activated by the pheromone encoded by the most distal pheromone precursor gene. The HD locus is apparently genetically unlinked to the P/R locus and encodes a single pair of divergently transcribed HD1 and HD2 transcription factors, both required for normal completion of the sexual cycle. Given the genetic makeup of P. rhodozyma MAT loci, we postulate that it is a primarily homothallic organism and we propose a model for the interplay of molecular interactions required for sexual development in this species. Phaffia rhodozyma is considered one of the most promising microbial source of the carotenoid astaxanthin. Further development of this yeast as an industrial organism will benefit from new insights regarding its sexual reproduction system. PMID:27327578

A platform for rapid prototyping of synthetic gene networks in mammalian cells

PubMed Central

Duportet, Xavier; Wroblewska, Liliana; Guye, Patrick; Li, Yinqing; Eyquem, Justin; Rieders, Julianne; Rimchala, Tharathorn; Batt, Gregory; Weiss, Ron

2014-01-01

Mammalian synthetic biology may provide novel therapeutic strategies, help decipher new paths for drug discovery and facilitate synthesis of valuable molecules. Yet, our capacity to genetically program cells is currently hampered by the lack of efficient approaches to streamline the design, construction and screening of synthetic gene networks. To address this problem, here we present a framework for modular and combinatorial assembly of functional (multi)gene expression vectors and their efficient and specific targeted integration into a well-defined chromosomal context in mammalian cells. We demonstrate the potential of this framework by assembling and integrating different functional mammalian regulatory networks including the largest gene circuit built and chromosomally integrated to date (6 transcription units, 27kb) encoding an inducible memory device. Using a library of 18 different circuits as a proof of concept, we also demonstrate that our method enables one-pot/single-flask chromosomal integration and screening of circuit libraries. This rapid and powerful prototyping platform is well suited for comparative studies of genetic regulatory elements, genes and multi-gene circuits as well as facile development of libraries of isogenic engineered cell lines. PMID:25378321

Sequence and analysis of chromosome 2 of the plant Arabidopsis thaliana.

PubMed

Lin, X; Kaul, S; Rounsley, S; Shea, T P; Benito, M I; Town, C D; Fujii, C Y; Mason, T; Bowman, C L; Barnstead, M; Feldblyum, T V; Buell, C R; Ketchum, K A; Lee, J; Ronning, C M; Koo, H L; Moffat, K S; Cronin, L A; Shen, M; Pai, G; Van Aken, S; Umayam, L; Tallon, L J; Gill, J E; Adams, M D; Carrera, A J; Creasy, T H; Goodman, H M; Somerville, C R; Copenhaver, G P; Preuss, D; Nierman, W C; White, O; Eisen, J A; Salzberg, S L; Fraser, C M; Venter, J C

1999-12-16

Arabidopsis thaliana (Arabidopsis) is unique among plant model organisms in having a small genome (130-140 Mb), excellent physical and genetic maps, and little repetitive DNA. Here we report the sequence of chromosome 2 from the Columbia ecotype in two gap-free assemblies (contigs) of 3.6 and 16 megabases (Mb). The latter represents the longest published stretch of uninterrupted DNA sequence assembled from any organism to date. Chromosome 2 represents 15% of the genome and encodes 4,037 genes, 49% of which have no predicted function. Roughly 250 tandem gene duplications were found in addition to large-scale duplications of about 0.5 and 4.5 Mb between chromosomes 2 and 1 and between chromosomes 2 and 4, respectively. Sequencing of nearly 2 Mb within the genetically defined centromere revealed a low density of recognizable genes, and a high density and diverse range of vestigial and presumably inactive mobile elements. More unexpected is what appears to be a recent insertion of a continuous stretch of 75% of the mitochondrial genome into chromosome 2.

Genetic conflicts: the usual suspects and beyond

PubMed Central

McLaughlin, Richard N.

2017-01-01

ABSTRACT Selfishness is pervasive and manifests at all scales of biology, from societies, to individuals, to genetic elements within a genome. The relentless struggle to seek evolutionary advantages drives perpetual cycles of adaptation and counter-adaptation, commonly referred to as Red Queen interactions. In this review, we explore insights gleaned from molecular and genetic studies of such genetic conflicts, both extrinsic (between genomes) and intrinsic (within genomes or cells). We argue that many different characteristics of selfish genetic elements can be distilled into two types of advantages: an over-replication advantage (e.g. mobile genetic elements in genomes) and a transmission distortion advantage (e.g. meiotic drivers in populations). These two general categories may help classify disparate types of selfish genetic elements. PMID:28057823

A genome-wide activity assessment of terminator regions in Saccharomyces cerevisiae provides a ″terminatome″ toolbox.

PubMed

Yamanishi, Mamoru; Ito, Yoichiro; Kintaka, Reiko; Imamura, Chie; Katahira, Satoshi; Ikeuchi, Akinori; Moriya, Hisao; Matsuyama, Takashi

2013-06-21

The terminator regions of eukaryotes encode functional elements in the 3' untranslated region (3'-UTR) that influence the 3'-end processing of mRNA, mRNA stability, and translational efficiency, which can modulate protein production. However, the contribution of these terminator regions to gene expression remains unclear, and therefore their utilization in metabolic engineering or synthetic genetic circuits has been limited. Here, we comprehensively evaluated the activity of 5302 terminator regions from a total of 5880 genes in the budding yeast Saccharomyces cerevisiae by inserting each terminator region downstream of the P TDH3 - green fluorescent protein (GFP) reporter gene and measuring the fluorescent intensity of GFP. Terminator region activities relative to that of the PGK1 standard terminator ranged from 0.036 to 2.52, with a mean of 0.87. We thus could isolate the most and least active terminator regions. The activities of the terminator regions showed a positive correlation with mRNA abundance, indicating that the terminator region is a determinant of mRNA abundance. The least active terminator regions tended to encode longer 3'-UTRs, suggesting the existence of active degradation mechanisms for those mRNAs. The terminator regions of ribosomal protein genes tended to be the most active, suggesting the existence of a common regulator of those genes. The ″terminatome″ (the genome-wide set of terminator regions) thus not only provides valuable information to understand the modulatory roles of terminator regions on gene expression but also serves as a useful toolbox for the development of metabolically and genetically engineered yeast.

Many Saccharomyces cerevisiae Cell Wall Protein Encoding Genes Are Coregulated by Mss11, but Cellular Adhesion Phenotypes Appear Only Flo Protein Dependent.

PubMed

Bester, Michael C; Jacobson, Dan; Bauer, Florian F

2012-01-01

The outer cell wall of the yeast Saccharomyces cerevisiae serves as the interface with the surrounding environment and directly affects cell-cell and cell-surface interactions. Many of these interactions are facilitated by specific adhesins that belong to the Flo protein family. Flo mannoproteins have been implicated in phenotypes such as flocculation, substrate adhesion, biofilm formation, and pseudohyphal growth. Genetic data strongly suggest that individual Flo proteins are responsible for many specific cellular adhesion phenotypes. However, it remains unclear whether such phenotypes are determined solely by the nature of the expressed FLO genes or rather as the result of a combination of FLO gene expression and other cell wall properties and cell wall proteins. Mss11 has been shown to be a central element of FLO1 and FLO11 gene regulation and acts together with the cAMP-PKA-dependent transcription factor Flo8. Here we use genome-wide transcription analysis to identify genes that are directly or indirectly regulated by Mss11. Interestingly, many of these genes encode cell wall mannoproteins, in particular, members of the TIR and DAN families. To examine whether these genes play a role in the adhesion properties associated with Mss11 expression, we assessed deletion mutants of these genes in wild-type and flo11Δ genetic backgrounds. This analysis shows that only FLO genes, in particular FLO1/10/11, appear to significantly impact on such phenotypes. Thus adhesion-related phenotypes are primarily dependent on the balance of FLO gene expression.

Regulation of gene expression in the mammalian eye and its relevance to eye disease.

PubMed

Scheetz, Todd E; Kim, Kwang-Youn A; Swiderski, Ruth E; Philp, Alisdair R; Braun, Terry A; Knudtson, Kevin L; Dorrance, Anne M; DiBona, Gerald F; Huang, Jian; Casavant, Thomas L; Sheffield, Val C; Stone, Edwin M

2006-09-26

We used expression quantitative trait locus mapping in the laboratory rat (Rattus norvegicus) to gain a broad perspective of gene regulation in the mammalian eye and to identify genetic variation relevant to human eye disease. Of >31,000 gene probes represented on an Affymetrix expression microarray, 18,976 exhibited sufficient signal for reliable analysis and at least 2-fold variation in expression among 120 F(2) rats generated from an SR/JrHsd x SHRSP intercross. Genome-wide linkage analysis with 399 genetic markers revealed significant linkage with at least one marker for 1,300 probes (alpha = 0.001; estimated empirical false discovery rate = 2%). Both contiguous and noncontiguous loci were found to be important in regulating mammalian eye gene expression. We investigated one locus of each type in greater detail and identified putative transcription-altering variations in both cases. We found an inserted cREL binding sequence in the 5' flanking sequence of the Abca4 gene associated with an increased expression level of that gene, and we found a mutation of the gene encoding thyroid hormone receptor beta2 associated with a decreased expression level of the gene encoding short-wavelength sensitive opsin (Opn1sw). In addition to these positional studies, we performed a pairwise analysis of gene expression to identify genes that are regulated in a coordinated manner and used this approach to validate two previously undescribed genes involved in the human disease Bardet-Biedl syndrome. These data and analytical approaches can be used to facilitate the discovery of additional genes and regulatory elements involved in human eye disease.

Diversity of the Tetracycline Mobilome within a Chinese Pig Manure Sample

PubMed Central

Leclercq, Sébastien Olivier; Wang, Chao; Zhu, Yaxin; Wu, Hai; Du, Xiaochen; Liu, Zhipei

2016-01-01

ABSTRACT Tetracycline antibiotics are widely used in livestock, and tetracycline resistance genes (TRG) are frequently reported in the manure of farmed animals. However, the diversity of TRG-carrying transposons in manure has still been rarely investigated. Using a culture-free functional metagenomic procedure, combined with large-insert library construction and sequencing, bioinformatic analyses, and functional experiments, we identified 17 distinct TRGs in a single pig manure sample, including two new tet genes: tet(59), encoding a tetracycline efflux pump, and tet(W/N/W), encoding mosaic ribosomal protection. Our study also revealed six new TRG-carrying putative nonconjugative transposons: Tn5706-like transposon Tn6298, IS200/605-related transposon Tn6303, Tn3 family transposon Tn6299, and three ISCR2-related transposons, Tn62300, Tn62301, and Tn62302. IMPORTANCE Fertilization of agricultural fields with animal manure is believed to play a major role in antibiotic resistance dissemination in the environment. There is growing concern for the possible spread of antibiotic resistance from the environment to humans since genetic resistance determinants may be located in transposons and other mobile genetic elements potentially transferable to pathogens. Among the various antibiotic resistance genes found in manure, tetracycline resistance genes (TRGs) are some of the most common. The present study provides a detailed snapshot of the tetracycline mobilome in a single pig manure sample, revealing an unappreciated diversity of TRGs and potential TRG mobility vectors. Our precise identification of the TRG-carrying units will enable us to investigate in more details their mobility effectiveness. PMID:27565618

Diversity of the Tetracycline Mobilome within a Chinese Pig Manure Sample.

PubMed

Leclercq, Sébastien Olivier; Wang, Chao; Zhu, Yaxin; Wu, Hai; Du, Xiaochen; Liu, Zhipei; Feng, Jie

2016-11-01

Tetracycline antibiotics are widely used in livestock, and tetracycline resistance genes (TRG) are frequently reported in the manure of farmed animals. However, the diversity of TRG-carrying transposons in manure has still been rarely investigated. Using a culture-free functional metagenomic procedure, combined with large-insert library construction and sequencing, bioinformatic analyses, and functional experiments, we identified 17 distinct TRGs in a single pig manure sample, including two new tet genes: tet(59), encoding a tetracycline efflux pump, and tet(W/N/W), encoding mosaic ribosomal protection. Our study also revealed six new TRG-carrying putative nonconjugative transposons: Tn5706-like transposon Tn6298, IS200/605-related transposon Tn6303, Tn3 family transposon Tn6299, and three ISCR2-related transposons, Tn62300, Tn62301, and Tn62302 IMPORTANCE: Fertilization of agricultural fields with animal manure is believed to play a major role in antibiotic resistance dissemination in the environment. There is growing concern for the possible spread of antibiotic resistance from the environment to humans since genetic resistance determinants may be located in transposons and other mobile genetic elements potentially transferable to pathogens. Among the various antibiotic resistance genes found in manure, tetracycline resistance genes (TRGs) are some of the most common. The present study provides a detailed snapshot of the tetracycline mobilome in a single pig manure sample, revealing an unappreciated diversity of TRGs and potential TRG mobility vectors. Our precise identification of the TRG-carrying units will enable us to investigate in more details their mobility effectiveness. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Interactive searching of facial image databases

NASA Astrophysics Data System (ADS)

Nicholls, Robert A.; Shepherd, John W.; Shepherd, Jean

1995-09-01

A set of psychological facial descriptors has been devised to enable computerized searching of criminal photograph albums. The descriptors have been used to encode image databased of up to twelve thousand images. Using a system called FACES, the databases are searched by translating a witness' verbal description into corresponding facial descriptors. Trials of FACES have shown that this coding scheme is more productive and efficient than searching traditional photograph albums. An alternative method of searching the encoded database using a genetic algorithm is currenly being tested. The genetic search method does not require the witness to verbalize a description of the target but merely to indicate a degree of similarity between the target and a limited selection of images from the database. The major drawback of FACES is that is requires a manual encoding of images. Research is being undertaken to automate the process, however, it will require an algorithm which can predict human descriptive values. Alternatives to human derived coding schemes exist using statistical classifications of images. Since databases encoded using statistical classifiers do not have an obvious direct mapping to human derived descriptors, a search method which does not require the entry of human descriptors is required. A genetic search algorithm is being tested for such a purpose.

Improved magnetic encoding device and method for making the same. [Patent application

DOEpatents

Fox, R.J.

A magnetic encoding device and method for making the same are provided for use as magnetic storage media in identification control applications that give output signals from a reader that are of shorter duration and substantially greater magnitude than those of the prior art. Magnetic encoding elements are produced by uniformly bending wire or strip stock of a magnetic material longitudinally about a common radius to exceed the elastic limit of the material and subsequently mounting the material so that it is restrained in an unbent position on a substrate of nonmagnetic material. The elements are spot weld attached to a substrate to form a binary coded array of elements according to a desired binary code. The coded substrate may be enclosed in a plastic laminate structure. Such devices may be used for security badges, key cards, and the like and may have many other applications. 7 figures.

Versatile protein recognition by the encoded display of multiple chemical elements on a constant macrocyclic scaffold

NASA Astrophysics Data System (ADS)

Li, Yizhou; De Luca, Roberto; Cazzamalli, Samuele; Pretto, Francesca; Bajic, Davor; Scheuermann, Jörg; Neri, Dario

2018-03-01

In nature, specific antibodies can be generated as a result of an adaptive selection and expansion of lymphocytes with suitable protein binding properties. We attempted to mimic antibody-antigen recognition by displaying multiple chemical diversity elements on a defined macrocyclic scaffold. Encoding of the displayed combinations was achieved using distinctive DNA tags, resulting in a library size of 35,393,112. Specific binders could be isolated against a variety of proteins, including carbonic anhydrase IX, horseradish peroxidase, tankyrase 1, human serum albumin, alpha-1 acid glycoprotein, calmodulin, prostate-specific antigen and tumour necrosis factor. Similar to antibodies, the encoded display of multiple chemical elements on a constant scaffold enabled practical applications, such as fluorescence microscopy procedures or the selective in vivo delivery of payloads to tumours. Furthermore, the versatile structure of the scaffold facilitated the generation of protein-specific chemical probes, as illustrated by photo-crosslinking.

Method for making an improved magnetic encoding device

DOEpatents

Fox, Richard J.

1981-01-01

A magnetic encoding device and method for making the same are provided for use as magnetic storage mediums in identification control applications which give output signals from a reader that are of shorter duration and substantially greater magnitude than those of the prior art. Magnetic encoding elements are produced by uniformly bending wire or strip stock of a magnetic material longitudinally about a common radius to exceed the elastic limit of the material and subsequently mounting the material so that it is restrained in an unbent position on a substrate of nonmagnetic material. The elements are spot weld attached to a substrate to form a binary coded array of elements according to a desired binary code. The coded substrate may be enclosed in a plastic laminate structure. Such devices may be used for security badges, key cards, and the like and may have many other applications.

MYB46 Modulates Disease Susceptibility to Botrytis cinerea in Arabidopsis12[W

PubMed Central

Ramírez, Vicente; Agorio, Astrid; Coego, Alberto; García-Andrade, Javier; Hernández, M. José; Balaguer, Begoña; Ouwerkerk, Pieter B.F.; Zarra, Ignacio; Vera, Pablo

2011-01-01

In this study, we show that the Arabidopsis (Arabidopsis thaliana) transcription factor MYB46, previously described to regulate secondary cell wall biosynthesis in the vascular tissue of the stem, is pivotal for mediating disease susceptibility to the fungal pathogen Botrytis cinerea. We identified MYB46 by its ability to bind to a new cis-element located in the 5′ promoter region of the pathogen-induced Ep5C gene, which encodes a type III cell wall-bound peroxidase. We present genetic and molecular evidence indicating that MYB46 modulates the magnitude of Ep5C gene induction following pathogenic insults. Moreover, we demonstrate that different myb46 knockdown mutant plants exhibit increased disease resistance to B. cinerea, a phenotype that is accompanied by selective transcriptional reprogramming of a set of genes encoding cell wall proteins and enzymes, of which extracellular type III peroxidases are conspicuous. In essence, our results substantiate that defense-related signaling pathways and cell wall integrity are interconnected and that MYB46 likely functions as a disease susceptibility modulator to B. cinerea through the integration of cell wall remodeling and downstream activation of secondary lines of defense. PMID:21282403

Identification of the sex genes in an early diverged fungus.

PubMed

Idnurm, Alexander; Walton, Felicia J; Floyd, Anna; Heitman, Joseph

2008-01-10

Sex determination in fungi is controlled by a small, specialized region of the genome in contrast to the large sex-specific chromosomes of animals and some plants. Different gene combinations reside at these mating-type (MAT) loci and confer sexual identity; invariably they encode homeodomain, alpha-box, or high mobility group (HMG)-domain transcription factors. So far, MAT loci have been characterized from a single monophyletic clade of fungi, the Dikarya (the ascomycetes and basidiomycetes), and the ancestral state and evolutionary history of these loci have remained a mystery. Mating in the basal members of the kingdom has been less well studied, and even their precise taxonomic inter-relationships are still obscure. Here we apply bioinformatic and genetic mapping to identify the sex-determining (sex) region in Phycomyces blakesleeanus (Zygomycota), which represents an early branch within the fungi. Each sex allele contains a single gene that encodes an HMG-domain protein, implicating the HMG-domain proteins as an earlier form of fungal MAT loci. Additionally, one allele also contains a copy of a unique, chromosome-specific repetitive element, suggesting a generalized mechanism for the earliest steps in the evolution of sex determination and sex chromosome structure in eukaryotes.

Erythro-megakaryocytic transcription factors associated with hereditary anemia

PubMed Central

Weiss, Mitchell J.

2014-01-01

Most heritable anemias are caused by mutations in genes encoding globins, red blood cell (RBC) membrane proteins, or enzymes in the glycolytic and hexose monophosphate shunt pathways. A less common class of genetic anemia is caused by mutations that alter the functions of erythroid transcription factors (TFs). Many TF mutations associated with heritable anemia cause truncations or amino acid substitutions, resulting in the production of functionally altered proteins. Characterization of these mutant proteins has provided insights into mechanisms of gene expression, hematopoietic development, and human disease. Mutations within promoter or enhancer regions that disrupt TF binding to essential erythroid genes also cause anemia and heritable variations in RBC traits, such as fetal hemoglobin content. Defining the latter may have important clinical implications for de-repressing fetal hemoglobin synthesis to treat sickle cell anemia and β thalassemia. Functionally important alterations in genes encoding TFs or their cognate cis elements are likely to occur more frequently than currently appreciated, a hypothesis that will soon be tested through ongoing genome-wide association studies and the rapidly expanding use of global genome sequencing for human diagnostics. Findings obtained through such studies of RBCs and associated diseases are likely generalizable to many human diseases and quantitative traits. PMID:24652993

Identification and characterization of tetracycline resistance in Lactococcus lactis isolated from Polish raw milk and fermented artisanal products.

PubMed

Zycka-Krzesinska, Joanna; Boguslawska, Joanna; Aleksandrzak-Piekarczyk, Tamara; Jopek, Jakub; Bardowski, Jacek K

2015-10-15

To assess the occurrence of antibiotic-resistant Lactic Acid Bacteria (LAB) in Polish raw milk and fermented artisanal products, a collection comprising 500 isolates from these products was screened. Among these isolates, six strains (IBB28, IBB160, IBB161, IBB224, IBB477 and IBB487) resistant to tetracycline were identified. The strains showing atypical tetracycline resistance were classified as Lactococcus lactis: three of them were identified as L. lactis subsp. cremoris (IBB224, IBB477 and IBB487) and the other three (IBB28, IBB160, IBB161) were identified as L. lactis subsp. lactis. The mechanism involving Ribosomal Protection Proteins (RPP) was identified as responsible for tetracycline resistance. Three of the tested strains (IBB28, IBB160 and IBB224) had genes encoding the TetS protein, whereas the remaining three (IBB161, IBB477 and IBB487) expressed TetM. The results also demonstrated that the genes encoding these proteins were located on genetic mobile elements. The tet(S) gene was found to be located on plasmids, whereas tet(M) was found within the Tn916 transposon. Copyright © 2015 Elsevier B.V. All rights reserved.

Genomics of high molecular weight plasmids isolated from an on-farm biopurification system.

PubMed

Martini, María C; Wibberg, Daniel; Lozano, Mauricio; Torres Tejerizo, Gonzalo; Albicoro, Francisco J; Jaenicke, Sebastian; van Elsas, Jan Dirk; Petroni, Alejandro; Garcillán-Barcia, M Pilar; de la Cruz, Fernando; Schlüter, Andreas; Pühler, Alfred; Pistorio, Mariano; Lagares, Antonio; Del Papa, María F

2016-06-20

The use of biopurification systems (BPS) constitutes an efficient strategy to eliminate pesticides from polluted wastewaters from farm activities. BPS environments contain a high microbial density and diversity facilitating the exchange of information among bacteria, mediated by mobile genetic elements (MGEs), which play a key role in bacterial adaptation and evolution in such environments. Here we sequenced and characterized high-molecular-weight plasmids from a bacterial collection of an on-farm BPS. The high-throughput-sequencing of the plasmid pool yielded a total of several Mb sequence information. Assembly of the sequence data resulted in six complete replicons. Using in silico analyses we identified plasmid replication genes whose encoding proteins represent 13 different Pfam families, as well as proteins involved in plasmid conjugation, indicating a large diversity of plasmid replicons and suggesting the occurrence of horizontal gene transfer (HGT) events within the habitat analyzed. In addition, genes conferring resistance to 10 classes of antimicrobial compounds and those encoding enzymes potentially involved in pesticide and aromatic hydrocarbon degradation were found. Global analysis of the plasmid pool suggest that the analyzed BPS represents a key environment for further studies addressing the dissemination of MGEs carrying catabolic genes and pathway assembly regarding degradation capabilities.

Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications

PubMed Central

Yeo, Chew Chieng; Abu Bakar, Fauziah; Chan, Wai Ting; Espinosa, Manuel; Harikrishna, Jennifer Ann

2016-01-01

Toxin-antitoxin (TA) systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies. PMID:26907343

Nature vs. nurture in human sociality: multi-level genomic analyses of social conformity.

PubMed

Chen, Biqing; Zhu, Zijian; Wang, Yingying; Ding, Xiaohu; Guo, Xiaobo; He, Mingguang; Fang, Wan; Zhou, Qin; Zhou, Shanbi; Lei, Han; Huang, Ailong; Chen, Tingmei; Ni, Dongsheng; Gu, Yuping; Liu, Jianing; Rao, Yi

2018-05-01

Social conformity is fundamental to human societies and has been studied for more than six decades, but our understanding of its mechanisms remains limited. Individual differences in conformity have been attributed to social and cultural environmental influences, but not to genes. Here we demonstrate a genetic contribution to conformity after analyzing 1,140 twins and single-nucleotide polymorphism (SNP)-based studies of 2,130 young adults. A two-step genome-wide association study (GWAS) revealed replicable associations in 9 genomic loci, and a meta-analysis of three GWAS with a sample size of ~2,600 further confirmed one locus, corresponding to the NAV3 (Neuron Navigator 3) gene which encodes a protein important for axon outgrowth and guidance. Further multi-level (haplotype, gene, pathway) GWAS strongly associated genes including NAV3, PTPRD (protein tyrosine phosphatase receptor type D), ARL10 (ADP ribosylation factor-like GTPase 10), and CTNND2 (catenin delta 2), with conformity. Magnetic resonance imaging of 64 subjects shows correlation of activation or structural features of brain regions with the SNPs of these genes, supporting their functional significance. Our results suggest potential moderate genetic influence on conformity, implicate several specific genetic elements in conformity and will facilitate further research on cellular and molecular mechanisms underlying human conformity.

Transposase-Mediated Excision, Conjugative Transfer, and Diversity of ICE6013 Elements in Staphylococcus aureus.

PubMed

Sansevere, Emily A; Luo, Xiao; Park, Joo Youn; Yoon, Sunghyun; Seo, Keun Seok; Robinson, D Ashley

2017-04-15

ICE 6013 represents one of two families of integrative conjugative elements (ICEs) identified in the pan-genome of the human and animal pathogen Staphylococcus aureus Here we investigated the excision and conjugation functions of ICE 6013 and further characterized the diversity of this element. ICE 6013 excision was not significantly affected by growth, temperature, pH, or UV exposure and did not depend on recA The IS 30 -like DDE transposase (Tpase; encoded by orf1 and orf2 ) of ICE 6013 must be uninterrupted for excision to occur, whereas disrupting three of the other open reading frames (ORFs) on the element significantly affects the level of excision. We demonstrate that ICE 6013 conjugatively transfers to different S. aureus backgrounds at frequencies approaching that of the conjugative plasmid pGO1. We found that excision is required for conjugation, that not all S. aureus backgrounds are successful recipients, and that transconjugants acquire the ability to transfer ICE 6013 Sequencing of chromosomal integration sites in serially passaged transconjugants revealed a significant integration site preference for a 15-bp AT-rich palindromic consensus sequence, which surrounds the 3-bp target site that is duplicated upon integration. A sequence analysis of ICE 6013 from different host strains of S. aureus and from eight other species of staphylococci identified seven divergent subfamilies of ICE 6013 that include sequences previously classified as a transposon, a plasmid, and various ICEs. In summary, these results indicate that the IS 30 -like Tpase functions as the ICE 6013 recombinase and that ICE 6013 represents a diverse family of mobile genetic elements that mediate conjugation in staphylococci. IMPORTANCE Integrative conjugative elements (ICEs) encode the abilities to integrate into and excise from bacterial chromosomes and plasmids and mediate conjugation between bacteria. As agents of horizontal gene transfer, ICEs may affect bacterial evolution. ICE 6013 represents one of two known families of ICEs in the pathogen Staphylococcus aureus , but its core functions of excision and conjugation are not well studied. Here, we show that ICE 6013 depends on its IS 30 -like DDE transposase for excision, which is unique among ICEs, and we demonstrate the conjugative transfer and integration site preference of ICE 6013 A sequence analysis revealed that ICE 6013 has diverged into seven subfamilies that are dispersed among staphylococci. Copyright © 2017 American Society for Microbiology.

PbrmiR397a regulates lignification during stone cell development in pear fruit.

PubMed

Xue, Cheng; Yao, Jia-Long; Qin, Meng-Fan; Zhang, Ming-Yue; Allan, Andrew C; Wang, De-Fu; Wu, Jun

2018-05-13

Lignified stone cells substantially reduce fruit quality. Therefore, it is desirable to inhibit stone cell development by using genetic technologies. However, the molecular mechanisms regulating lignification are poorly understood in fruit stone cells. In this study, we have shown that microRNA (miR) miR397a regulates fruit cell lignification by inhibiting laccase (LAC) genes that encode key lignin biosynthesis enzymes. Transient overexpression of PbrmiR397a, which is the miR397a of Chinese pear (Pyrus bretschneideri), and simultaneous silencing of three LAC genes reduced the lignin content and stone cell number in pear fruit. A single nucleotide polymorphism (SNP) identified in the promoter of the PbrmiR397a gene was found to associate with low levels of fruit lignin, after analysis of the genome sequences of sixty pear varieties. This SNP created a TCA-element that responded to salicylic acid (SA) to induce gene expression as confirmed using a cell-based assay system. Furthermore, stable overexpression of PbrmiR397a in transgenic tobacco plants reduced the expression of target LAC genes and decreased the content of lignin but did not change the ratio of syringyl and guaiacyl lignin monomers. Consistent with reduction of lignin content, the transgenic plants showed fewer numbers of vessel elements and thinner secondary walls in the remaining elements compared to wild-type control plants. This study has advanced our understanding of the regulation of lignin biosynthesis and provided useful molecular genetic information for improving pear fruit quality. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Genetics Home Reference: malonyl-CoA decarboxylase deficiency

MedlinePlus

... decarboxylase malonic aciduria malonyl-coenzyme A decarboxylase deficiency MCD deficiency Related Information How are genetic conditions and ... Morrell JC, Wanders RJ, Matalon R, Gould SJ. MCD encodes peroxisomal and cytoplasmic forms of malonyl-CoA ...

Cloud-based uniform ChIP-Seq processing tools for modENCODE and ENCODE.

PubMed

Trinh, Quang M; Jen, Fei-Yang Arthur; Zhou, Ziru; Chu, Kar Ming; Perry, Marc D; Kephart, Ellen T; Contrino, Sergio; Ruzanov, Peter; Stein, Lincoln D

2013-07-22

Funded by the National Institutes of Health (NIH), the aim of the Model Organism ENCyclopedia of DNA Elements (modENCODE) project is to provide the biological research community with a comprehensive encyclopedia of functional genomic elements for both model organisms C. elegans (worm) and D. melanogaster (fly). With a total size of just under 10 terabytes of data collected and released to the public, one of the challenges faced by researchers is to extract biologically meaningful knowledge from this large data set. While the basic quality control, pre-processing, and analysis of the data has already been performed by members of the modENCODE consortium, many researchers will wish to reinterpret the data set using modifications and enhancements of the original protocols, or combine modENCODE data with other data sets. Unfortunately this can be a time consuming and logistically challenging proposition. In recognition of this challenge, the modENCODE DCC has released uniform computing resources for analyzing modENCODE data on Galaxy (https://github.com/modENCODE-DCC/Galaxy), on the public Amazon Cloud (http://aws.amazon.com), and on the private Bionimbus Cloud for genomic research (http://www.bionimbus.org). In particular, we have released Galaxy workflows for interpreting ChIP-seq data which use the same quality control (QC) and peak calling standards adopted by the modENCODE and ENCODE communities. For convenience of use, we have created Amazon and Bionimbus Cloud machine images containing Galaxy along with all the modENCODE data, software and other dependencies. Using these resources provides a framework for running consistent and reproducible analyses on modENCODE data, ultimately allowing researchers to use more of their time using modENCODE data, and less time moving it around.

Cloud-based uniform ChIP-Seq processing tools for modENCODE and ENCODE

PubMed Central

2013-01-01

Background Funded by the National Institutes of Health (NIH), the aim of the Model Organism ENCyclopedia of DNA Elements (modENCODE) project is to provide the biological research community with a comprehensive encyclopedia of functional genomic elements for both model organisms C. elegans (worm) and D. melanogaster (fly). With a total size of just under 10 terabytes of data collected and released to the public, one of the challenges faced by researchers is to extract biologically meaningful knowledge from this large data set. While the basic quality control, pre-processing, and analysis of the data has already been performed by members of the modENCODE consortium, many researchers will wish to reinterpret the data set using modifications and enhancements of the original protocols, or combine modENCODE data with other data sets. Unfortunately this can be a time consuming and logistically challenging proposition. Results In recognition of this challenge, the modENCODE DCC has released uniform computing resources for analyzing modENCODE data on Galaxy (https://github.com/modENCODE-DCC/Galaxy), on the public Amazon Cloud (http://aws.amazon.com), and on the private Bionimbus Cloud for genomic research (http://www.bionimbus.org). In particular, we have released Galaxy workflows for interpreting ChIP-seq data which use the same quality control (QC) and peak calling standards adopted by the modENCODE and ENCODE communities. For convenience of use, we have created Amazon and Bionimbus Cloud machine images containing Galaxy along with all the modENCODE data, software and other dependencies. Conclusions Using these resources provides a framework for running consistent and reproducible analyses on modENCODE data, ultimately allowing researchers to use more of their time using modENCODE data, and less time moving it around. PMID:23875683

Selfish genetic elements and the gene’s-eye view of evolution

PubMed Central

2016-01-01

During the last few decades, we have seen an explosion in the influx of details about the biology of selfish genetic elements. Ever since the early days of the field, the gene’s-eye view of Richard Dawkins, George Williams, and others, has been instrumental to make sense of new empirical observations and to the generation of new hypotheses. However, the close association between selfish genetic elements and the gene’s-eye view has not been without critics and several other conceptual frameworks have been suggested. In particular, proponents of multilevel selection models have used selfish genetic elements to criticize the gene’s-eye view. In this paper, I first trace the intertwined histories of the study of selfish genetic elements and the gene’s-eye view and then discuss how their association holds up when compared with other proposed frameworks. Next, using examples from transposable elements and the major transitions, I argue that different models highlight separate aspects of the evolution of selfish genetic elements and that the productive way forward is to maintain a plurality of perspectives. Finally, I discuss how the empirical study of selfish genetic elements has implications for other conceptual issues associated with the gene’s-eye view, such as agential thinking, adaptationism, and the role of fitness maximizing models in evolution. PMID:29491953

Elemental concentrations in the seed of mutants and natural variants of Arabidopsis thaliana grown under varying soil conditions.

PubMed

McDowell, Stephen C; Akmakjian, Garo; Sladek, Chris; Mendoza-Cozatl, David; Morrissey, Joe B; Saini, Nick; Mittler, Ron; Baxter, Ivan; Salt, David E; Ward, John M; Schroeder, Julian I; Guerinot, Mary Lou; Harper, Jeffrey F

2013-01-01

The concentrations of mineral nutrients in seeds are critical to both the life cycle of plants as well as human nutrition. These concentrations are strongly influenced by soil conditions, as shown here by quantifying the concentration of 14 elements in seeds from Arabidopsis thaliana plants grown under four different soil conditions: standard, or modified with NaCl, heavy metals, or alkali. Each of the modified soils resulted in a unique change to the seed ionome (the mineral nutrient content of the seeds). To help identify the genetic networks regulating the seed ionome, changes in elemental concentrations were evaluated using mutants corresponding to 760 genes as well as 10 naturally occurring accessions. The frequency of ionomic phenotypes supports an estimate that as much as 11% of the A. thaliana genome encodes proteins of functional relevance to ion homeostasis in seeds. A subset of mutants were analyzed with two independent alleles, providing five examples of genes important for regulation of the seed ionome: SOS2, ABH1, CCC, At3g14280 and CNGC2. In a comparison of nine different accessions to a Col-0 reference, eight accessions were observed to have reproducible differences in elemental concentrations, seven of which were dependent on specific soil conditions. These results indicate that the A. thaliana seed ionome is distinct from the vegetative ionome, and that elemental analysis is a sensitive approach to identify genes controlling ion homeostasis, including those that regulate gene expression, phospho-regulation, and ion transport.

A linear-encoding model explains the variability of the target morphology in regeneration

PubMed Central

Lobo, Daniel; Solano, Mauricio; Bubenik, George A.; Levin, Michael

2014-01-01

A fundamental assumption of today's molecular genetics paradigm is that complex morphology emerges from the combined activity of low-level processes involving proteins and nucleic acids. An inherent characteristic of such nonlinear encodings is the difficulty of creating the genetic and epigenetic information that will produce a given self-assembling complex morphology. This ‘inverse problem’ is vital not only for understanding the evolution, development and regeneration of bodyplans, but also for synthetic biology efforts that seek to engineer biological shapes. Importantly, the regenerative mechanisms in deer antlers, planarian worms and fiddler crabs can solve an inverse problem: their target morphology can be altered specifically and stably by injuries in particular locations. Here, we discuss the class of models that use pre-specified morphological goal states and propose the existence of a linear encoding of the target morphology, making the inverse problem easy for these organisms to solve. Indeed, many model organisms such as Drosophila, hydra and Xenopus also develop according to nonlinear encodings producing linear encodings of their final morphologies. We propose the development of testable models of regeneration regulation that combine emergence with a top-down specification of shape by linear encodings of target morphology, driving transformative applications in biomedicine and synthetic bioengineering. PMID:24402915

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

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator.

PubMed

Rossano, Adam J; Romero, Michael F

2017-08-11

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH (pHi) is influenced by many ion transporters and thus monitoring pHi is a useful tool for assessing transporter activity. Modern Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of pHi in intact cells on a cellular and subcellular scale. This protocol describes real-time quantification of cellular pHi regulation in Malpighian Tubules (MTs) of Drosophila melanogaster through ex vivo live-imaging of pHerry, a pseudo-ratiometric GEpHI with a pKa well-suited to track pH changes in the cytosol. Extracted adult fly MTs are composed of morphologically and functionally distinct sections of single-cell layer epithelia, and can serve as an accessible and genetically tractable model for investigation of epithelial transport. GEpHIs offer several advantages over conventional pH-sensitive fluorescent dyes and ion-selective electrodes. GEpHIs can label distinct cell populations provided appropriate promoter elements are available. This labeling is particularly useful in ex vivo, in vivo, and in situ preparations, which are inherently heterogeneous. GEpHIs also permit quantification of pHi in intact tissues over time without need for repeated dye treatment or tissue externalization. The primary drawback of current GEpHIs is the tendency to aggregate in cytosolic inclusions in response to tissue damage and construct over-expression. These shortcomings, their solutions, and the inherent advantages of GEpHIs are demonstrated in this protocol through assessment of basolateral proton (H + ) transport in functionally distinct principal and stellate cells of extracted fly MTs. The techniques and analysis described are readily adaptable to a wide variety of vertebrate and invertebrate preparations, and the sophistication of the assay can be scaled from teaching labs to intricate determination of ion flux via specific transporters.

Genetic background of novel sequence types of CTX-M-8- and CTX-M-15-producing Escherichia coli and Klebsiella pneumoniae from public wastewater treatment plants in São Paulo, Brazil.

PubMed

Dropa, Milena; Lincopan, Nilton; Balsalobre, Livia C; Oliveira, Danielle E; Moura, Rodrigo A; Fernandes, Miriam Rodriguez; da Silva, Quézia Moura; Matté, Glavur R; Sato, Maria I Z; Matté, Maria H

2016-03-01

The release of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae to the environment is a public health issue worldwide. The aim of this study was to investigate the genetic background of genes encoding ESBLs in wastewater treatment plants (WWTPs) in São Paulo, southeastern Brazil. In 2009, during a local surveillance study, seven ESBL-producing Enterobacteriaceae strains were recovered from five WWTPs and screened for ESBL genes and mobile genetic elements. Multilocus sequence typing (MLST) was carried out, and wild plasmids were transformed into electrocompetent Escherichia coli. S1-PFGE technique was used to verify the presence of high molecular weight plasmids in wild-type strains and in bla ESBL-containing E. coli transformants. Strains harbored bla CTX-M-8, bla CTX-M-15, and/or bla SHV-28. Sequencing results showed that bla CTX-M-8 and bla CTX-M-15 genes were associated with IS26. MLST revealed new sequence types for E. coli (ST4401, ST4402, ST4403, and ST4445) and Klebsiella pneumoniae (ST1574), except for one K. pneumoniae from ST307 and Enterobacter cloacae from ST131. PCR and S1-PFGE results showed CTX-M-producing E. coli transformants carried heavy plasmids sizing 48.5-209 kb, which belonged to IncI1, IncF, and IncM1 incompatibility groups. This is the first report of CTX-M-8 and SHV-28 enzymes in environmental samples, and the present results demonstrate the plasmid-mediated spread of CTX-M-encoding genes through five WWTPs in São Paulo, Brazil, suggesting WWTPs are hotspots for the transfer of ESBL genes and confirming the urgent need to improve the management of sewage in order to minimize the dissemination of resistance genes to the environment.

Targeted next-generation sequencing helps to decipher the genetic and phenotypic heterogeneity of hypertrophic cardiomyopathy

PubMed Central

Cecconi, Massimiliano; Parodi, Maria I.; Formisano, Francesco; Spirito, Paolo; Autore, Camillo; Musumeci, Maria B.; Favale, Stefano; Forleo, Cinzia; Rapezzi, Claudio; Biagini, Elena; Davì, Sabrina; Canepa, Elisabetta; Pennese, Loredana; Castagnetta, Mauro; Degiorgio, Dario; Coviello, Domenico A.

2016-01-01

Hypertrophic cardiomyopathy (HCM) is mainly associated with myosin, heavy chain 7 (MYH7) and myosin binding protein C, cardiac (MYBPC3) mutations. In order to better explain the clinical and genetic heterogeneity in HCM patients, in this study, we implemented a target-next generation sequencing (NGS) assay. An Ion AmpliSeq™ Custom Panel for the enrichment of 19 genes, of which 9 of these did not encode thick/intermediate and thin myofilament (TTm) proteins and, among them, 3 responsible of HCM phenocopy, was created. Ninety-two DNA samples were analyzed by the Ion Personal Genome Machine: 73 DNA samples (training set), previously genotyped in some of the genes by Sanger sequencing, were used to optimize the NGS strategy, whereas 19 DNA samples (discovery set) allowed the evaluation of NGS performance. In the training set, we identified 72 out of 73 expected mutations and 15 additional mutations: the molecular diagnosis was achieved in one patient with a previously wild-type status and the pre-excitation syndrome was explained in another. In the discovery set, we identified 20 mutations, 5 of which were in genes encoding non-TTm proteins, increasing the diagnostic yield by approximately 20%: a single mutation in genes encoding non-TTm proteins was identified in 2 out of 3 borderline HCM patients, whereas co-occuring mutations in genes encoding TTm and galactosidase alpha (GLA) altered proteins were characterized in a male with HCM and multiorgan dysfunction. Our combined targeted NGS-Sanger sequencing-based strategy allowed the molecular diagnosis of HCM with greater efficiency than using the conventional (Sanger) sequencing alone. Mutant alleles encoding non-TTm proteins may aid in the complete understanding of the genetic and phenotypic heterogeneity of HCM: co-occuring mutations of genes encoding TTm and non-TTm proteins could explain the wide variability of the HCM phenotype, whereas mutations in genes encoding only the non-TTm proteins are identifiable in patients with a milder HCM status. PMID:27600940

Genetic conflicts: the usual suspects and beyond.

PubMed

McLaughlin, Richard N; Malik, Harmit S

2017-01-01

Selfishness is pervasive and manifests at all scales of biology, from societies, to individuals, to genetic elements within a genome. The relentless struggle to seek evolutionary advantages drives perpetual cycles of adaptation and counter-adaptation, commonly referred to as Red Queen interactions. In this review, we explore insights gleaned from molecular and genetic studies of such genetic conflicts, both extrinsic (between genomes) and intrinsic (within genomes or cells). We argue that many different characteristics of selfish genetic elements can be distilled into two types of advantages: an over-replication advantage (e.g. mobile genetic elements in genomes) and a transmission distortion advantage (e.g. meiotic drivers in populations). These two general categories may help classify disparate types of selfish genetic elements. © 2017. Published by The Company of Biologists Ltd.

Creutzfeldt-Jakob disease and mad cows: lessons learnt from yeast cells.

PubMed

Hofmann, J; Wolf, H; Grassmann, A; Arndt, V; Graham, J; Vorberg, I

2012-01-24

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases that affect mammals including humans. The proteinaceous nature of the infectious agent, the prion, and its propagation, challenge established dogmas in biology. It is now widely accepted that prion diseases are caused by unconventional agents principally composed of a misfolded host-encoded protein, PrP. Surprisingly, major break-throughs in prion research came from studies on functionally unrelated proteins in yeast and filamentous fungi. Aggregates composed of these proteins act as epigenetic elements of inheritance that can propagate their alternative states by a conformational switch into an ordered ß-sheet rich polymer just like mammalian prions. Since their discovery prions of lower eukaryotes have provided invaluable insights into all aspects of prion biogenesis. Importantly, yeast prions provide proof-of-principle that distinct protein conformers can be infectious and can serve as genetic elements that have the capacity to encipher strain specific information. As a powerful and tractable model system, yeast prions will continue to increase our understanding of prion-host cell interaction and potential mechanisms of protein-based epigenetic inheritance.

Unusual Structure of the attB Site of the Site-Specific Recombination System of Lactobacillus delbrueckii Bacteriophage mv4

PubMed Central

Auvray, Frédéric; Coddeville, Michèle; Ordonez, Romy Catoira; Ritzenthaler, Paul

1999-01-01

The temperate phage mv4 integrates its genome into the chromosome of Lactobacillus delbrueckii subsp. bulgaricus by site-specific recombination within the 3′ end of a tRNASer gene. Recombination is catalyzed by the phage-encoded integrase and occurs between the phage attP site and the bacterial attB site. In this study, we show that the mv4 integrase functions in vivo in Escherichia coli and we characterize the bacterial attB site with a site-specific recombination test involving compatible plasmids carrying the recombination sites. The importance of particular nucleotides within the attB sequence was determined by site-directed mutagenesis. The structure of the attB site was found to be simple but rather unusual. A 16-bp DNA fragment was sufficient for function. Unlike most genetic elements that integrate their DNA into tRNA genes, none of the dyad symmetry elements of the tRNASer gene were present within the minimal attB site. No inverted repeats were detected within this site either, in contrast to the lambda site-specific recombination model. PMID:10572145

Cryo-EM Structures Reveal Mechanism and Inhibition of DNA Targeting by a CRISPR-Cas Surveillance Complex.

PubMed

Guo, Tai Wei; Bartesaghi, Alberto; Yang, Hui; Falconieri, Veronica; Rao, Prashant; Merk, Alan; Eng, Edward T; Raczkowski, Ashleigh M; Fox, Tara; Earl, Lesley A; Patel, Dinshaw J; Subramaniam, Sriram

2017-10-05

Prokaryotic cells possess CRISPR-mediated adaptive immune systems that protect them from foreign genetic elements, such as invading viruses. A central element of this immune system is an RNA-guided surveillance complex capable of targeting non-self DNA or RNA for degradation in a sequence- and site-specific manner analogous to RNA interference. Although the complexes display considerable diversity in their composition and architecture, many basic mechanisms underlying target recognition and cleavage are highly conserved. Using cryoelectron microscopy (cryo-EM), we show that the binding of target double-stranded DNA (dsDNA) to a type I-F CRISPR system yersinia (Csy) surveillance complex leads to large quaternary and tertiary structural changes in the complex that are likely necessary in the pathway leading to target dsDNA degradation by a trans-acting helicase-nuclease. Comparison of the structure of the surveillance complex before and after dsDNA binding, or in complex with three virally encoded anti-CRISPR suppressors that inhibit dsDNA binding, reveals mechanistic details underlying target recognition and inhibition. Published by Elsevier Inc.

Inhibition Mechanism of an Anti-CRISPR Suppressor AcrIIA4 Targeting SpyCas9.

PubMed

Yang, Hui; Patel, Dinshaw J

2017-07-06

Prokaryotic CRISPR-Cas adaptive immune systems utilize sequence-specific RNA-guided endonucleases to defend against infection by viruses, bacteriophages, and mobile elements, while these foreign genetic elements evolve diverse anti-CRISPR proteins to overcome the CRISPR-Cas-mediated defense of the host. Recently, AcrIIA2 and AcrIIA4, encoded by Listeria monocytogene prophages, were shown to block the endonuclease activity of type II-A Streptococcus pyogene Cas9 (SpyCas9). We now report the crystal structure of AcrIIA4 in complex with single-guide RNA-bound SpyCas9, thereby establishing that AcrIIA4 preferentially targets critical residues essential for PAM duplex recognition, as well as blocks target DNA access to key catalytic residues lining the RuvC pocket. These structural insights, validated by biochemical assays on key mutants, demonstrate that AcrIIA4 competitively occupies both PAM-interacting and non-target DNA strand cleavage catalytic pockets. Our studies provide insights into anti-CRISPR-mediated suppression mechanisms for inactivating SpyCas9, thereby broadening the applicability of CRISPR-Cas regulatory tools for genome editing. Published by Elsevier Inc.

Minos as a novel Tc1/mariner-type transposable element for functional genomic analysis in Aspergillus nidulans.

PubMed

Evangelinos, Minoas; Anagnostopoulos, Gerasimos; Karvela-Kalogeraki, Iliana; Stathopoulou, Panagiota M; Scazzocchio, Claudio; Diallinas, George

2015-08-01

Transposons constitute powerful genetic tools for gene inactivation, exon or promoter trapping and genome analyses. The Minos element from Drosophila hydei, a Tc1/mariner-like transposon, has proved as a very efficient tool for heterologous transposition in several metazoa. In filamentous fungi, only a handful of fungal-specific transposable elements have been exploited as genetic tools, with the impala Tc1/mariner element from Fusarium oxysporum being the most successful. Here, we developed a two-component transposition system to manipulate Minos transposition in Aspergillus nidulans (AnMinos). Our system allows direct selection of transposition events based on re-activation of niaD, a gene necessary for growth on nitrate as a nitrogen source. On average, among 10(8) conidiospores, we obtain up to ∼0.8×10(2) transposition events leading to the expected revertant phenotype (niaD(+)), while ∼16% of excision events lead to AnMinos loss. Characterized excision footprints consisted of the four terminal bases of the transposon flanked by the TA target duplication and led to no major DNA rearrangements. AnMinos transposition depends on the presence of its homologous transposase. Its frequency was not significantly affected by temperature, UV irradiation or the transcription status of the original integration locus (niaD). Importantly, transposition is dependent on nkuA, encoding an enzyme essential for non-homologous end joining of DNA in double-strand break repair. AnMinos proved to be an efficient tool for functional analysis as it seems to transpose in different genomic loci positions in all chromosomes, including a high proportion of integration events within or close to genes. We have used Minos to obtain morphological and toxic analogue resistant mutants. Interestingly, among morphological mutants some seem to be due to Minos-elicited over-expression of specific genes, rather than gene inactivation. Copyright © 2015 Elsevier Inc. All rights reserved.

Identification and characterization of mobile genetic elements LINEs from Brassica genome.

PubMed

Nouroz, Faisal; Noreen, Shumaila; Khan, Muhammad Fiaz; Ahmed, Shehzad; Heslop-Harrison, J S Pat

2017-09-05

Among transposable elements (TEs), the LTR retrotransposons are abundant followed by non-LTR retrotransposons in plant genomes, the lateral being represented by LINEs and SINEs. Computational and molecular approaches were used for the characterization of Brassica LINEs, their diversity and phylogenetic relationships. Four autonomous and four non-autonomous LINE families were identified and characterized from Brassica. Most of the autonomous LINEs displayed two open reading frames, ORF1 and ORF2, where ORF1 is a gag protein domain, while ORF2 encodes endonuclease (EN) and a reverse transcriptase (RT). Three of four families encoded an additional RNase H (RH) domain in pol gene common to 'R' and 'I' type of LINEs. The PCR analyses based on LINEs RT fragments indicate their high diversity and widespread occurrence in tested 40 Brassica cultivars. Database searches revealed the homology in LINE sequences in closely related genera Arabidopsis indicating their origin from common ancestors predating their separation. The alignment of 58 LINEs RT sequences from Brassica, Arabidopsis and other plants depicted 4 conserved domains (domain II-V) showing similarity to previously detected domains. Based on RT alignment of Brassica and 3 known LINEs from monocots, Brassicaceae LINEs clustered in separate clade, further resolving 4 Brassica-Arabidopsis specific families in 2 sub-clades. High similarities were observed in RT sequences in the members of same family, while low homology was detected in members across the families. The investigation led to the characterization of Brassica specific LINE families and their diversity across Brassica species and their cultivars. Copyright © 2017 Elsevier B.V. All rights reserved.

An Enterotoxin-Bearing Pathogenicity Island in Staphylococcus epidermidis▿†

PubMed Central

Madhusoodanan, Jyoti; Seo, Keun Seok; Remortel, Brian; Park, Joo Youn; Hwang, Sun Young; Fox, Lawrence K.; Park, Yong Ho; Deobald, Claudia F.; Wang, Dan; Liu, Song; Daugherty, Sean C.; Gill, Ann Lindley; Bohach, Gregory A.; Gill, Steven R.

2011-01-01

Cocolonization of human mucosal surfaces causes frequent encounters between various staphylococcal species, creating opportunities for the horizontal acquisition of mobile genetic elements. The majority of Staphylococcus aureus toxins and virulence factors are encoded on S. aureus pathogenicity islands (SaPIs). Horizontal movement of SaPIs between S. aureus strains plays a role in the evolution of virulent clinical isolates. Although there have been reports of the production of toxic shock syndrome toxin 1 (TSST-1), enterotoxin, and other superantigens by coagulase-negative staphylococci, no associated pathogenicity islands have been found in the genome of Staphylococcus epidermidis, a generally less virulent relative of S. aureus. We show here the first evidence of a composite S. epidermidis pathogenicity island (SePI), the product of multiple insertions in the genome of a clinical isolate. The taxonomic placement of S. epidermidis strain FRI909 was confirmed by a number of biochemical tests and multilocus sequence typing. The genome sequence of this strain was analyzed for other unique gene clusters and their locations. This pathogenicity island encodes and expresses staphylococcal enterotoxin C3 (SEC3) and staphylococcal enterotoxin-like toxin L (SElL), as confirmed by quantitative reverse transcription-PCR (qRT-PCR) and immunoblotting. We present here an initial characterization of this novel pathogenicity island, and we establish that it is stable, expresses enterotoxins, and is not obviously transmissible by phage transduction. We also describe the genome sequence, excision, replication, and packaging of a novel bacteriophage in S. epidermidis FRI909, as well as attempts to mobilize the SePI element by this phage. PMID:21317317

Structural Insight into the Clostridium difficile Ethanolamine Utilisation Microcompartment

PubMed Central

Faulds-Pain, Alexandra; Lewis, Richard J.; Marles-Wright, Jon

2012-01-01

Bacterial microcompartments form a protective proteinaceous barrier around metabolic enzymes that process unstable or toxic chemical intermediates. The genome of the virulent, multidrug-resistant Clostridium difficile 630 strain contains an operon, eut, encoding a bacterial microcompartment with genes for the breakdown of ethanolamine and its utilisation as a source of reduced nitrogen and carbon. The C. difficile eut operon displays regulatory genetic elements and protein encoding regions in common with homologous loci found in the genomes of other bacteria, including the enteric pathogens Salmonella enterica and Enterococcus faecalis. The crystal structures of two microcompartment shell proteins, CD1908 and CD1918, and an uncharacterised protein with potential enzymatic activity, CD1925, were determined by X-ray crystallography. CD1908 and CD1918 display the same protein fold, though the order of secondary structure elements is permuted in CD1908 and this protein displays an N-terminal β-strand extension. These proteins form hexamers with molecules related by crystallographic and non-crystallographic symmetry. The structure of CD1925 has a cupin β-barrel fold and a putative active site that is distinct from the metal-ion dependent catalytic cupins. Thin-section transmission electron microscopy of Escherichia coli over-expressing eut proteins indicates that CD1918 is capable of self-association into arrays, suggesting an organisational role for CD1918 in the formation of this microcompartment. The work presented provides the basis for further study of the architecture and function of the C. difficile eut microcompartment, its role in metabolism and the wider consequences of intestinal colonisation and virulence in this pathogen. PMID:23144756

Designed Proteins Induce the Formation of Nanocage-containing Extracellular Vesicles

PubMed Central

Votteler, Jörg; Ogohara, Cassandra; Yi, Sue; Hsia, Yang; Nattermann, Una; Belnap, David M.; King, Neil P.; Sundquist, Wesley I.

2017-01-01

Complex biological processes are often performed by self-organizing nanostructures comprising multiple classes of macromolecules, such as ribosomes (proteins and RNA) or enveloped viruses (proteins, nucleic acids, and lipids). Approaches have been developed for designing self-assembling structures consisting of either nucleic acids1,2 or proteins3–5, but strategies for engineering hybrid biological materials are only beginning to emerge6,7. Here, we describe the design of self-assembling protein nanocages that direct their own release from human cells inside small vesicles in a manner that resembles some viruses. We refer to these hybrid biomaterials as Enveloped Protein Nanocages (EPNs). Robust EPN biogenesis required protein sequence elements that encode three distinct functions: membrane binding, self-assembly, and recruitment of the Endosomal Sorting Complexes Required for Transport (ESCRT) machinery8. A variety of synthetic proteins with these functional elements induced EPN biogenesis, highlighting the modularity and generality of the design strategy. Biochemical and electron cryomicroscopic (cryo-EM) analyses revealed that one design, EPN-01, comprised small (~100 nm) vesicles containing multiple protein nanocages that closely matched the structure of the designed 60-subunit self-assembling scaffold9. EPNs that incorporated the vesicular stomatitis viral glycoprotein (VSV-G) could fuse with target cells and deliver their contents, thereby transferring cargoes from one cell to another. These studies show how proteins can be programmed to direct the formation of hybrid biological materials that perform complex tasks, and establish EPNs as a novel class of designed, modular, genetically-encoded nanomaterials that can transfer molecules between cells. PMID:27919066

Novel Applications of Multi-task Learning and Multiple Output Regression to Multiple Genetic Trait Prediction

USDA-ARS?s Scientific Manuscript database

Given a set of biallelic molecular markers, such as SNPs, with genotype values encoded numerically on a collection of plant, animal or human samples, the goal of genetic trait prediction is to predict the quantitative trait values by simultaneously modeling all marker effects. Genetic trait predicti...

The genome analysis of Oleiphilus messinensis ME102 (DSM 13489T) reveals backgrounds of its obligate alkane-devouring marine lifestyle.

PubMed

Toshchakov, Stepan V; Korzhenkov, Alexei A; Chernikova, Tatyana N; Ferrer, Manuel; Golyshina, Olga V; Yakimov, Michail M; Golyshin, Peter N

2017-12-01

Marine bacterium Oleiphilus messinensis ME102 (DSM 13489 T ) isolated from the sediments of the harbor of Messina (Italy) is a member of the order Oceanospirillales, class Gammaproteobacteria, representing the physiological group of marine obligate hydrocarbonoclastic bacteria (OHCB) alongside the members of the genera Alcanivorax, Oleispira, Thalassolituus, Cycloclasticus and Neptunomonas. These organisms play a crucial role in the natural environmental cleanup in marine systems. Despite having the largest genome (6.379.281bp) among OHCB, O. messinensis exhibits a very narrow substrate profile. The alkane metabolism is pre-determined by three loci encoding for two P450 family monooxygenases, one of which formed a cassette with ferredoxin and alcohol dehydrogenase encoding genes and alkane monoxygenase (AlkB) gene clustered with two genes for rubredoxins and NAD + -dependent rubredoxin reductase. Its genome contains the largest numbers of genomic islands (15) and mobile genetic elements (140), as compared with more streamlined genomes of its OHCB counterparts. Among hydrocarbon-degrading Oceanospirillales, O. messinensis encodes the largest array of proteins involved in the signal transduction for sensing and responding to the environmental stimuli (345 vs 170 in Oleispira antarctica, the bacterium with the second highest number). This must be an important trait to adapt to the conditions in marine sediments with a high physico-chemical patchiness and heterogeneity as compared to those in the water column. Copyright © 2017. Published by Elsevier B.V.

Regression Analysis of Combined Gene Expression Regulation in Acute Myeloid Leukemia

PubMed Central

Li, Yue; Liang, Minggao; Zhang, Zhaolei

2014-01-01

Gene expression is a combinatorial function of genetic/epigenetic factors such as copy number variation (CNV), DNA methylation (DM), transcription factors (TF) occupancy, and microRNA (miRNA) post-transcriptional regulation. At the maturity of microarray/sequencing technologies, large amounts of data measuring the genome-wide signals of those factors became available from Encyclopedia of DNA Elements (ENCODE) and The Cancer Genome Atlas (TCGA). However, there is a lack of an integrative model to take full advantage of these rich yet heterogeneous data. To this end, we developed RACER (Regression Analysis of Combined Expression Regulation), which fits the mRNA expression as response using as explanatory variables, the TF data from ENCODE, and CNV, DM, miRNA expression signals from TCGA. Briefly, RACER first infers the sample-specific regulatory activities by TFs and miRNAs, which are then used as inputs to infer specific TF/miRNA-gene interactions. Such a two-stage regression framework circumvents a common difficulty in integrating ENCODE data measured in generic cell-line with the sample-specific TCGA measurements. As a case study, we integrated Acute Myeloid Leukemia (AML) data from TCGA and the related TF binding data measured in K562 from ENCODE. As a proof-of-concept, we first verified our model formalism by 10-fold cross-validation on predicting gene expression. We next evaluated RACER on recovering known regulatory interactions, and demonstrated its superior statistical power over existing methods in detecting known miRNA/TF targets. Additionally, we developed a feature selection procedure, which identified 18 regulators, whose activities clustered consistently with cytogenetic risk groups. One of the selected regulators is miR-548p, whose inferred targets were significantly enriched for leukemia-related pathway, implicating its novel role in AML pathogenesis. Moreover, survival analysis using the inferred activities identified C-Fos as a potential AML prognostic marker. Together, we provided a novel framework that successfully integrated the TCGA and ENCODE data in revealing AML-specific regulatory program at global level. PMID:25340776

Selfish genetic elements, genetic conflict, and evolutionary innovation.

PubMed

Werren, John H

2011-06-28

Genomes are vulnerable to selfish genetic elements (SGEs), which enhance their own transmission relative to the rest of an individual's genome but are neutral or harmful to the individual as a whole. As a result, genetic conflict occurs between SGEs and other genetic elements in the genome. There is growing evidence that SGEs, and the resulting genetic conflict, are an important motor for evolutionary change and innovation. In this review, the kinds of SGEs and their evolutionary consequences are described, including how these elements shape basic biological features, such as genome structure and gene regulation, evolution of new genes, origin of new species, and mechanisms of sex determination and development. The dynamics of SGEs are also considered, including possible "evolutionary functions" of SGEs.

Selfish genetic elements, genetic conflict, and evolutionary innovation

PubMed Central

Werren, John H.

2011-01-01

Genomes are vulnerable to selfish genetic elements (SGEs), which enhance their own transmission relative to the rest of an individual's genome but are neutral or harmful to the individual as a whole. As a result, genetic conflict occurs between SGEs and other genetic elements in the genome. There is growing evidence that SGEs, and the resulting genetic conflict, are an important motor for evolutionary change and innovation. In this review, the kinds of SGEs and their evolutionary consequences are described, including how these elements shape basic biological features, such as genome structure and gene regulation, evolution of new genes, origin of new species, and mechanisms of sex determination and development. The dynamics of SGEs are also considered, including possible “evolutionary functions” of SGEs. PMID:21690392

The molecular genetics of Usher syndrome.

PubMed

Ahmed, Z M; Riazuddin, S; Riazuddin, S; Wilcox, E R

2003-06-01

Association of sensorineural deafness and progressive retinitis pigmentosa with and without a vestibular abnormality is the hallmark of Usher syndrome and involves at least 12 loci among three different clinical subtypes. Genes identified for the more commonly inherited loci are USH2A (encoding usherin), MYO7A (encoding myosin VIIa), CDH23 (encoding cadherin 23), PCDH15 (encoding protocadherin 15), USH1C (encoding harmonin), USH3A (encoding clarin 1), and USH1G (encoding SANS). Transcripts from all these genes are found in many tissues/cell types other than the inner ear and retina, but all are uniquely critical for retinal and cochlear cell function. Many of these protein products have been demonstrated to have direct interactions with each other and perform an essential role in stereocilia homeostasis.

Genetically Encoded Photoactuators and Photosensors for Characterization and Manipulation of Pluripotent Stem Cells

PubMed Central

Pomeroy, Jordan E.; Nguyen, Hung X.; Hoffman, Brenton D.; Bursac, Nenad

2017-01-01

Our knowledge of pluripotent stem cell biology has advanced considerably in the past four decades, but it has yet to deliver on the great promise of regenerative medicine. The slow progress can be mainly attributed to our incomplete understanding of the complex biologic processes regulating the dynamic developmental pathways from pluripotency to fully-differentiated states of functional somatic cells. Much of the difficulty arises from our lack of specific tools to query, or manipulate, the molecular scale circuitry on both single-cell and organismal levels. Fortunately, the last two decades of progress in the field of optogenetics have produced a variety of genetically encoded, light-mediated tools that enable visualization and control of the spatiotemporal regulation of cellular function. The merging of optogenetics and pluripotent stem cell biology could thus be an important step toward realization of the clinical potential of pluripotent stem cells. In this review, we have surveyed available genetically encoded photoactuators and photosensors, a rapidly expanding toolbox, with particular attention to those with utility for studying pluripotent stem cells. PMID:28912894

Genetically encoded proton sensors reveal activity-dependent pH changes in neurons.

PubMed

Raimondo, Joseph V; Irkle, Agnese; Wefelmeyer, Winnie; Newey, Sarah E; Akerman, Colin J

2012-01-01

The regulation of hydrogen ion concentration (pH) is fundamental to cell viability, metabolism, and enzymatic function. Within the nervous system, the control of pH is also involved in diverse and dynamic processes including development, synaptic transmission, and the control of network excitability. As pH affects neuronal activity, and can also itself be altered by neuronal activity, the existence of tools to accurately measure hydrogen ion fluctuations is important for understanding the role pH plays under physiological and pathological conditions. Outside of their use as a marker of synaptic release, genetically encoded pH sensors have not been utilized to study hydrogen ion fluxes associated with network activity. By combining whole-cell patch clamp with simultaneous two-photon or confocal imaging, we quantified the amplitude and time course of neuronal, intracellular, acidic transients evoked by epileptiform activity in two separate in vitro models of temporal lobe epilepsy. In doing so, we demonstrate the suitability of three genetically encoded pH sensors: deGFP4, E(2)GFP, and Cl-sensor for investigating activity-dependent pH changes at the level of single neurons.

A new optimized GA-RBF neural network algorithm.

PubMed

Jia, Weikuan; Zhao, Dean; Shen, Tian; Su, Chunyang; Hu, Chanli; Zhao, Yuyan

2014-01-01

When confronting the complex problems, radial basis function (RBF) neural network has the advantages of adaptive and self-learning ability, but it is difficult to determine the number of hidden layer neurons, and the weights learning ability from hidden layer to the output layer is low; these deficiencies easily lead to decreasing learning ability and recognition precision. Aiming at this problem, we propose a new optimized RBF neural network algorithm based on genetic algorithm (GA-RBF algorithm), which uses genetic algorithm to optimize the weights and structure of RBF neural network; it chooses new ways of hybrid encoding and optimizing simultaneously. Using the binary encoding encodes the number of the hidden layer's neurons and using real encoding encodes the connection weights. Hidden layer neurons number and connection weights are optimized simultaneously in the new algorithm. However, the connection weights optimization is not complete; we need to use least mean square (LMS) algorithm for further leaning, and finally get a new algorithm model. Using two UCI standard data sets to test the new algorithm, the results show that the new algorithm improves the operating efficiency in dealing with complex problems and also improves the recognition precision, which proves that the new algorithm is valid.

Sieve element occlusion (SEO) genes encode structural phloem proteins involved in wound sealing of the phloem.

PubMed

Ernst, Antonia M; Jekat, Stephan B; Zielonka, Sascia; Müller, Boje; Neumann, Ulla; Rüping, Boris; Twyman, Richard M; Krzyzanek, Vladislav; Prüfer, Dirk; Noll, Gundula A

2012-07-10

The sieve element occlusion (SEO) gene family originally was delimited to genes encoding structural components of forisomes, which are specialized crystalloid phloem proteins found solely in the Fabaceae. More recently, SEO genes discovered in various non-Fabaceae plants were proposed to encode the common phloem proteins (P-proteins) that plug sieve plates after wounding. We carried out a comprehensive characterization of two tobacco (Nicotiana tabacum) SEO genes (NtSEO). Reporter genes controlled by the NtSEO promoters were expressed specifically in immature sieve elements, and GFP-SEO fusion proteins formed parietal agglomerates in intact sieve elements as well as sieve plate plugs after wounding. NtSEO proteins with and without fluorescent protein tags formed agglomerates similar in structure to native P-protein bodies when transiently coexpressed in Nicotiana benthamiana, and the analysis of these protein complexes by electron microscopy revealed ultrastructural features resembling those of native P-proteins. NtSEO-RNA interference lines were essentially devoid of P-protein structures and lost photoassimilates more rapidly after injury than control plants, thus confirming the role of P-proteins in sieve tube sealing. We therefore provide direct evidence that SEO genes in tobacco encode P-protein subunits that affect translocation. We also found that peptides recently identified in fascicular phloem P-protein plugs from squash (Cucurbita maxima) represent cucurbit members of the SEO family. Our results therefore suggest a common evolutionary origin for P-proteins found in the sieve elements of all dicotyledonous plants and demonstrate the exceptional status of extrafascicular P-proteins in cucurbits.

Co-occurrence of mobile genetic elements and antibiotic resistance genes in municipal solid waste landfill leachates: A preliminary insight into the role of landfill age.

PubMed

Yu, Zhuofeng; He, Pinjing; Shao, Liming; Zhang, Hua; Lü, Fan

2016-12-01

Since municipal solid waste (MSW) landfill harbours miscellaneous wastes, pollutants and microorganisms, it gradually becomes a huge potential reservoir for breeding antibiotic resistance genes (ARGs). The objective of this study was to determine the prevalence and diversity of ARGs associated with various mobile genetic elements (MGEs) in MSW landfill leachates. The relationship of ARGs with leachate characteristics was also studied to explore the influence of landfill age. Seven sulfonamides (sulfapyridine, sulfadiazine, sulfathiazole, sulfamethoxazole, sulfamerazine, sulfamethazine and sulfaquinoxaline), three encoded ARGs (sul-I, sul-II and sul-III) and four types of MGEs (plasmids, transposons, integrons and insertion sequences) were quantified in leachates with landfill ages ranging from 3 months-6 years. ARGs increased to an absolute concentration of 10 6 copies/μL and were positively correlated (p < 0.05) to MGEs. Significant correlations (p < 0.05) were also discovered among ARGs and the increasing humic acids, heavy metals (Zn, Cu and Co) and antibiotics (except for sulfathiazole and sulfaquinoxaline), implying landfilling might contribute to the enrichment of ARGs in the long-term. Non-target full scans revealed the role of persistent unknown compounds in stimulating the ARGs dissemination. Overall, this study demonstrates the exacerbation of ARGs pollution in landfill environment and a detailed delineation of the complex inter-relationships between ARGs and the substances harbouring in landfills is badly required. Copyright © 2016 Elsevier Ltd. All rights reserved.

Endogenous Retroviruses: With Us and Against Us

NASA Astrophysics Data System (ADS)

Meyer, Thomas J.; Rosenkrantz, Jimi L.; Carbone, Lucia; Chavez, Shawn L.

2017-04-01

Mammalian genomes are scattered with thousands of copies of endogenous retroviruses (ERVs), mobile genetic elements that are relics of ancient retroviral infections. After inserting copies into the germ line of a host, most ERVs accumulate mutations that prevent the normal assembly of infectious viral particles, becoming trapped in host genomes and unable to leave to infect other cells. While most copies of ERVs are inactive, some are transcribed and encode the proteins needed to generate new insertions at novel loci. In some cases, old copies are removed via recombination and other mechanisms. This creates a shifting landscape of ERV copies within host genomes. New insertions can disrupt normal expression of nearby genes via directly inserting into key regulatory elements or by containing regulatory motifs within their sequences. Further, the transcriptional silencing of ERVs via epigenetic modification may result in changes to the epigenetic regulation of adjacent genes. In these ways, ERVs can be potent sources of regulatory disruption as well as genetic innovation. Here, we provide a brief review of the association between ERVs and gene expression, especially as observed in pre-implantation development and placentation. Moreover, we will describe the roles ERVs may play in somatic tissues, mostly in the context of human disease, including cancer, neurodegenerative disorders, and schizophrenia. Lastly, we discuss the recent discovery that some ERVs may have been pressed into the service of their host genomes to aid in the innate immune response to exogenous viral infections.

Evolution of Sphingomonad Gene Clusters Related to Pesticide Catabolism Revealed by Genome Sequence and Mobilomics of Sphingobium herbicidovorans MH.

PubMed

Nielsen, Tue Kjærgaard; Rasmussen, Morten; Demanèche, Sandrine; Cecillon, Sébastien; Vogel, Timothy M; Hansen, Lars Hestbjerg

2017-09-01

Bacterial degraders of chlorophenoxy herbicides have been isolated from various ecosystems, including pristine environments. Among these degraders, the sphingomonads constitute a prominent group that displays versatile xenobiotic-degradation capabilities. Four separate sequencing strategies were required to provide the complete sequence of the complex and plastic genome of the canonical chlorophenoxy herbicide-degrading Sphingobium herbicidovorans MH. The genome has an intricate organization of the chlorophenoxy-herbicide catabolic genes sdpA, rdpA, and cadABCD that encode the (R)- and (S)-enantiomer-specific 2,4-dichlorophenoxypropionate dioxygenases and four subunits of a Rieske non-heme iron oxygenase involved in 2-methyl-chlorophenoxyacetic acid degradation, respectively. Several major genomic rearrangements are proposed to help understand the evolution and mobility of these important genes and their genetic context. Single-strain mobilomic sequence analysis uncovered plasmids and insertion sequence-associated circular intermediates in this environmentally important bacterium and enabled the description of evolutionary models for pesticide degradation in strain MH and related organisms. The mobilome presented a complex mosaic of mobile genetic elements including four plasmids and several circular intermediate DNA molecules of insertion-sequence elements and transposons that are central to the evolution of xenobiotics degradation. Furthermore, two individual chromosomally integrated prophages were shown to excise and form free circular DNA molecules. This approach holds great potential for improving the understanding of genome plasticity, evolution, and microbial ecology. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

A functional (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase exhibits diurnal regulation of expression in Stevia rebaudiana (Bertoni).

PubMed

Kumar, Hitesh; Kumar, Sanjay

2013-09-15

The leaves of stevia [Stevia rebaudiana (Bertoni)] are a rich source of steviol glycosides that are used as non-calorific sweetener in many countries around the world. Steviol moiety of steviol glycosides is synthesized via plastidial 2C-methyl-D-erythritol 4-phosphate pathway, where (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) is the key enzyme. HDR catalyzes the simultaneous conversion of (E)-4-hydroxy-3-methylbut-2-enyl diphosphate into five carbon isoprenoid units, isopentenyl diphosphate and dimethylallyl diphosphate. Stevia HDR (SrHDR) successfully rescued HDR lethal mutant strain MG1655 ara<>ispH upon genetic complementation, suggesting SrHDR to encode a functional protein. The gene exhibited diurnal variation in expression. To identify the possible regulatory elements, upstream region of the gene was cloned and putative cis-acting elements were detected by in silico analysis. Electrophoretic mobility shift assay, using a putative light responsive element GATA showed the binding of nuclear proteins (NP) isolated from leaves during light period of the day, but not with the NP from leaves during the dark period. Data suggested the involvement of GATA box in light mediated gene regulation of SrHDR in stevia. Copyright © 2013 Elsevier B.V. All rights reserved.

The Change of a Medically Important Genus: Worldwide Occurrence of Genetically Diverse Novel Brucella Species in Exotic Frogs.

PubMed

Scholz, Holger C; Mühldorfer, Kristin; Shilton, Cathy; Benedict, Suresh; Whatmore, Adrian M; Blom, Jochen; Eisenberg, Tobias

2016-01-01

The genus Brucella comprises various species of both veterinary and human medical importance. All species are genetically highly related to each other, sharing intra-species average nucleotide identities (ANI) of > 99%. Infections occur among various warm-blooded animal species, marine mammals, and humans. Until recently, amphibians had not been recognized as a host for Brucella. In this study, however, we show that novel Brucella species are distributed among exotic frogs worldwide. Comparative recA gene analysis of 36 frog isolates from various continents and different frog species revealed an unexpected high genetic diversity, not observed among classical Brucella species. In phylogenetic reconstructions the isolates consequently formed various clusters and grouped together with atypical more distantly related brucellae, like B. inopinata, strain BO2, and Australian isolates from rodents, some of which were isolated as human pathogens. Of one frog isolate (10RB9215) the genome sequence was determined. Comparative genome analysis of this isolate and the classical Brucella species revealed additional genetic material, absent from classical Brucella species but present in Ochrobactrum, the closest genetic neighbor of Brucella, and in other soil associated genera of the Alphaproteobacteria. The presence of gene clusters encoding for additional metabolic functions, flanked by tRNAs and mobile genetic elements, as well as by bacteriophages is suggestive for a different ecology compared to classical Brucella species. Furthermore it suggests that amphibian isolates may represent a link between free living soil saprophytes and the pathogenic Brucella with a preferred intracellular habitat. We therefore assume that brucellae from frogs have a reservoir in soil and, in contrast to classical brucellae, undergo extensive horizontal gene transfer.

Mavericks, a novel class of giant transposable elements widespread in eukaryotes and related to DNA viruses.

PubMed

Pritham, Ellen J; Putliwala, Tasneem; Feschotte, Cédric

2007-04-01

We previously identified a group of atypical mobile elements designated Mavericks from the nematodes Caenorhabditis elegans and C. briggsae and the zebrafish Danio rerio. Here we present the results of comprehensive database searches of the genome sequences available, which reveal that Mavericks are widespread in invertebrates and non-mammalian vertebrates but show a patchy distribution in non-animal species, being present in the fungi Glomus intraradices and Phakopsora pachyrhizi and in several single-celled eukaryotes such as the ciliate Tetrahymena thermophila, the stramenopile Phytophthora infestans and the trichomonad Trichomonas vaginalis, but not detectable in plants. This distribution, together with comparative and phylogenetic analyses of Maverick-encoded proteins, is suggestive of an ancient origin of these elements in eukaryotes followed by lineage-specific losses and/or recurrent episodes of horizontal transmission. In addition, we report that Maverick elements have amplified recently to high copy numbers in T. vaginalis where they now occupy as much as 30% of the genome. Sequence analysis confirms that most Mavericks encode a retroviral-like integrase, but lack other open reading frames typically found in retroelements. Nevertheless, the length and conservation of the target site duplication created upon Maverick insertion (5- or 6-bp) is consistent with a role of the integrase-like protein in the integration of a double-stranded DNA transposition intermediate. Mavericks also display long terminal-inverted repeats but do not contain ORFs similar to proteins encoded by DNA transposons. Instead, Mavericks encode a conserved set of 5 to 9 genes (in addition to the integrase) that are predicted to encode proteins with homology to replication and packaging proteins of some bacteriophages and diverse eukaryotic double-stranded DNA viruses, including a DNA polymerase B homolog and putative capsid proteins. Based on these and other structural similarities, we speculate that Mavericks represent an evolutionary missing link between seemingly disparate invasive DNA elements that include bacteriophages, adenoviruses and eukaryotic linear plasmids.

The ribosomal protein genes and Minute loci of Drosophila melanogaster

PubMed Central

Marygold, Steven J; Roote, John; Reuter, Gunter; Lambertsson, Andrew; Ashburner, Michael; Millburn, Gillian H; Harrison, Paul M; Yu, Zhan; Kenmochi, Naoya; Kaufman, Thomas C; Leevers, Sally J; Cook, Kevin R

2007-01-01

Background Mutations in genes encoding ribosomal proteins (RPs) have been shown to cause an array of cellular and developmental defects in a variety of organisms. In Drosophila melanogaster, disruption of RP genes can result in the 'Minute' syndrome of dominant, haploinsufficient phenotypes, which include prolonged development, short and thin bristles, and poor fertility and viability. While more than 50 Minute loci have been defined genetically, only 15 have so far been characterized molecularly and shown to correspond to RP genes. Results We combined bioinformatic and genetic approaches to conduct a systematic analysis of the relationship between RP genes and Minute loci. First, we identified 88 genes encoding 79 different cytoplasmic RPs (CRPs) and 75 genes encoding distinct mitochondrial RPs (MRPs). Interestingly, nine CRP genes are present as duplicates and, while all appear to be functional, one member of each gene pair has relatively limited expression. Next, we defined 65 discrete Minute loci by genetic criteria. Of these, 64 correspond to, or very likely correspond to, CRP genes; the single non-CRP-encoding Minute gene encodes a translation initiation factor subunit. Significantly, MRP genes and more than 20 CRP genes do not correspond to Minute loci. Conclusion This work answers a longstanding question about the molecular nature of Minute loci and suggests that Minute phenotypes arise from suboptimal protein synthesis resulting from reduced levels of cytoribosomes. Furthermore, by identifying the majority of haplolethal and haplosterile loci at the molecular level, our data will directly benefit efforts to attain complete deletion coverage of the D. melanogaster genome. PMID:17927810

A Surrogate Approach to Study the Evolution of Noncoding DNA Elements That Organize Eukaryotic Genomes

PubMed Central

Vermaak, Danielle; Bayes, Joshua J.

2009-01-01

Comparative genomics provides a facile way to address issues of evolutionary constraint acting on different elements of the genome. However, several important DNA elements have not reaped the benefits of this new approach. Some have proved intractable to current day sequencing technology. These include centromeric and heterochromatic DNA, which are essential for chromosome segregation as well as gene regulation, but the highly repetitive nature of the DNA sequences in these regions make them difficult to assemble into longer contigs. Other sequences, like dosage compensation X chromosomal sites, origins of DNA replication, or heterochromatic sequences that encode piwi-associated RNAs, have proved difficult to study because they do not have recognizable DNA features that allow them to be described functionally or computationally. We have employed an alternate approach to the direct study of these DNA elements. By using proteins that specifically bind these noncoding DNAs as surrogates, we can indirectly assay the evolutionary constraints acting on these important DNA elements. We review the impact that such “surrogate strategies” have had on our understanding of the evolutionary constraints shaping centromeres, origins of DNA replication, and dosage compensation X chromosomal sites. These have begun to reveal that in contrast to the view that such structural DNA elements are either highly constrained (under purifying selection) or free to drift (under neutral evolution), some of them may instead be shaped by adaptive evolution and genetic conflicts (these are not mutually exclusive). These insights also help to explain why the same elements (e.g., centromeres and replication origins), which are so complex in some eukaryotic genomes, can be simple and well defined in other where similar conflicts do not exist. PMID:19635763

The developmental transcriptome of Drosophila melanogaster

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

University of Connecticut; Graveley, Brenton R.; Brooks, Angela N.

Drosophila melanogaster is one of the most well studied genetic model organisms; nonetheless, its genome still contains unannotated coding and non-coding genes, transcripts, exons and RNA editing sites. Full discovery and annotation are pre-requisites for understanding how the regulation of transcription, splicing and RNA editing directs the development of this complex organism. Here we used RNA-Seq, tiling microarrays and cDNA sequencing to explore the transcriptome in 30 distinct developmental stages. We identified 111,195 new elements, including thousands of genes, coding and non-coding transcripts, exons, splicing and editing events, and inferred protein isoforms that previously eluded discovery using established experimental, predictionmore » and conservation-based approaches. These data substantially expand the number of known transcribed elements in the Drosophila genome and provide a high-resolution view of transcriptome dynamics throughout development. Drosophila melanogaster is an important non-mammalian model system that has had a critical role in basic biological discoveries, such as identifying chromosomes as the carriers of genetic information and uncovering the role of genes in development. Because it shares a substantial genic content with humans, Drosophila is increasingly used as a translational model for human development, homeostasis and disease. High-quality maps are needed for all functional genomic elements. Previous studies demonstrated that a rich collection of genes is deployed during the life cycle of the fly. Although expression profiling using microarrays has revealed the expression of, 13,000 annotated genes, it is difficult to map splice junctions and individual base modifications generated by RNA editing using such approaches. Single-base resolution is essential to define precisely the elements that comprise the Drosophila transcriptome. Estimates of the number of transcript isoforms are less accurate than estimates of the number of genes. Whereas, 20% of Drosophila genes are annotated as encoding alternatively spliced premRNAs, splice-junction microarray experiments indicate that this number is at least 40% (ref. 7). Determining the diversity of mRNAs generated by alternative promoters, alternative splicing and RNA editing will substantially increase the inferred protein repertoire. Non-coding RNA genes (ncRNAs) including short interfering RNAs (siRNAs) and microRNAS (miRNAs) (reviewed in ref. 10), and longer ncRNAs such as bxd (ref. 11) and rox (ref. 12), have important roles in gene regulation, whereas others such as small nucleolar RNAs (snoRNAs)and small nuclear RNAs (snRNAs) are important components of macromolecular machines such as the ribosome and spliceosome. The transcription and processing of these ncRNAs must also be fully documented and mapped. As part of the modENCODE project to annotate the functional elements of the D. melanogaster and Caenorhabditis elegans genomes, we used RNA-Seq and tiling microarrays to sample the Drosophila transcriptome at unprecedented depth throughout development from early embryo to ageing male and female adults. We report on a high-resolution view of the discovery, structure and dynamic expression of the D. melanogaster transcriptome.« less

Assessment of the Requirements for Magnesium Transporters in Bacillus subtilis

PubMed Central

Wakeman, Catherine A.; Goodson, Jonathan R.; Zacharia, Vineetha M.

2014-01-01

Magnesium is the most abundant divalent metal in cells and is required for many structural and enzymatic functions. For bacteria, at least three families of proteins function as magnesium transporters. In recent years, it has been shown that a subset of these transport proteins is regulated by magnesium-responsive genetic control elements. In this study, we investigated the cellular requirements for magnesium homeostasis in the model microorganism Bacillus subtilis. Putative magnesium transporter genes were mutationally disrupted, singly and in combination, in order to assess their general importance. Mutation of only one of these genes resulted in strong dependency on supplemental extracellular magnesium. Notably, this transporter gene, mgtE, is known to be under magnesium-responsive genetic regulatory control. This suggests that the identification of magnesium-responsive genetic mechanisms may generally denote primary transport proteins for bacteria. To investigate whether B. subtilis encodes yet additional classes of transport mechanisms, suppressor strains that permitted the growth of a transporter-defective mutant were identified. Several of these strains were sequenced to determine the genetic basis of the suppressor phenotypes. None of these mutations occurred in transport protein homologues; instead, they affected housekeeping functions, such as signal recognition particle components and ATP synthase machinery. From these aggregate data, we speculate that the mgtE protein provides the primary route of magnesium import in B. subtilis and that the other putative transport proteins are likely to be utilized for more-specialized growth conditions. PMID:24415722

In Silico Detection of Sequence Variations Modifying Transcriptional Regulation

PubMed Central

Andersen, Malin C; Engström, Pär G; Lithwick, Stuart; Arenillas, David; Eriksson, Per; Lenhard, Boris; Wasserman, Wyeth W; Odeberg, Jacob

2008-01-01

Identification of functional genetic variation associated with increased susceptibility to complex diseases can elucidate genes and underlying biochemical mechanisms linked to disease onset and progression. For genes linked to genetic diseases, most identified causal mutations alter an encoded protein sequence. Technological advances for measuring RNA abundance suggest that a significant number of undiscovered causal mutations may alter the regulation of gene transcription. However, it remains a challenge to separate causal genetic variations from linked neutral variations. Here we present an in silico driven approach to identify possible genetic variation in regulatory sequences. The approach combines phylogenetic footprinting and transcription factor binding site prediction to identify variation in candidate cis-regulatory elements. The bioinformatics approach has been tested on a set of SNPs that are reported to have a regulatory function, as well as background SNPs. In the absence of additional information about an analyzed gene, the poor specificity of binding site prediction is prohibitive to its application. However, when additional data is available that can give guidance on which transcription factor is involved in the regulation of the gene, the in silico binding site prediction improves the selection of candidate regulatory polymorphisms for further analyses. The bioinformatics software generated for the analysis has been implemented as a Web-based application system entitled RAVEN (regulatory analysis of variation in enhancers). The RAVEN system is available at http://www.cisreg.ca for all researchers interested in the detection and characterization of regulatory sequence variation. PMID:18208319

A Guide to Fluorescent Protein FRET Pairs

PubMed Central

Bajar, Bryce T.; Wang, Emily S.; Zhang, Shu; Lin, Michael Z.; Chu, Jun

2016-01-01

Förster or fluorescence resonance energy transfer (FRET) technology and genetically encoded FRET biosensors provide a powerful tool for visualizing signaling molecules in live cells with high spatiotemporal resolution. Fluorescent proteins (FPs) are most commonly used as both donor and acceptor fluorophores in FRET biosensors, especially since FPs are genetically encodable and live-cell compatible. In this review, we will provide an overview of methods to measure FRET changes in biological contexts, discuss the palette of FP FRET pairs developed and their relative strengths and weaknesses, and note important factors to consider when using FPs for FRET studies. PMID:27649177

Genetically encoded sensors and fluorescence microscopy for anticancer research

NASA Astrophysics Data System (ADS)

Zagaynova, Elena V.; Shirmanova, Marina V.; Sergeeva, Tatiana F.; Klementieva, Natalia V.; Mishin, Alexander S.; Gavrina, Alena I.; Zlobovskay, Olga A.; Furman, Olga E.; Dudenkova, Varvara V.; Perelman, Gregory S.; Lukina, Maria M.; Lukyanov, Konstantin A.

2017-02-01

Early response of cancer cells to chemical compounds and chemotherapeutic drugs were studied using novel fluorescence tools and microscopy techniques. We applied confocal microscopy, two-photon fluorescence lifetime imaging microscopy and super-resolution localization-based microscopy to assess structural and functional changes in cancer cells in vitro. The dynamics of energy metabolism, intracellular pH, caspase-3 activation during staurosporine-induced apoptosis as well as actin cytoskeleton rearrangements under chemotherapy were evaluated. We have showed that new genetically encoded sensors and advanced fluorescence microscopy methods provide an efficient way for multiparameter analysis of cell activities

Insight into the mobilome of Aeromonas strains.

PubMed

Piotrowska, Marta; Popowska, Magdalena

2015-01-01

The mobilome is a pool of genes located within mobile genetic elements (MGE), such as plasmids, IS elements, transposons, genomic/pathogenicity islands, and integron-associated gene cassettes. These genes are often referred to as "flexible" and may encode virulence factors, toxic compounds as well as resistance to antibiotics. The phenomenon of MGE transfer between bacteria, known as horizontal gene transfer (HGT), is well documented. The genes present on MGE are subject to continuous processes of evolution and environmental changes, largely induced or significantly accelerated by man. For bacteria, the only chance of survival in an environment contaminated with toxic chemicals, heavy metals and antibiotics is the acquisition of genes providing the ability to survive in such conditions. The process of acquiring and spreading antibiotic resistance genes (ARG) is of particular significance, as it is important for the health of humans and animals. Therefore, it is important to thoroughly study the mobilome of Aeromonas spp. that is widely distributed in various environments, causing many diseases in fishes and humans. This review discusses the recently published information on MGE prevalent in Aeromonas spp. with special emphasis on plasmids belonging to different incompatibility groups, i.e., IncA/C, IncU, IncQ, IncF, IncI, and ColE-type. The vast majority of plasmids carry a number of different transposons (Tn3, Tn21, Tn1213, Tn1721, Tn4401), the 1st, 2nd, or 3rd class of integrons, IS elements (e.g., IS26, ISPa12, ISPa13, ISKpn8, ISKpn6) and encode determinants such as antibiotic and mercury resistance genes, as well as virulence factors. Although the actual role of Aeromonas spp. as a human pathogen remains controversial, species of this genus may pose a serious risk to human health. This is due to the considerable potential of their mobilome, particularly in terms of antibiotic resistance and the possibility of the horizontal transfer of resistance genes.

Insight into the mobilome of Aeromonas strains

PubMed Central

Piotrowska, Marta; Popowska, Magdalena

2015-01-01

The mobilome is a pool of genes located within mobile genetic elements (MGE), such as plasmids, IS elements, transposons, genomic/pathogenicity islands, and integron-associated gene cassettes. These genes are often referred to as “flexible” and may encode virulence factors, toxic compounds as well as resistance to antibiotics. The phenomenon of MGE transfer between bacteria, known as horizontal gene transfer (HGT), is well documented. The genes present on MGE are subject to continuous processes of evolution and environmental changes, largely induced or significantly accelerated by man. For bacteria, the only chance of survival in an environment contaminated with toxic chemicals, heavy metals and antibiotics is the acquisition of genes providing the ability to survive in such conditions. The process of acquiring and spreading antibiotic resistance genes (ARG) is of particular significance, as it is important for the health of humans and animals. Therefore, it is important to thoroughly study the mobilome of Aeromonas spp. that is widely distributed in various environments, causing many diseases in fishes and humans. This review discusses the recently published information on MGE prevalent in Aeromonas spp. with special emphasis on plasmids belonging to different incompatibility groups, i.e., IncA/C, IncU, IncQ, IncF, IncI, and ColE-type. The vast majority of plasmids carry a number of different transposons (Tn3, Tn21, Tn1213, Tn1721, Tn4401), the 1st, 2nd, or 3rd class of integrons, IS elements (e.g., IS26, ISPa12, ISPa13, ISKpn8, ISKpn6) and encode determinants such as antibiotic and mercury resistance genes, as well as virulence factors. Although the actual role of Aeromonas spp. as a human pathogen remains controversial, species of this genus may pose a serious risk to human health. This is due to the considerable potential of their mobilome, particularly in terms of antibiotic resistance and the possibility of the horizontal transfer of resistance genes. PMID:26074893

Localization of Action of the Is50-Encoded Transposase Protein

PubMed Central

Phadnis, Suhas H.; Sasakawa, Chihiro; Berg, Douglas E.

1986-01-01

The movement of the bacterial insertion sequence IS50 and of composite elements containing direct terminal repeats of IS50 involves the two ends of IS50, designated O (outside) and I (inside), which are weakly matched in DNA sequence, and an IS50 encoded protein, transposase, which recognizes the O and I ends and acts preferentially in cis. Previous data had suggested that, initially, transposase interacts preferentially with the O end sequence and then, in a second step, with either an O or an I end. To better understand the cis action of transposase and how IS50 ends are selected, we generated a series of composite transposons which contain direct repeats of IS50 elements. In each transposon, one IS50 element encoded transposase (tnp +), and the other contained a null (tnp-) allele. In each of the five sets of composite transposons studied, the transposon for which the tnp+ IS50 element contained its O end was more active than a complementary transposon for which the tnp - IS50 element contained its O end. This pattern of O end use suggests models in which the cis action of transposase and its choice of ends is determined by protein tracking along DNA molecules. PMID:3007274

Genetically encoded fluorescent tags

PubMed Central

Thorn, Kurt

2017-01-01

Genetically encoded fluorescent tags are protein sequences that can be fused to a protein of interest to render it fluorescent. These tags have revolutionized cell biology by allowing nearly any protein to be imaged by light microscopy at submicrometer spatial resolution and subsecond time resolution in a live cell or organism. They can also be used to measure protein abundance in thousands to millions of cells using flow cytometry. Here I provide an introduction to the different genetic tags available, including both intrinsically fluorescent proteins and proteins that derive their fluorescence from binding of either endogenous or exogenous fluorophores. I discuss their optical and biological properties and guidelines for choosing appropriate tags for an experiment. Tools for tagging nucleic acid sequences and reporter molecules that detect the presence of different biomolecules are also briefly discussed. PMID:28360214

[Plasticity of bacterial genomes: pathogenicity islands and the locus of enterocyte effacement (LEE)].

PubMed

Kirsch, Petra; Jores, Jörg; Wieler, Lothar H

2004-01-01

Many bacterial virulence attributes, like toxins, adhesins, invasins, iron uptake systems, are encoded within specific regions of the bacterial genome. These in size varying regions are termed pathogenicity islands (PAIs) since they confer pathogenic properties to the respective micro-organism. Per definition PAIs are exclusively found in pathogenic strains and are often inserted near transfer-RNA genes. Nevertheless, non-pathogenic bacteria also possess foreign DNA elements that confer advantageous features, leading to improved fitness. These additional DNA elements as well as PAIs are termed genomic islands and were acquired during bacterial evolution. Significant G+C content deviation in pathogenicity islands with respect to the rest of the genome, the presence of direct repeat sequences at the flanking regions, the presence of integrase gene determinants as other mobility features,the particular insertion site (tRNA gene) as well as the observed genetic instability suggests that pathogenicity islands were acquired by horizontal gene transfer. PAIs are the fascinating proof of the plasticity of bacterial genomes. PAIs were originally described in human pathogenic Escherichia (E.) coli strains. In the meantime PAIs have been found in various pathogenic bacteria of humans, animals and even plants. The Locus of Enterocyte Effacement (LEE) is one particular widely distributed PAI of E coli. In addition, it also confers pathogenicity to the related species Citrobacter (C.) rodentium and Escherichia (E.) alvei. The LEE is an important virulence feature of several animal pathogens. It is an obligate PAI of all animal and human enteropathogenic E. coli (EPEC), and most enterohaemorrhegic E. coli (EHEC) also harbor the LEE. The LEE encodes a type III secretion system, an adhesion (intimin) that mediates the intimate contact between the bacterium and the epithelial cell, as well as various proteins which are secreted via the type III secretion system. The LEE encoded virulence features are responsible for the formation of so called attaching and effacing (AE) lesions in the intestinal epithelium. Due to its wide distribution in animal pathogens, LEE encoded antigens are suitable vaccine antigens. Acquisition and structure of the LEE pathogenicity island is the crucial point of numerous investigations. However, the evolution of the LEE, its origin and further spread in E. coli, are far from being resolved.

Reverse Genetics for Mammalian Orthoreovirus.

PubMed

Stuart, Johnasha D; Phillips, Matthew B; Boehme, Karl W

2017-01-01

Reverse genetics allows introduction of specific alterations into a viral genome. Studies performed with mutant viruses generated using reverse genetics approaches have contributed immeasurably to our understanding of viral replication and pathogenesis, and also have led to development of novel vaccines and virus-based vectors. Here, we describe the reverse genetics system that allows for production and recovery of mammalian orthoreovirus, a double-stranded (ds) RNA virus, from plasmids that encode the viral genome.

Quetzal: a transposon of the Tc1 family in the mosquito Anopheles albimanus.

PubMed

Ke, Z; Grossman, G L; Cornel, A J; Collins, F H

1996-10-01

A member of the Tc1 family of transposable elements has been identified in the Central and South American mosquito Anopheles albimanus. The full-length Quetzal element is 1680 base pairs (bp) in length, possesses 236 bp inverted terminal repeats (ITRs), and has a single open reading frame (ORF) with the potential of encoding a 341-amino-acid (aa) protein that is similar to the transposases of other members of the Tc1 family, particularly elements described from three different Drosophila species. The approximately 10-12 copies per genome of Quetzal are found in the euchromatin of all three chromosomes of A. albimanus. One full-length clone, Que27, appears capable of encoding a complete transposase and may represent a functional copy of this element.

Production Of Cellulase In Plastids Of Transgenic Plants

DOEpatents

Lamppa, Gayle

2002-08-06

A genetic construct encoding a fusion protein including endogluconase E1 and a transit peptide is used to transform plants. The plants produce cellulase by expressing the genetic construct. The cellulase is targeted to plastids and can be collected and purified.

Complete genome sequence of the chromate-reducing bacterium Thermoanaerobacter thermohydrosulfuricus strain BSB-33

DOE PAGES

Bhattacharya, Pamela; Barnebey, Adam; Zemla, Marcin; ...

2015-10-05

Thermoanaerobacter thermohydrosulfuricus BSB-33 is a thermophilic gram positive obligate anaerobe isolated from a hot spring in West Bengal, India. Unlike other T. thermohydrosulfuricus strains, BSB-33 is able to anaerobically reduce Fe(III) and Cr(VI) optimally at 60 °C. BSB-33 is the first Cr(VI) reducing T. thermohydrosulfuricus genome sequenced and of particular interest for bioremediation of environmental chromium contaminations. Here we discuss features of T. thermohydrosulfuricus BSB-33 and the unique genetic elements that may account for the peculiar metal reducing properties of this organism. The T. thermohydrosulfuricus BSB-33 genome comprises 2597606 bp encoding 2581 protein genes, 12 rRNA, 193 pseudogenes and hasmore » a G + C content of 34.20 %. Lastly, putative chromate reductases were identified by comparative analyses with other Thermoanaerobacter and chromate-reducing bacteria.« less

Physiological impact of transposable elements encoding DDE transposases in the environmental adaptation of Streptococcus agalactiae.

PubMed

Fléchard, Maud; Gilot, Philippe

2014-07-01

We have referenced and described Streptococcus agalactiae transposable elements encoding DDE transposases. These elements belonged to nine families of insertion sequences (ISs) and to a family of conjugative transposons (TnGBSs). An overview of the physiological impact of the insertion of all these elements is provided. DDE-transposable elements affect S. agalactiae in a number of aspects of its capability to adapt to various environments and modulate the expression of several virulence genes, the scpB-lmB genomic region and the genes involved in capsule expression and haemolysin transport being the targets of several different mobile elements. The referenced mobile elements modify S. agalactiae behaviour by transferring new gene(s) to its genome, by modifying the expression of neighbouring genes at the integration site or by promoting genomic rearrangements. Transposition of some of these elements occurs in vivo, suggesting that by dynamically regulating some adaptation and/or virulence genes, they improve the ability of S. agalactiae to reach different niches within its host and ensure the 'success' of the infectious process. © 2014 The Authors.

Screening for ATM Mutations in an African-American Population to Identify a Predictor of Breast Cancer Susceptibility

DTIC Science & Technology

2006-07-01

ATM genetic variant identified affects radiosensitivity and levels of the protein encoded by the ATM gene for each mutation examined. 15. SUBJECT...women without breast cancer. An additional objective is to determine the functional impact upon the protein encoded by the ATM gene for each mutation ...each ATM variant identified affects radiosensitivity and levels of the protein encoded by the ATM gene for mutations identified. Body STATEMENT

Nucleotide sequences encoding a thermostable alkaline protease

DOEpatents

Wilson, David B.; Lao, Guifang

1998-01-01

Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium.

A high-accuracy optical linear algebra processor for finite element applications

NASA Technical Reports Server (NTRS)

Casasent, D.; Taylor, B. K.

1984-01-01

Optical linear processors are computationally efficient computers for solving matrix-matrix and matrix-vector oriented problems. Optical system errors limit their dynamic range to 30-40 dB, which limits their accuray to 9-12 bits. Large problems, such as the finite element problem in structural mechanics (with tens or hundreds of thousands of variables) which can exploit the speed of optical processors, require the 32 bit accuracy obtainable from digital machines. To obtain this required 32 bit accuracy with an optical processor, the data can be digitally encoded, thereby reducing the dynamic range requirements of the optical system (i.e., decreasing the effect of optical errors on the data) while providing increased accuracy. This report describes a new digitally encoded optical linear algebra processor architecture for solving finite element and banded matrix-vector problems. A linear static plate bending case study is described which quantities the processor requirements. Multiplication by digital convolution is explained, and the digitally encoded optical processor architecture is advanced.

Intracellular pH imaging in cancer cells in vitro and tumors in vivo using the new genetically encoded sensor SypHer2.

PubMed

Shirmanova, Marina V; Druzhkova, Irina N; Lukina, Maria M; Matlashov, Mikhail E; Belousov, Vsevolod V; Snopova, Ludmila B; Prodanetz, Natalia N; Dudenkova, Varvara V; Lukyanov, Sergey A; Zagaynova, Elena V

2015-09-01

Measuring intracellular pH (pHi) in tumors is essential for the monitoring of cancer progression and the response of cancer cells to various treatments. The purpose of the study was to develop a method for pHi mapping in living cancer cells in vitro and in tumors in vivo, using the novel genetically encoded indicator, SypHer2. A HeLa Kyoto cell line stably expressing SypHer2 in the cytoplasm was used, to perform ratiometric (dual excitation) imaging of the probe in cell culture, in 3D tumor spheroids and in tumor xenografts in living mice. Using SypHer2, pHi was demonstrated to be 7.34±0.11 in monolayer HeLa cells in vitro under standard cultivation conditions. An increasing pHi gradient from the center to the periphery of the spheroids was displayed. We obtained fluorescence ratio maps for HeLa tumors in vivo and ex vivo. Comparison of the map with the pathomorphology and with hypoxia staining of the tumors revealed a correspondence of the zones with higher pHi to the necrotic and hypoxic areas. Our results demonstrate that pHi imaging with the genetically encoded pHi indicator, SypHer2, can be a valuable tool for evaluating tumor progression in xenograft models. We have demonstrated, for the first time, the possibility of using the genetically encoded sensor SypHer2 for ratiometric pH imaging in cancer cells in vitro and in tumors in vivo. SypHer2 shows great promise as an instrument for pHi monitoring able to provide high accuracy and spatiotemporal resolution. Copyright © 2015 Elsevier B.V. All rights reserved.

Development and implementation of an 84-channel matrix gradient coil.

PubMed

Littin, Sebastian; Jia, Feng; Layton, Kelvin J; Kroboth, Stefan; Yu, Huijun; Hennig, Jürgen; Zaitsev, Maxim

2018-02-01

Design, implement, integrate, and characterize a customized coil system that allows for generating spatial encoding magnetic fields (SEMs) in a highly-flexible fashion. A gradient coil with a high number of individual elements was designed. Dimensions of the coil were chosen to mimic a whole-body gradient system, scaled down to a head insert. Mechanical shape and wire layout of each element were optimized to increase the local gradient strength while minimizing eddy current effects and simultaneously considering manufacturing constraints. Resulting wire layout and mechanical design is presented. A prototype matrix gradient coil with 12 × 7 = 84 elements consisting of two element types was realized and characterized. Measured eddy currents are <1% of the original field. The coil is shown to be capable of creating nonlinear, and linear SEMs. In a DSV of 0.22 m gradient strengths between 24 mT∕m and 78 mT∕m could be realized locally with maximum currents of 150 A. Initial proof-of-concept imaging experiments using linear and nonlinear encoding fields are demonstrated. A shielded matrix gradient coil setup capable of generating encoding fields in a highly-flexible manner was designed and implemented. The presented setup is expected to serve as a basis for validating novel imaging techniques that rely on nonlinear spatial encoding fields. Magn Reson Med 79:1181-1191, 2018. © 2017 International Society for Magnetic Resonance in Medicine. © 2017 International Society for Magnetic Resonance in Medicine.

Imaging Voltage in Genetically Defined Neuronal Subpopulations with a Cre Recombinase-Targeted Hybrid Voltage Sensor.

PubMed

Bayguinov, Peter O; Ma, Yihe; Gao, Yu; Zhao, Xinyu; Jackson, Meyer B

2017-09-20

Genetically encoded voltage indicators create an opportunity to monitor electrical activity in defined sets of neurons as they participate in the complex patterns of coordinated electrical activity that underlie nervous system function. Taking full advantage of genetically encoded voltage indicators requires a generalized strategy for targeting the probe to genetically defined populations of cells. To this end, we have generated a mouse line with an optimized hybrid voltage sensor (hVOS) probe within a locus designed for efficient Cre recombinase-dependent expression. Crossing this mouse with Cre drivers generated double transgenics expressing hVOS probe in GABAergic, parvalbumin, and calretinin interneurons, as well as hilar mossy cells, new adult-born neurons, and recently active neurons. In each case, imaging in brain slices from male or female animals revealed electrically evoked optical signals from multiple individual neurons in single trials. These imaging experiments revealed action potentials, dynamic aspects of dendritic integration, and trial-to-trial fluctuations in response latency. The rapid time response of hVOS imaging revealed action potentials with high temporal fidelity, and enabled accurate measurements of spike half-widths characteristic of each cell type. Simultaneous recording of rapid voltage changes in multiple neurons with a common genetic signature offers a powerful approach to the study of neural circuit function and the investigation of how neural networks encode, process, and store information. SIGNIFICANCE STATEMENT Genetically encoded voltage indicators hold great promise in the study of neural circuitry, but realizing their full potential depends on targeting the sensor to distinct cell types. Here we present a new mouse line that expresses a hybrid optical voltage sensor under the control of Cre recombinase. Crossing this line with Cre drivers generated double-transgenic mice, which express this sensor in targeted cell types. In brain slices from these animals, single-trial hybrid optical voltage sensor recordings revealed voltage changes with submillisecond resolution in multiple neurons simultaneously. This imaging tool will allow for the study of the emergent properties of neural circuits and permit experimental tests of the roles of specific types of neurons in complex circuit activity. Copyright © 2017 the authors 0270-6474/17/379305-15$15.00/0.

Three new insertion sequence elements ISLdl2, ISLdl3, and ISLdl4 in Lactobacillus delbrueckii: isolation, molecular characterization, and potential use for strain identification.

PubMed

Ravin, Victor; Alatossava, Tapani

2003-05-01

A group of new insertion sequence (IS) elements, ISLdl2, ISLdl3, and ISLdl4, from Lactobacillus delbrueckii subsp. lactis ATCC 15808 was isolated, characterized, and used for strain identification together with ISLdl1, recently characterized as an L. delbrueckii IS element belonging to the ISL3 family. ISLdl2 was 1367 bp in size and had a 24 bp IR and an 8 bp DR. The single ORF of ISLdl2 encoded a protein of 392 aa similar to transposases of the IS256 family. ISLdl3 had a single ORF encoding a protein of 343 aa similar to transposases of the IS30 family. Finally, ISLdl4 had a single ORF encoding a protein of 406 aa and displayed homology to the transposases of the IS110 family. ISLdl4 was only slight different from ISL4 (Accession No. AY040213). ISLdl1, ISLdl2, and ISLdl4 were present in all of the 10 L. delbrueckii subsp. lactis and subsp. delbrueckii strains tested, as well as in three of the 11 L. delbrueckii subsp. bulgaricus strains tested. ISLdl3 was present only in four closely related strains of L. delbrueckii subsp. lactis. These IS elements were not observed in Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus helveticus, or Lactobacillus plantarum. A cluster of IS elements, ISLdl1, ISLdl2, ISLdl3, ISLdl4, and ISL6, was observed in L. delbrueckii subsp. lactis strain ATCC 15808. Within this cluster, ISLdl4 was inserted into ISLdl1 between the left IR and the start codon of ORF455, encoding a putative transposase. Most of the integration sites of the IS elements were strain-specific. We have observed that IS elements can migrate from one strain to another as integral parts of bacterial DNA by using phage LL-H as a vehicle. We demonstrate for the first time that inverse PCR and vectorette PCR methods with primers based on sequences of the IS elements could be used for identification of L. delbrueckii strains.

Secretion Trap Tagging of Secreted and Membrane-Spanning Proteins Using Arabidopsis Gene Traps

Treesearch

Andrew T. Groover; Joseph R. Fontana; Juana M. Arroyo; Cristina Yordan; W. Richard McCombie; Robert A. Martienssen

2003-01-01

Secreted and membrane-spanning proteins play fundamental roles in plant development but pose challenges for genetic identification and characterization. We describe a "secretion trap" screen for gene trap insertions in genes encoding proteins routed through the secretory pathway. The gene trap transposon encodes a ß-glucuronidase reporter enzyme...

Genealogical evidence for epidemics of selfish genes.

PubMed

Ingvarsson, Par K; Taylor, Douglas R

2002-08-20

Some genetic elements spread infectiously in populations by increasing their rate of genetic transmission at the expense of other genes in the genome. These so-called selfish genetic elements comprise a substantial portion of eukaryotic genomes and have long been viewed as a potent evolutionary force. Despite this view, little is known about the evolutionary history of selfish genetic elements in natural populations, or their genetic effects on other portions of the genome. Here we use nuclear and chloroplast gene genealogies in two species of Silene to show the historical pattern of selection on a well known selfish genetic element, cytoplasmic male sterility. We provide evidence that evolution of cytoplasmic male sterility has been characterized by frequent turnovers of mutations in natural populations, thus supporting an epidemic model for the evolution of selfish genes, where new mutations repeatedly arise and rapidly sweep through populations.

Genomic Diversification in Strains of Rickettsia felis Isolated from Different Arthropods

PubMed Central

Gillespie, Joseph J.; Driscoll, Timothy P.; Verhoeve, Victoria I.; Utsuki, Tadanobu; Husseneder, Claudia; Chouljenko, Vladimir N.; Azad, Abdu F.; Macaluso, Kevin R.

2015-01-01

Rickettsia felis (Alphaproteobacteria: Rickettsiales) is the causative agent of an emerging flea-borne rickettsiosis with worldwide occurrence. Originally described from the cat flea, Ctenocephalides felis, recent reports have identified R. felis from other flea species, as well as other insects and ticks. This diverse host range for R. felis may indicate an underlying genetic variability associated with host-specific strains. Accordingly, to determine a potential genetic basis for host specialization, we sequenced the genome of R. felis str. LSU-Lb, which is an obligate mutualist of the parthenogenic booklouse Liposcelis bostrychophila (Insecta: Psocoptera). We also sequenced the genome of R. felis str. LSU, the second genome sequence for cat flea-associated strains (cf. R. felis str. URRWXCal2), which are presumably facultative parasites of fleas. Phylogenomics analysis revealed R. felis str. LSU-Lb diverged from the flea-associated strains. Unexpectedly, R. felis str. LSU was found to be divergent from R. felis str. URRWXCal2, despite sharing similar hosts. Although all three R. felis genomes contain the pRF plasmid, R. felis str. LSU-Lb carries an additional unique plasmid, pLbaR (plasmid of L. bostrychophila associated Rickettsia), nearly half of which encodes a unique 23-gene integrative conjugative element. Remarkably, pLbaR also encodes a repeats-in-toxin-like type I secretion system and associated toxin, heretofore unknown from other Rickettsiales genomes, which likely originated from lateral gene transfer with another obligate intracellular parasite of arthropods, Cardinium (Bacteroidetes). Collectively, our study reveals unexpected genomic diversity across three R. felis strains and identifies several diversifying factors that differentiate facultative parasites of fleas from obligate mutualists of booklice. PMID:25477419

Lhx6 and Lhx8 promote palate development through negative regulation of a cell cycle inhibitor gene, p57Kip2

PubMed Central

Cesario, Jeffry M.; Landin Malt, Andre; Deacon, Lindsay J.; Sandberg, Magnus; Vogt, Daniel; Tang, Zuojian; Zhao, Yangu; Brown, Stuart; Rubenstein, John L.; Jeong, Juhee

2015-01-01

Cleft palate is a common birth defect in humans. Therefore, understanding the molecular genetics of palate development is important from both scientific and medical perspectives. Lhx6 and Lhx8 encode LIM homeodomain transcription factors, and inactivation of both genes in mice resulted in profound craniofacial defects including cleft secondary palate. The initial outgrowth of the palate was severely impaired in the mutant embryos, due to decreased cell proliferation. Through genome-wide transcriptional profiling, we discovered that p57Kip2 (Cdkn1c), encoding a cell cycle inhibitor, was up-regulated in the prospective palate of Lhx6−/−;Lhx8−/− mutants. p57Kip2 has been linked to Beckwith–Wiedemann syndrome and IMAGe syndrome in humans, which are developmental disorders with increased incidents of palate defects among the patients. To determine the molecular mechanism underlying the regulation of p57Kip2 by the Lhx genes, we combined chromatin immunoprecipitation, in silico search for transcription factor-binding motifs, and in vitro reporter assays with putative cis-regulatory elements. The results of these experiments indicated that LHX6 and LHX8 regulated p57Kip2 via both direct and indirect mechanisms, with the latter mediated by Forkhead box (FOX) family transcription factors. Together, our findings uncovered a novel connection between the initiation of palate development and a cell cycle inhibitor via LHX. We propose a model in which Lhx6 and Lhx8 negatively regulate p57Kip2 expression in the prospective palate area to allow adequate levels of cell proliferation and thereby promote normal palate development. This is the first report elucidating a molecular genetic pathway downstream of Lhx in palate development. PMID:26071365

Regulation of gene expression in the mammalian eye and its relevance to eye disease

PubMed Central

Scheetz, Todd E.; Kim, Kwang-Youn A.; Swiderski, Ruth E.; Philp, Alisdair R.; Braun, Terry A.; Knudtson, Kevin L.; Dorrance, Anne M.; DiBona, Gerald F.; Huang, Jian; Casavant, Thomas L.; Sheffield, Val C.; Stone, Edwin M.

2006-01-01

We used expression quantitative trait locus mapping in the laboratory rat (Rattus norvegicus) to gain a broad perspective of gene regulation in the mammalian eye and to identify genetic variation relevant to human eye disease. Of >31,000 gene probes represented on an Affymetrix expression microarray, 18,976 exhibited sufficient signal for reliable analysis and at least 2-fold variation in expression among 120 F2 rats generated from an SR/JrHsd × SHRSP intercross. Genome-wide linkage analysis with 399 genetic markers revealed significant linkage with at least one marker for 1,300 probes (α = 0.001; estimated empirical false discovery rate = 2%). Both contiguous and noncontiguous loci were found to be important in regulating mammalian eye gene expression. We investigated one locus of each type in greater detail and identified putative transcription-altering variations in both cases. We found an inserted cREL binding sequence in the 5′ flanking sequence of the Abca4 gene associated with an increased expression level of that gene, and we found a mutation of the gene encoding thyroid hormone receptor β2 associated with a decreased expression level of the gene encoding short-wavelength sensitive opsin (Opn1sw). In addition to these positional studies, we performed a pairwise analysis of gene expression to identify genes that are regulated in a coordinated manner and used this approach to validate two previously undescribed genes involved in the human disease Bardet–Biedl syndrome. These data and analytical approaches can be used to facilitate the discovery of additional genes and regulatory elements involved in human eye disease. PMID:16983098

In Situ Formation of an Azo Bridge on Proteins Controllable by Visible Light.

PubMed

Hoppmann, Christian; Maslennikov, Innokentiy; Choe, Senyon; Wang, Lei

2015-09-09

Optical modulation of proteins provides superior spatiotemporal resolution for understanding biological processes, and photoswitches built on light-sensitive proteins have been significantly advancing neuronal and cellular studies. Small molecule photoswitches could complement protein-based switches by mitigating potential interference and affording high specificity for modulation sites. However, genetic encodability and responsiveness to nonultraviolet light, two desired properties possessed by protein photoswitches, are challenging to be engineered into small molecule photoswitches. Here we developed a small molecule photoswitch that can be genetically installed onto proteins in situ and controlled by visible light. A pentafluoro azobenzene-based photoswitchable click amino acid (F-PSCaa) was designed to isomerize in response to visible light. After genetic incorporation into proteins via the expansion of the genetic code, F-PSCaa reacts with a nearby cysteine within the protein generating an azo bridge in situ. The resultant bridge is switchable by visible light and allows conformation and binding of CaM to be regulated by such light. This photoswitch should prove valuable in optobiology for its minimal interference, site flexibility, genetic encodability, and response to the more biocompatible visible light.

Genetic Dissection of Anopheles gambiae Gut Epithelial Responses to Serratia marcescens

PubMed Central

Stathopoulos, Stavros; Neafsey, Daniel E.; Lawniczak, Mara K. N.; Muskavitch, Marc A. T.; Christophides, George K.

2014-01-01

Genetic variation in the mosquito Anopheles gambiae profoundly influences its ability to transmit malaria. Mosquito gut bacteria are shown to influence the outcome of infections with Plasmodium parasites and are also thought to exert a strong drive on genetic variation through natural selection; however, a link between antibacterial effects and genetic variation is yet to emerge. Here, we combined SNP genotyping and expression profiling with phenotypic analyses of candidate genes by RNAi-mediated silencing and 454 pyrosequencing to investigate this intricate biological system. We identified 138 An. gambiae genes to be genetically associated with the outcome of Serratia marcescens infection, including the peptidoglycan recognition receptor PGRPLC that triggers activation of the antibacterial IMD/REL2 pathway and the epidermal growth factor receptor EGFR. Silencing of three genes encoding type III fibronectin domain proteins (FN3Ds) increased the Serratia load and altered the gut microbiota composition in favor of Enterobacteriaceae. These data suggest that natural genetic variation in immune-related genes can shape the bacterial population structure of the mosquito gut with high specificity. Importantly, FN3D2 encodes a homolog of the hypervariable pattern recognition receptor Dscam, suggesting that pathogen-specific recognition may involve a broader family of immune factors. Additionally, we showed that silencing the gene encoding the gustatory receptor Gr9 that is also associated with the Serratia infection phenotype drastically increased Serratia levels. The Gr9 antibacterial activity appears to be related to mosquito feeding behavior and to mostly rely on changes of neuropeptide F expression, together suggesting a behavioral immune response following Serratia infection. Our findings reveal that the mosquito response to oral Serratia infection comprises both an epithelial and a behavioral immune component. PMID:24603764

A lanthipeptide library used to identify a protein-protein interaction inhibitor.

PubMed

Yang, Xiao; Lennard, Katherine R; He, Chang; Walker, Mark C; Ball, Andrew T; Doigneaux, Cyrielle; Tavassoli, Ali; van der Donk, Wilfred A

2018-04-01

In this article we describe the production and screening of a genetically encoded library of 10 6 lanthipeptides in Escherichia coli using the substrate-tolerant lanthipeptide synthetase ProcM. This plasmid-encoded library was combined with a bacterial reverse two-hybrid system for the interaction of the HIV p6 protein with the UEV domain of the human TSG101 protein, which is a critical protein-protein interaction for HIV budding from infected cells. Using this approach, we identified an inhibitor of this interaction from the lanthipeptide library, whose activity was verified in vitro and in cell-based virus-like particle-budding assays. Given the variety of lanthipeptide backbone scaffolds that may be produced with ProcM, this method may be used for the generation of genetically encoded libraries of natural product-like lanthipeptides containing substantial structural diversity. Such libraries may be combined with any cell-based assay to identify lanthipeptides with new biological activities.

Elemental Concentrations in the Seed of Mutants and Natural Variants of Arabidopsis thaliana Grown under Varying Soil Conditions

PubMed Central

McDowell, Stephen C.; Akmakjian, Garo; Sladek, Chris; Mendoza-Cozatl, David; Morrissey, Joe B.; Saini, Nick; Mittler, Ron; Baxter, Ivan; Salt, David E.; Ward, John M.; Schroeder, Julian I.; Guerinot, Mary Lou; Harper, Jeffrey F.

2013-01-01

The concentrations of mineral nutrients in seeds are critical to both the life cycle of plants as well as human nutrition. These concentrations are strongly influenced by soil conditions, as shown here by quantifying the concentration of 14 elements in seeds from Arabidopsis thaliana plants grown under four different soil conditions: standard, or modified with NaCl, heavy metals, or alkali. Each of the modified soils resulted in a unique change to the seed ionome (the mineral nutrient content of the seeds). To help identify the genetic networks regulating the seed ionome, changes in elemental concentrations were evaluated using mutants corresponding to 760 genes as well as 10 naturally occurring accessions. The frequency of ionomic phenotypes supports an estimate that as much as 11% of the A. thaliana genome encodes proteins of functional relevance to ion homeostasis in seeds. A subset of mutants were analyzed with two independent alleles, providing five examples of genes important for regulation of the seed ionome: SOS2, ABH1, CCC, At3g14280 and CNGC2. In a comparison of nine different accessions to a Col-0 reference, eight accessions were observed to have reproducible differences in elemental concentrations, seven of which were dependent on specific soil conditions. These results indicate that the A. thaliana seed ionome is distinct from the vegetative ionome, and that elemental analysis is a sensitive approach to identify genes controlling ion homeostasis, including those that regulate gene expression, phospho-regulation, and ion transport. PMID:23671651

Design of sparse Halbach magnet arrays for portable MRI using a genetic algorithm.

PubMed

Cooley, Clarissa Zimmerman; Haskell, Melissa W; Cauley, Stephen F; Sappo, Charlotte; Lapierre, Cristen D; Ha, Christopher G; Stockmann, Jason P; Wald, Lawrence L

2018-01-01

Permanent magnet arrays offer several attributes attractive for the development of a low-cost portable MRI scanner for brain imaging. They offer the potential for a relatively lightweight, low to mid-field system with no cryogenics, a small fringe field, and no electrical power requirements or heat dissipation needs. The cylindrical Halbach array, however, requires external shimming or mechanical adjustments to produce B 0 fields with standard MRI homogeneity levels (e.g., 0.1 ppm over FOV), particularly when constrained or truncated geometries are needed, such as a head-only magnet where the magnet length is constrained by the shoulders. For portable scanners using rotation of the magnet for spatial encoding with generalized projections, the spatial pattern of the field is important since it acts as the encoding field. In either a static or rotating magnet, it will be important to be able to optimize the field pattern of cylindrical Halbach arrays in a way that retains construction simplicity. To achieve this, we present a method for designing an optimized cylindrical Halbach magnet using the genetic algorithm to achieve either homogeneity (for standard MRI applications) or a favorable spatial encoding field pattern (for rotational spatial encoding applications). We compare the chosen designs against a standard, fully populated sparse Halbach design, and evaluate optimized spatial encoding fields using point-spread-function and image simulations. We validate the calculations by comparing to the measured field of a constructed magnet. The experimentally implemented design produced fields in good agreement with the predicted fields, and the genetic algorithm was successful in improving the chosen metrics. For the uniform target field, an order of magnitude homogeneity improvement was achieved compared to the un-optimized, fully populated design. For the rotational encoding design the resolution uniformity is improved by 95% compared to a uniformly populated design.

Distribution of genetic polymorphisms of genes encoding drug metabolizing enzymes & drug transporters - a review with Indian perspective.

PubMed

Umamaheswaran, Gurusamy; Kumar, Dhakchinamoorthi Krishna; Adithan, Chandrasekaran

2014-01-01

Phase I and II drug metabolizing enzymes (DME) and drug transporters are involved in the absorption, distribution, metabolism as well as elimination of many therapeutic agents, toxins and various pollutants. Presence of genetic polymorphisms in genes encoding these proteins has been associated with marked inter-individual variability in their activity that could result in variation in drug response, toxicity as well as in disease predisposition. The emergent field pharmacogenetics and pharmacogenomics (PGx) is a promising discipline, as it predicts disease risk, selection of proper medication with regard to response and toxicity, and appropriate drug dosage guidance based on an individual's genetic make-up. Consequently, genetic variations are essential to understand the ethnic differences in disease occurrence, development, prognosis, therapeutic response and toxicity. For that reason, it is necessary to establish the normative frequency of these genes in a particular population before unraveling the genotype-phenotype associations. Although a fair amount of allele frequency data are available in Indian populations, the existing pharmacogenetic data have not been compiled into a database. This review was intended to compile the normative frequency distribution of the variants of genes encoding DMEs (CYP450s, TPMT, GSTs, COMT, SULT1A1, NAT2 and UGTs) and transporter proteins (MDR1, OCT1 and SLCO1B1) with Indian perspective.

Nucleotide sequences encoding a thermostable alkaline protease

DOEpatents

Wilson, D.B.; Lao, G.

1998-01-06

Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium. 3 figs.

The Drosophila pigmentation gene pink (p) encodes a homologue of human Hermansky-Pudlak syndrome 5 (HPS5).

PubMed

Falcón-Pérez, Juan M; Romero-Calderón, Rafael; Brooks, Elizabeth S; Krantz, David E; Dell'Angelica, Esteban C

2007-02-01

Lysosome-related organelles comprise a group of specialized intracellular compartments that include melanosomes and platelet dense granules (in mammals) and eye pigment granules (in insects). In humans, the biogenesis of these organelles is defective in genetic disorders collectively known as Hermansky-Pudlak syndrome (HPS). Patients with HPS-2, and two murine HPS models, carry mutations in genes encoding subunits of adaptor protein (AP)-3. Other genes mutated in rodent models include those encoding VPS33A and Rab38. Orthologs of all of these genes in Drosophila melanogaster belong to the 'granule group' of eye pigmentation genes. Other genes associated with HPS encode subunits of three complexes of unknown function, named biogenesis of lysosome-related organelles complex (BLOC)-1, -2 and -3, for which the Drosophila counterparts had not been characterized. Here, we report that the gene encoding the Drosophila ortholog of the HPS5 subunit of BLOC-2 is identical to the granule group gene pink (p), which was first studied in 1910 but had not been identified at the molecular level. The phenotype of pink mutants was exacerbated by mutations in AP-3 subunits or in the orthologs of VPS33A and Rab38. These results validate D. melanogaster as a genetic model to study the function of the BLOCs.

Staphylococci on ICE: Overlooked agents of horizontal gene transfer.

PubMed

Sansevere, Emily A; Robinson, D Ashley

2017-01-01

Horizontal gene transfer plays a significant role in spreading antimicrobial resistance and virulence genes throughout the genus Staphylococcus , which includes species of clinical relevance to humans and animals. While phages and plasmids are the most well-studied agents of horizontal gene transfer in staphylococci, the contribution of integrative conjugative elements (ICEs) has been mostly overlooked. Experimental work demonstrating the activity of ICEs in staphylococci remained frozen for years after initial work in the 1980s that showed Tn 916 was capable of transfer from Enterococcus to Staphylococcus . However, recent work has begun to thaw this field. To date, 2 families of ICEs have been identified among staphylococci - Tn 916 that includes the Tn 5801 subfamily, and ICE 6013 that includes at least 7 subfamilies. Both Tn 5801 and ICE 6013 commonly occur in clinical strains of S. aureus . Tn 5801 is the most studied of the Tn 916 family elements in staphylococci and encodes tetracycline resistance and a protein that, when expressed in Escherichia coli , inhibits restriction barriers to incoming DNA. ICE 6013 is among the shortest known ICEs, but it still includes many uncharacterized open reading frames. This element uses an IS 30 -like transposase as its recombinase, providing some versatility in integration sites. ICE 6013 also conjugatively transfers among receptive S. aureus strains at relatively higher frequency than Tn 5801 . Continued study of these mobile genetic elements may reveal the full extent to which ICEs impact horizontal gene transfer and the evolution of staphylococci.

[Molecular epidemiology of methicillin-resistant Staphylococcus aureus isolates with toxic shock syndrome toxin and staphylococcal enterotoxin C genes].

PubMed

Kim, Jae Seok; Kim, Han Sung; Song, Wonkeun; Cho, Hyoun Chan; Lee, Kyu Man; Kim, Eui Chong

2007-04-01

Many methicillin-resistant Staphylococcus aureus (MRSA) isolates in Korea possess a specific profile of staphylococcal enterotoxins in that the toxic shock syndrome toxin gene (tst) coexists with the staphylococcal enterotoxin C gene (sec). Because the analysis of staphylococcal cassette chromosome mec (SCCmec), a mobile genetic element mecA gene encoding methicillin resistance, showed that majority of these are SCCmec type II, these MRSA isolates with tst and sec may be genetically related with each other. This study was performed to investigate the genetic relatedness of tstand sec-harboring MRSA strains isolated in Korea by using pulsed-field gel electrophoresis (PFGE). A total of 59 strains of MRSA isolates of SCCmec type II possessing tst and sec were selected for PFGE and phylogenetic analyses. These isolates were collected from 13 health care facilities during nationwide surveillance of antimicrobial resistance in 2002. The 59 MRSA isolates were clustered into 11 PFGE types, including one major group of 26 strains (44.1%) isolated from 7 healthcare facilities. Seven PFGE types contained 2 or more isolates each, comprising 55 isolates in total. Most of SCCmec type II MRSA isolates containing tst and sec showed closely related PFGE patterns. Moreover, MRSA isolates collected from different healthcare facilities showed identical PFGE patterns. These findings suggested a clonal spread of MRSA strains possessing tst and sec in Korean hospitals.

Towards the discovery of novel genetic component involved in stress resistance in Arabidopsis thaliana.

PubMed

Juraniec, Michal; Lequeux, Hélène; Hermans, Christian; Willems, Glenda; Nordborg, Magnus; Schneeberger, Korbinian; Salis, Pietrino; Vromant, Maud; Lutts, Stanley; Verbruggen, Nathalie

2014-02-01

The exposure of plants to high concentrations of trace metallic elements such as copper involves a remodeling of the root system, characterized by a primary root growth inhibition and an increase in the lateral root density. These characteristics constitute easy and suitable markers for screening mutants altered in their response to copper excess. A forward genetic approach was undertaken in order to discover novel genetic factors involved in the response to copper excess. A Cu(2+) -sensitive mutant named copper modified resistance1 (cmr1) was isolated and a causative mutation in the CMR1 gene was identified by using positional cloning and next-generation sequencing. CMR1 encodes a plant-specific protein of unknown function. The analysis of the cmr1 mutant indicates that the CMR1 protein is required for optimal growth under normal conditions and has an essential role in the stress response. Impairment of the CMR1 activity alters root growth through aberrant activity of the root meristem, and modifies potassium concentration and hormonal balance (ethylene production and auxin accumulation). Our data support a putative role for CMR1 in cell division regulation and meristem maintenance. Research on the role of CMR1 will contribute to the understanding of the plasticity of plants in response to changing environments. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

Engineering Genetically Encoded FRET Sensors

PubMed Central

Lindenburg, Laurens; Merkx, Maarten

2014-01-01

Förster Resonance Energy Transfer (FRET) between two fluorescent proteins can be exploited to create fully genetically encoded and thus subcellularly targetable sensors. FRET sensors report changes in energy transfer between a donor and an acceptor fluorescent protein that occur when an attached sensor domain undergoes a change in conformation in response to ligand binding. The design of sensitive FRET sensors remains challenging as there are few generally applicable design rules and each sensor must be optimized anew. In this review we discuss various strategies that address this shortcoming, including rational design approaches that exploit self-associating fluorescent domains and the directed evolution of FRET sensors using high-throughput screening. PMID:24991940

The Genetics of Asthma and Allergic Disease: A 21st Century Perspective

PubMed Central

Ober, Carole; Yao, Tsung-Chieh

2011-01-01

Summary Asthma and allergy are common conditions with complex etiologies involving both genetic and environmental contributions. Recent genome-wide association studies (GWAS) and meta-analyses of GWAS have begun to shed light on both common and distinct pathways that contribute to asthma and allergic diseases. Associations with variation in genes encoding the epithelial cell-derived cytokines, interleukin-33 (IL-33) and thymic stromal lymphopoietin (TSLP), and the IL1RL1 gene encoding the IL-33 receptor, ST2, highlight the central roles for innate immune response pathways that promote the activation and differentiation of T-helper 2 (Th2) cells in the pathogenesis of both asthma and allergic diseases. In contrast, variation at the 17q21 asthma locus, encoding the ORMDL3 and GSDML genes, is specifically associated with risk for childhood onset asthma. These and other genetic findings are providing a list of well-validated asthma and allergy susceptibility genes that are expanding our understanding of the common and unique biological pathways that are dysregulated in these related conditions. Ongoing studies will continue to broaden our understanding of asthma and allergy and unravel the mechanisms for the development of these complex traits. PMID:21682736

Imaging of Intracellular pH in Tumor Spheroids Using Genetically Encoded Sensor SypHer2.

PubMed

Zagaynova, Elena V; Druzhkova, Irina N; Mishina, Natalia M; Ignatova, Nadezhda I; Dudenkova, Varvara V; Shirmanova, Marina V

2017-01-01

Intracellular pH (pHi) is one of the most important parameters that regulate the physiological state of cells and tissues. pHi homeostasis is crucial for normal cell functioning. Cancer cells are characterized by having a higher (neutral to slightly alkaline) pHi and lower (acidic) extracellular pH (pHe) compared to normal cells. This is referred to as a "reversed" pH gradient, and is essential in supporting their accelerated growth rate, invasion and migration, and in suppressing anti-tumor immunity, the promotion of metabolic coupling with fibroblasts and in preventing apoptosis. Moreover, abnormal pH, both pHi and pHe, contribute to drug resistance in cancers. Therefore, the development of methods for measuring pH in living tumor cells is likely to lead to better understanding of tumor biology and to open new ways for cancer treatment. Genetically encoded, fluorescent, pH-sensitive probes represent promising instruments enabling the subcellular measurement of pHi with unrivaled specificity and high accuracy. Here, we describe a protocol for pHi imaging at a microscopic level in HeLa tumor spheroids, using the genetically encoded ratiometric (dual-excitation) pHi indicator, SypHer2.

Voltage imaging to understand connections and functions of neuronal circuits.

PubMed

Antic, Srdjan D; Empson, Ruth M; Knöpfel, Thomas

2016-07-01

Understanding of the cellular mechanisms underlying brain functions such as cognition and emotions requires monitoring of membrane voltage at the cellular, circuit, and system levels. Seminal voltage-sensitive dye and calcium-sensitive dye imaging studies have demonstrated parallel detection of electrical activity across populations of interconnected neurons in a variety of preparations. A game-changing advance made in recent years has been the conceptualization and development of optogenetic tools, including genetically encoded indicators of voltage (GEVIs) or calcium (GECIs) and genetically encoded light-gated ion channels (actuators, e.g., channelrhodopsin2). Compared with low-molecular-weight calcium and voltage indicators (dyes), the optogenetic imaging approaches are 1) cell type specific, 2) less invasive, 3) able to relate activity and anatomy, and 4) facilitate long-term recordings of individual cells' activities over weeks, thereby allowing direct monitoring of the emergence of learned behaviors and underlying circuit mechanisms. We highlight the potential of novel approaches based on GEVIs and compare those to calcium imaging approaches. We also discuss how novel approaches based on GEVIs (and GECIs) coupled with genetically encoded actuators will promote progress in our knowledge of brain circuits and systems. Copyright © 2016 the American Physiological Society.

Voltage imaging to understand connections and functions of neuronal circuits

PubMed Central

Antic, Srdjan D.; Empson, Ruth M.

2016-01-01

Understanding of the cellular mechanisms underlying brain functions such as cognition and emotions requires monitoring of membrane voltage at the cellular, circuit, and system levels. Seminal voltage-sensitive dye and calcium-sensitive dye imaging studies have demonstrated parallel detection of electrical activity across populations of interconnected neurons in a variety of preparations. A game-changing advance made in recent years has been the conceptualization and development of optogenetic tools, including genetically encoded indicators of voltage (GEVIs) or calcium (GECIs) and genetically encoded light-gated ion channels (actuators, e.g., channelrhodopsin2). Compared with low-molecular-weight calcium and voltage indicators (dyes), the optogenetic imaging approaches are 1) cell type specific, 2) less invasive, 3) able to relate activity and anatomy, and 4) facilitate long-term recordings of individual cells' activities over weeks, thereby allowing direct monitoring of the emergence of learned behaviors and underlying circuit mechanisms. We highlight the potential of novel approaches based on GEVIs and compare those to calcium imaging approaches. We also discuss how novel approaches based on GEVIs (and GECIs) coupled with genetically encoded actuators will promote progress in our knowledge of brain circuits and systems. PMID:27075539

Auto-FPFA: An Automated Microscope for Characterizing Genetically Encoded Biosensors.

PubMed

Nguyen, Tuan A; Puhl, Henry L; Pham, An K; Vogel, Steven S

2018-05-09

Genetically encoded biosensors function by linking structural change in a protein construct, typically tagged with one or more fluorescent proteins, to changes in a biological parameter of interest (such as calcium concentration, pH, phosphorylation-state, etc.). Typically, the structural change triggered by alterations in the bio-parameter is monitored as a change in either fluorescent intensity, or lifetime. Potentially, other photo-physical properties of fluorophores, such as fluorescence anisotropy, molecular brightness, concentration, and lateral and/or rotational diffusion could also be used. Furthermore, while it is likely that multiple photo-physical attributes of a biosensor might be altered as a function of the bio-parameter, standard measurements monitor only a single photo-physical trait. This limits how biosensors are designed, as well as the accuracy and interpretation of biosensor measurements. Here we describe the design and construction of an automated multimodal-microscope. This system can autonomously analyze 96 samples in a micro-titer dish and for each sample simultaneously measure intensity (photon count), fluorescence lifetime, time-resolved anisotropy, molecular brightness, lateral diffusion time, and concentration. We characterize the accuracy and precision of this instrument, and then demonstrate its utility by characterizing three types of genetically encoded calcium sensors as well as a negative control.

Comparative performance of a genetically-encoded voltage indicator and a blue voltage sensitive dye for large scale cortical voltage imaging

PubMed Central

Mutoh, Hiroki; Mishina, Yukiko; Gallero-Salas, Yasir; Knöpfel, Thomas

2015-01-01

Traditional small molecule voltage sensitive dye indicators have been a powerful tool for monitoring large scale dynamics of neuronal activities but have several limitations including the lack of cell class specific targeting, invasiveness and difficulties in conducting longitudinal studies. Recent advances in the development of genetically-encoded voltage indicators have successfully overcome these limitations. Genetically-encoded voltage indicators (GEVIs) provide sufficient sensitivity to map cortical representations of sensory information and spontaneous network activities across cortical areas and different brain states. In this study, we directly compared the performance of a prototypic GEVI, VSFP2.3, with that of a widely used small molecule voltage sensitive dye (VSD), RH1691, in terms of their ability to resolve mesoscopic scale cortical population responses. We used three synchronized CCD cameras to simultaneously record the dual emission ratiometric fluorescence signal from VSFP2.3 and RH1691 fluorescence. The results show that VSFP2.3 offers more stable and less invasive recording conditions, while the signal-to-noise level and the response dynamics to sensory inputs are comparable to RH1691 recordings. PMID:25964738

Crystallization and preliminary X-ray characterization of the genetically encoded fluorescent calcium indicator protein GCaMP2

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

Rodríguez Guilbe, María M.; Protein Research and Development Center, University of Puerto Rico; Alfaro Malavé, Elisa C.

The genetically encoded fluorescent calcium-indicator protein GCaMP2 was crystallized in the calcium-saturated form. X-ray diffraction data were collected to 2.0 Å resolution and the structure was solved by molecular replacement. Fluorescent proteins and their engineered variants have played an important role in the study of biology. The genetically encoded calcium-indicator protein GCaMP2 comprises a circularly permuted fluorescent protein coupled to the calcium-binding protein calmodulin and a calmodulin target peptide, M13, derived from the intracellular calmodulin target myosin light-chain kinase and has been used to image calcium transients in vivo. To aid rational efforts to engineer improved variants of GCaMP2, thismore » protein was crystallized in the calcium-saturated form. X-ray diffraction data were collected to 2.0 Å resolution. The crystals belong to space group C2, with unit-cell parameters a = 126.1, b = 47.1, c = 68.8 Å, β = 100.5° and one GCaMP2 molecule in the asymmetric unit. The structure was phased by molecular replacement and refinement is currently under way.« less

Metabolic basis for the self-referential genetic code.

PubMed

Guimarães, Romeu Cardoso

2011-08-01

An investigation of the biosynthesis pathways producing glycine and serine was necessary to clarify an apparent inconsistency between the self-referential model (SRM) for the formation of the genetic code and the model of coevolution of encodings and of amino acid biosynthesis routes. According to the SRM proposal, glycine was the first amino acid encoded, followed by serine. The coevolution model does not state precisely which the first encodings were, only presenting a list of about ten early assignments including the derivation of glycine from serine-this being derived from the glycolysis intermediate glycerate, which reverses the order proposed by the self-referential model. Our search identified the glycine-serine pathway of syntheses based on one-carbon sources, involving activities of the glycine decarboxylase complex and its associated serine hydroxymethyltransferase, which is consistent with the order proposed by the self-referential model and supports its rationale for the origin of the genetic code: protein synthesis was developed inside an early metabolic system, serving the function of a sink of amino acids; the first peptides were glycine-rich and fit for the function of building the early ribonucleoproteins; glycine consumption in proteins drove the fixation of the glycine-serine pathway.

Genetically encoded multispectral labeling of proteins with polyfluorophores on a DNA backbone.

PubMed

Singh, Vijay; Wang, Shenliang; Chan, Ke Min; Clark, Spencer A; Kool, Eric T

2013-04-24

Genetically encoded methods for protein conjugation are of high importance as biological tools. Here we describe the development of a new class of dyes for genetically encoded tagging that add new capabilities for protein reporting and detection via HaloTag methodology. Oligodeoxyfluorosides (ODFs) are short DNA-like oligomers in which the natural nucleic acid bases are replaced by interacting fluorescent chromophores, yielding a broad range of emission colors using a single excitation wavelength. We describe the development of an alkyl halide dehalogenase-compatible chloroalkane linker phosphoramidite derivative that enables the rapid automated synthesis of many possible dyes for protein conjugation. Experiments to test the enzymatic self-conjugation of nine different DNA-like dyes to proteins with HaloTag domains in vitro were performed, and the data confirmed the rapid and efficient covalent labeling of the proteins. Notably, a number of the ODF dyes were found to increase in brightness or change color upon protein conjugation. Tests in mammalian cellular settings revealed that the dyes are functional in multiple cellular contexts, both on the cell surface and within the cytoplasm, allowing protein localization to be imaged in live cells by epifluorescence and laser confocal microscopy.

Evolution of Hsp70 Gene Expression: A Role for Changes in AT-Richness within Promoters

PubMed Central

Ma, Ronghui; Zhang, Bo; Kang, Le

2011-01-01

In disparate organisms adaptation to thermal stress has been linked to changes in the expression of genes encoding heat-shock proteins (Hsp). The underlying genetics, however, remain elusive. We show here that two AT-rich sequence elements in the promoter region of the hsp70 gene of the fly Liriomyza sativae that are absent in the congeneric species, Liriomyza huidobrensis, have marked cis-regulatory consequences. We studied the cis-regulatory consequences of these elements (called ATRS1 and ATRS2) by measuring the constitutive and heat-shock-induced luciferase luminescence that they drive in cells transfected with constructs carrying them modified, deleted, or intact, in the hsp70 promoter fused to the luciferase gene. The elements affected expression level markedly and in different ways: Deleting ATRS1 augmented both the constitutive and the heat-shock-induced luminescence, suggesting that this element represses transcription. Interestingly, replacing the element with random sequences of the same length and A+T content delivered the wild-type luminescence pattern, proving that the element's high A+T content is crucial for its effects. Deleting ATRS2 decreased luminescence dramatically and almost abolished heat-shock inducibility and so did replacing the element with random sequences matching the element's length and A+T content, suggesting that ATRS2's effects on transcription and heat-shock inducibility involve a common mechanism requiring at least in part the element's specific primary structure. Finally, constitutive and heat-shock luminescence were reduced strongly when two putative binding sites for the Zeste transcription factor identified within ATRS2 were altered through site-directed mutagenesis, and the heat-shock-induced luminescence increased when Zeste was over-expressed, indicating that Zeste participates in the effects mapped to ATRS2 at least in part. AT-rich sequences are common in promoters and our results suggest that they should play important roles in regulatory evolution since they can affect expression markedly and constrain promoter DNA in at least two different ways. PMID:21655251

Performance evaluation of matrix gradient coils.

PubMed

Jia, Feng; Schultz, Gerrit; Testud, Frederik; Welz, Anna Masako; Weber, Hans; Littin, Sebastian; Yu, Huijun; Hennig, Jürgen; Zaitsev, Maxim

2016-02-01

In this paper, we present a new performance measure of a matrix coil (also known as multi-coil) from the perspective of efficient, local, non-linear encoding without explicitly considering target encoding fields. An optimization problem based on a joint optimization for the non-linear encoding fields is formulated. Based on the derived objective function, a figure of merit of a matrix coil is defined, which is a generalization of a previously known resistive figure of merit for traditional gradient coils. A cylindrical matrix coil design with a high number of elements is used to illustrate the proposed performance measure. The results are analyzed to reveal novel features of matrix coil designs, which allowed us to optimize coil parameters, such as number of coil elements. A comparison to a scaled, existing multi-coil is also provided to demonstrate the use of the proposed performance parameter. The assessment of a matrix gradient coil profits from using a single performance parameter that takes the local encoding performance of the coil into account in relation to the dissipated power.

Spatially Fourier-encoded photoacoustic microscopy using a digital micromirror device.

PubMed

Liang, Jinyang; Gao, Liang; Li, Chiye; Wang, Lihong V

2014-02-01

We have developed spatially Fourier-encoded photoacoustic (PA) microscopy using a digital micromirror device. The spatial intensity distribution of laser pulses is Fourier-encoded, and a series of such encoded PA measurements allows one to decode the spatial distribution of optical absorption. The throughput and Fellgett advantages were demonstrated by imaging a chromium target. By using 63 spatial elements, the signal-to-noise ratio in the recovered PA signal was enhanced by ∼4×. The system was used to image two biological targets, a monolayer of red blood cells and melanoma cells.

Spatially Fourier-encoded photoacoustic microscopy using a digital micromirror device

PubMed Central

Liang, Jinyang; Gao, Liang; Li, Chiye; Wang, Lihong V.

2014-01-01

We have developed spatially Fourier-encoded photoacoustic microscopy using a digital micromirror device. The spatial intensity distribution of laser pulses is Fourier-encoded, and a series of such encoded photoacoustic measurements allows one to decode the spatial distribution of optical absorption. The throughput and Fellgett advantages were demonstrated by imaging a chromium target. By using 63 spatial elements, the signal-to-noise ratio in the recovered photoacoustic signal was enhanced by ~4×. The system was used to image two biological targets, a monolayer of red blood cells and melanoma cells. PMID:24487832

Defense Against Cannibalism: The SdpI Family of Bacterial Immunity/Signal Transduction Proteins

PubMed Central

Povolotsky, Tatyana Leonidovna; Orlova, Ekaterina; Tamang, Dorjee G.

2010-01-01

The SdpI family consists of putative bacterial toxin immunity and signal transduction proteins. One member of the family in Bacillus subtilis, SdpI, provides immunity to cells from cannibalism in times of nutrient limitation. SdpI family members are transmembrane proteins with 3, 4, 5, 6, 7, 8, or 12 putative transmembrane α-helical segments (TMSs). These varied topologies appear to be genuine rather than artifacts due to sequencing or annotation errors. The basic and most frequently occurring element of the SdpI family has 6 TMSs. Homologues of all topological types were aligned to determine the homologous TMSs and loop regions, and the positive-inside rule was used to determine sidedness. The two most conserved motifs were identified between TMSs 1 and 2 and TMSs 4 and 5 of the 6 TMS proteins. These showed significant sequence similarity, leading us to suggest that the primordial precursor of these proteins was a 3 TMS–encoding genetic element that underwent intragenic duplication. Various deletional and fusional events, as well as intragenic duplications and inversions, may have yielded SdpI homologues with topologies of varying numbers and positions of TMSs. We propose a specific evolutionary pathway that could have given rise to these distantly related bacterial immunity proteins. We further show that genes encoding SdpI homologues often appear in operons with genes for homologues of SdpR, SdpI’s autorepressor. Our analyses allow us to propose structure–function relationships that may be applicable to most family members. Electronic supplementary material The online version of this article (doi:10.1007/s00232-010-9260-7) contains supplementary material, which is available to authorized users. PMID:20563570

Translational Redefinition of UGA Codons Is Regulated by Selenium Availability*

PubMed Central

Howard, Michael T.; Carlson, Bradley A.; Anderson, Christine B.; Hatfield, Dolph L.

2013-01-01

Incorporation of selenium into ∼25 mammalian selenoproteins occurs by translational recoding whereby in-frame UGA codons are redefined to encode the selenium containing amino acid, selenocysteine (Sec). Here we applied ribosome profiling to examine the effect of dietary selenium levels on the translational mechanisms controlling selenoprotein synthesis in mouse liver. Dietary selenium levels were shown to control gene-specific selenoprotein expression primarily at the translation level by differential regulation of UGA redefinition and Sec incorporation efficiency, although effects on translation initiation and mRNA abundance were also observed. Direct evidence is presented that increasing dietary selenium causes a vast increase in ribosome density downstream of UGA-Sec codons for a subset of selenoprotein mRNAs and that the selenium-dependent effects on Sec incorporation efficiency are mediated in part by the degree of Sec-tRNA[Ser]Sec Um34 methylation. Furthermore, we find evidence for translation in the 5′-UTRs for a subset of selenoproteins and for ribosome pausing near the UGA-Sec codon in those mRNAs encoding the selenoproteins most affected by selenium availability. These data illustrate how dietary levels of the trace element selenium can alter the readout of the genetic code to affect the expression of an entire class of proteins. PMID:23696641

batman Interacts with polycomb and trithorax group genes and encodes a BTB/POZ protein that is included in a complex containing GAGA factor.

PubMed

Faucheux, M; Roignant, J-Y; Netter, S; Charollais, J; Antoniewski, C; Théodore, L

2003-02-01

Polycomb and trithorax group genes maintain the appropriate repressed or activated state of homeotic gene expression throughout Drosophila melanogaster development. We have previously identified the batman gene as a Polycomb group candidate since its function is necessary for the repression of Sex combs reduced. However, our present genetic analysis indicates functions of batman in both activation and repression of homeotic genes. The 127-amino-acid Batman protein is almost reduced to a BTB/POZ domain, an evolutionary conserved protein-protein interaction domain found in a large protein family. We show that this domain is involved in the interaction between Batman and the DNA binding GAGA factor encoded by the Trithorax-like gene. The GAGA factor and Batman codistribute on polytene chromosomes, coimmunoprecipitate from nuclear embryonic and larval extracts, and interact in the yeast two-hybrid assay. Batman, together with the GAGA factor, binds to MHS-70, a 70-bp fragment of the bithoraxoid Polycomb response element. This binding, like that of the GAGA factor, requires the presence of d(GA)n sequences. Together, our results suggest that batman belongs to a subset of the Polycomb/trithorax group of genes that includes Trithorax-like, whose products are involved in both activation and repression of homeotic genes.

Disruption of the nitrogen regulatory gene AcareA in Acremonium chrysogenum leads to reduction of cephalosporin production and repression of nitrogen metabolism.

PubMed

Li, Jinyang; Pan, Yuanyuan; Liu, Gang

2013-12-01

AcareA, encoding a homologue of the fungal nitrogen regulatory GATA zinc-finger proteins, was cloned from Acremonium chrysogenum. Gene disruption and genetic complementation revealed that AcareA was required for nitrogen metabolism and cephalosporin production. Disruption of AcareA resulted in growth defect in the medium using nitrate, uric acid and low concentration of ammonium, glutamine or urea as sole nitrogen source. Transcriptional analysis showed that the transcription of niaD/niiA was increased drastically when induced with nitrate in the wild-type and AcareA complemented strains but not in AcareA disruption mutant. Consistent with the reduction of cephalosporin production, the transcription of pcbAB, cefD2, cefEF and cefG encoding the enzymes for cephalosporin production was reduced in AcareA disruption mutant. Band shift assays showed that AcAREA bound to the promoter regions of niaD, niiA and the bidirectional promoter region of pcbAB-pcbC. Sequence analysis showed that all the AcAREA binding sites contain the consensus GATA elements. These results indicated that AcAREA plays an important role both in the regulation of nitrogen metabolism and cephalosporin production in A. chrysogenum. Copyright © 2013 Elsevier Inc. All rights reserved.

batman Interacts with Polycomb and trithorax Group Genes and Encodes a BTB/POZ Protein That Is Included in a Complex Containing GAGA Factor

PubMed Central

Faucheux, M.; Roignant, J.-Y.; Netter, S.; Charollais, J.; Antoniewski, C.; Théodore, L.

2003-01-01

Polycomb and trithorax group genes maintain the appropriate repressed or activated state of homeotic gene expression throughout Drosophila melanogaster development. We have previously identified the batman gene as a Polycomb group candidate since its function is necessary for the repression of Sex combs reduced. However, our present genetic analysis indicates functions of batman in both activation and repression of homeotic genes. The 127-amino-acid Batman protein is almost reduced to a BTB/POZ domain, an evolutionary conserved protein-protein interaction domain found in a large protein family. We show that this domain is involved in the interaction between Batman and the DNA binding GAGA factor encoded by the Trithorax-like gene. The GAGA factor and Batman codistribute on polytene chromosomes, coimmunoprecipitate from nuclear embryonic and larval extracts, and interact in the yeast two-hybrid assay. Batman, together with the GAGA factor, binds to MHS-70, a 70-bp fragment of the bithoraxoid Polycomb response element. This binding, like that of the GAGA factor, requires the presence of d(GA)n sequences. Together, our results suggest that batman belongs to a subset of the Polycomb/trithorax group of genes that includes Trithorax-like, whose products are involved in both activation and repression of homeotic genes. PMID:12556479

An Archaeal Immune System Can Detect Multiple Protospacer Adjacent Motifs (PAMs) to Target Invader DNA*

PubMed Central

Fischer, Susan; Maier, Lisa-Katharina; Stoll, Britta; Brendel, Jutta; Fischer, Eike; Pfeiffer, Friedhelm; Dyall-Smith, Mike; Marchfelder, Anita

2012-01-01

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system provides adaptive and heritable immunity against foreign genetic elements in most archaea and many bacteria. Although this system is widespread and diverse with many subtypes, only a few species have been investigated to elucidate the precise mechanisms for the defense of viruses or plasmids. Approximately 90% of all sequenced archaea encode CRISPR/Cas systems, but their molecular details have so far only been examined in three archaeal species: Sulfolobus solfataricus, Sulfolobus islandicus, and Pyrococcus furiosus. Here, we analyzed the CRISPR/Cas system of Haloferax volcanii using a plasmid-based invader assay. Haloferax encodes a type I-B CRISPR/Cas system with eight Cas proteins and three CRISPR loci for which the identity of protospacer adjacent motifs (PAMs) was unknown until now. We identified six different PAM sequences that are required upstream of the protospacer to permit target DNA recognition. This is only the second archaeon for which PAM sequences have been determined, and the first CRISPR group with such a high number of PAM sequences. Cells could survive the plasmid challenge if their CRISPR/Cas system was altered or defective, e.g. by deletion of the cas gene cassette. Experimental PAM data were supplemented with bioinformatics data on Haloferax and Haloquadratum. PMID:22767603

Efflux pumps as antimicrobial resistance mechanisms.

PubMed

Poole, Keith

2007-01-01

Antibiotic resistance continues to hamper antimicrobial chemotherapy of infectious disease, and while biocide resistance outside of the laboratory is as yet unrealized, in vitro and in vivo episodes of reduced biocide susceptibility are not uncommon. Efflux mechanisms, both drug-specific and multidrug, are important determinants of intrinsic and/or acquired resistance to these antimicrobials in important human pathogens. Multidrug efflux mechanisms are generally chromosome-encoded, with their expression typically resultant from mutations in regulatory genes, while drug-specific efflux mechanisms are encoded by mobile genetic elements whose acquisition is sufficient for resistance. While it has been suggested that drug-specific efflux systems originated from efflux determinants of self-protection in antibiotic-producing Actinomycetes, chromosomal multidrug efflux determinants, at least in Gram-negative bacteria, are appreciated as having an intended housekeeping function unrelated to drug export and resistance. Thus, it will be important to elucidate the intended natural function of these efflux mechanisms in order, for example, to anticipate environmental conditions or circumstances that might promote their expression and, so, compromise antimicrobial chemotherapy. Given the clinical significance of antimicrobial exporters, it is clear that efflux must be considered in formulating strategies for treatment of drug-resistant infections, both in the development of new agents, for example, less impacted by efflux or in targeting efflux directly with efflux inhibitors.

An Extracytoplasmic Function Sigma Factor-Mediated Cell Surface Signaling System in Pseudomonas syringae pv. tomato DC3000 Regulates Gene Expression in Response to Heterologous Siderophores ▿ †

PubMed Central

Markel, Eric; Maciak, Charlene; Butcher, Bronwyn G.; Myers, Christopher R.; Stodghill, Paul; Bao, Zhongmeng; Cartinhour, Sam; Swingle, Bryan

2011-01-01

The diversity of regulatory systems encoded by bacteria provides an indication of the variety of stresses and interactions that these organisms encounter in nature. We have been investigating how the plant pathogen Pseudomonas syringae pv. tomato DC3000 responds to iron limitation and have focused on the iron starvation (IS) sigma factors to identify regulon members and to explore the mechanistic details of genetic control for this class of regulators. In the study described in this report, we used chromatin immunoprecipitation paired with high-throughput sequencing (ChIP-Seq) to screen the genome for locations associated with binding of the P. syringae IS sigma factor PSPTO_1203. We used multiple methods to demonstrate differential regulation of two genes identified in the ChIP-Seq screen and characterize the promoter elements that facilitate PSPTO_1203-dependent regulation. The genes regulated by PSPTO_1203 encode a TonB-dependent transducer (PSPTO_1206) and a cytoplasmic membrane protein (PSPTO_2145), which is located in the P. syringae pyoverdine cluster. Additionally, we identified siderophores that induce the activity of PSPTO_1203 and used this information to investigate the functional components of the signal transduction cascade. PMID:21840980

Antibiotic resistance in Staphylococcus aureus. Current status and future prospects.

PubMed

Foster, Timothy J

2017-05-01

The major targets for antibiotics in staphylococci are (i) the cell envelope, (ii) the ribosome and (iii) nucleic acids. Several novel targets emerged from recent targeted drug discovery programmes including the ClpP protease and FtsZ from the cell division machinery. Resistance can either develop by horizontal transfer of resistance determinants encoded by mobile genetic elements viz plasmids, transposons and the staphylococcal cassette chromosome or by mutations in chromosomal genes. Horizontally acquired resistance can occur by one of the following mechanisms: (i) enzymatic drug modification and inactivation, (ii) enzymatic modification of the drug binding site, (iii) drug efflux, (iv) bypass mechanisms involving acquisition of a novel drug-resistant target, (v) displacement of the drug to protect the target. Acquisition of resistance by mutation can result from (i) alteration of the drug target that prevents the inhibitor from binding, (ii) derepression of chromosomally encoded multidrug resistance efflux pumps and (iii) multiple stepwise mutations that alter the structure and composition of the cell wall and/or membrane to reduce drug access to its target. This review focuses on development of resistance to currently used antibiotics and examines future prospects for new antibiotics and informed use of drug combinations. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The paradox of MHC-DRB exon/intron evolution: alpha-helix and beta-sheet encoding regions diverge while hypervariable intronic simple repeats coevolve with beta-sheet codons.

PubMed

Schwaiger, F W; Weyers, E; Epplen, C; Brün, J; Ruff, G; Crawford, A; Epplen, J T

1993-09-01

Twenty-one different caprine and 13 ovine MHC-DRB exon 2 sequences were determined including part of the adjacent introns containing simple repetitive (gt)n(ga)m elements. The positions for highly polymorphic DRB amino acids vary slightly among ungulates and other mammals. From man and mouse to ungulates the basic (gt)n(ga)m structure is fixed in evolution for 7 x 10(7) years whereas ample variations exist in the tandem (gt)n and (ga)m dinucleotides and especially their "degenerated" derivatives. Phylogenetic trees for the alpha-helices and beta-pleated sheets of the ungulate DRB sequences suggest different evolutionary histories. In hoofed animals as well as in humans DRB beta-sheet encoding sequences and adjacent intronic repeats can be assembled into virtually identical groups suggesting coevolution of noncoding as well as coding DNA. In contrast alpha-helices and C-terminal parts of the first DRB domain evolve distinctly. In the absence of a defined mechanism causing specific, site-directed mutations, double-recombination or gene-conversion-like events would readily explain this fact. The role of the intronic simple (gt)n(ga)m repeat is discussed with respect to these genetic exchange mechanisms during evolution.

Tyramine and phenylethylamine biosynthesis by food bacteria.

PubMed

Marcobal, Angela; De las Rivas, Blanca; Landete, José María; Tabera, Laura; Muñoz, Rosario

2012-01-01

Tyramine poisoning is caused by the ingestion of food containing high levels of tyramine, a biogenic amine. Any foods containing free tyrosine are subject to tyramine formation if poor sanitation and low quality foods are used or if the food is subject to temperature abuse or extended storage time. Tyramine is generated by decarboxylation of the tyrosine through tyrosine decarboxylase (TDC) enzymes derived from the bacteria present in the food. Bacterial TDC have been only unequivocally identified and characterized in Gram-positive bacteria, especially in lactic acid bacteria. Pyridoxal phosphate (PLP)-dependent TDC encoding genes (tyrDC) appeared flanked by a similar genetic organization in several species of lactic acid bacteria, suggesting a common origin by a single mobile genetic element. Bacterial TDC are also able to decarboxylate phenylalanine to produce phenylethylamine (PEA), another biogenic amine. The molecular knowledge of the genes involved in tyramine production has led to the development of molecular methods for the detection of bacteria able to produce tyramine and PEA. These rapid and simple methods could be used for the analysis of the ability to form tyramine by bacteria in order to evaluate the potential risk of tyramine biosynthesis in food products.

Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site.

PubMed

Hatoum-Aslan, Asma; Maniv, Inbal; Marraffini, Luciano A

2011-12-27

Precise RNA processing is fundamental to all small RNA-mediated interference pathways. In prokaryotes, clustered, regularly interspaced, short palindromic repeats (CRISPR) loci encode small CRISPR RNAs (crRNAs) that protect against invasive genetic elements by antisense targeting. CRISPR loci are transcribed as a long precursor that is cleaved within repeat sequences by CRISPR-associated (Cas) proteins. In many organisms, this primary processing generates crRNA intermediates that are subject to additional nucleolytic trimming to render mature crRNAs of specific lengths. The molecular mechanisms underlying this maturation event remain poorly understood. Here, we defined the genetic requirements for crRNA primary processing and maturation in Staphylococcus epidermidis. We show that changes in the position of the primary processing site result in extended or diminished maturation to generate mature crRNAs of constant length. These results indicate that crRNA maturation occurs by a ruler mechanism anchored at the primary processing site. We also show that maturation is mediated by specific cas genes distinct from those genes involved in primary processing, showing that this event is directed by CRISPR/Cas loci.

tRNA acceptor-stem and anticodon bases embed separate features of amino acid chemistry

PubMed Central

Carter, Charles W.; Wolfenden, Richard

2016-01-01

abstract The universal genetic code is a translation table by which nucleic acid sequences can be interpreted as polypeptides with a wide range of biological functions. That information is used by aminoacyl-tRNA synthetases to translate the code. Moreover, amino acid properties dictate protein folding. We recently reported that digital correlation techniques could identify patterns in tRNA identity elements that govern recognition by synthetases. Our analysis, and the functionality of truncated synthetases that cannot recognize the tRNA anticodon, support the conclusion that the tRNA acceptor stem houses an independent code for the same 20 amino acids that likely functioned earlier in the emergence of genetics. The acceptor-stem code, related to amino acid size, is distinct from a code in the anticodon that is related to amino acid polarity. Details of the acceptor-stem code suggest that it was useful in preserving key properties of stereochemically-encoded peptides that had developed the capacity to interact catalytically with RNA. The quantitative embedding of the chemical properties of amino acids into tRNA bases has implications for the origins of molecular biology. PMID:26595350

An Efficient and Versatile System for Visualization and Genetic Modification of Dopaminergic Neurons in Transgenic Mice

PubMed Central

Kramer, Edgar R.

2015-01-01

Background & Aims The brain dopaminergic (DA) system is involved in fine tuning many behaviors and several human diseases are associated with pathological alterations of the DA system such as Parkinson’s disease (PD) and drug addiction. Because of its complex network integration, detailed analyses of physiological and pathophysiological conditions are only possible in a whole organism with a sophisticated tool box for visualization and functional modification. Methods & Results Here, we have generated transgenic mice expressing the tetracycline-regulated transactivator (tTA) or the reverse tetracycline-regulated transactivator (rtTA) under control of the tyrosine hydroxylase (TH) promoter, TH-tTA (tet-OFF) and TH-rtTA (tet-ON) mice, to visualize and genetically modify DA neurons. We show their tight regulation and efficient use to overexpress proteins under the control of tet-responsive elements or to delete genes of interest with tet-responsive Cre. In combination with mice encoding tet-responsive luciferase, we visualized the DA system in living mice progressively over time. Conclusion These experiments establish TH-tTA and TH-rtTA mice as a powerful tool to generate and monitor mouse models for DA system diseases. PMID:26291828

Genetic and biochemical characterization of an acquired subgroup B3 metallo-β-lactamase gene, blaAIM-1, and its unique genetic context in Pseudomonas aeruginosa from Australia.

PubMed

Yong, Dongeun; Toleman, Mark A; Bell, Jan; Ritchie, Brett; Pratt, Rachael; Ryley, Henry; Walsh, Timothy R

2012-12-01

Three clinical Pseudomonas aeruginosa isolates (WCH2677, WCH2813, and WCH2837) isolated from the Women's and Children's Hospital, Adelaide, Australia, produced a metallo-β-lactamase (MBL)-positive Etest result. All isolates were PCR negative for known MBL genes. A gene bank was created, and an MBL gene, designated bla(AIM-1), was cloned and fully characterized. The encoded enzyme, AIM-1, is a group B3 MBL that has the highest level of identity to THIN-B and L1. It is chromosomal and flanked by two copies (one intact and one truncated) of an ISCR element, ISCR15. Southern hybridization studies indicated the movement of both ISCR15 and bla(AIM-1) within the three different clinical isolates. AIM-1 hydrolyzes most β-lactams, with the exception of aztreonam and, to a lesser extent, ceftazidime; however, it possesses significantly higher k(cat) values for cefepime and carbapenems than most other MBLs. AIM-1 was the first mobile group B3 enzyme detected and signals further problems for already beleaguered antimicrobial regimes to treat serious P. aeruginosa and other Gram-negative infections.

Chromosome-level genome map provides insights into diverse defense mechanisms in the medicinal fungus Ganoderma sinense

PubMed Central

Zhu, Yingjie; Xu, Jiang; Sun, Chao; Zhou, Shiguo; Xu, Haibin; Nelson, David R.; Qian, Jun; Song, Jingyuan; Luo, Hongmei; Xiang, Li; Li, Ying; Xu, Zhichao; Ji, Aijia; Wang, Lizhi; Lu, Shanfa; Hayward, Alice; Sun, Wei; Li, Xiwen; Schwartz, David C.; Wang, Yitao; Chen, Shilin

2015-01-01

Fungi have evolved powerful genomic and chemical defense systems to protect themselves against genetic destabilization and other organisms. However, the precise molecular basis involved in fungal defense remain largely unknown in Basidiomycetes. Here the complete genome sequence, as well as DNA methylation patterns and small RNA transcriptomes, was analyzed to provide a holistic overview of secondary metabolism and defense processes in the model medicinal fungus, Ganoderma sinense. We reported the 48.96 Mb genome sequence of G. sinense, consisting of 12 chromosomes and encoding 15,688 genes. More than thirty gene clusters involved in the biosynthesis of secondary metabolites, as well as a large array of genes responsible for their transport and regulation were highlighted. In addition, components of genome defense mechanisms, namely repeat-induced point mutation (RIP), DNA methylation and small RNA-mediated gene silencing, were revealed in G. sinense. Systematic bioinformatic investigation of the genome and methylome suggested that RIP and DNA methylation combinatorially maintain G. sinense genome stability by inactivating invasive genetic material and transposable elements. The elucidation of the G. sinense genome and epigenome provides an unparalleled opportunity to advance our understanding of secondary metabolism and fungal defense mechanisms. PMID:26046933

Korean, Japanese, and Chinese populations featured similar genes encoding drug-metabolizing enzymes and transporters: a DMET Plus microarray assessment.

PubMed

Yi, SoJeong; An, Hyungmi; Lee, Howard; Lee, Sangin; Ieiri, Ichiro; Lee, Youngjo; Cho, Joo-Youn; Hirota, Takeshi; Fukae, Masato; Yoshida, Kenji; Nagatsuka, Shinichiro; Kimura, Miyuki; Irie, Shin; Sugiyama, Yuichi; Shin, Dong Wan; Lim, Kyoung Soo; Chung, Jae-Yong; Yu, Kyung-Sang; Jang, In-Jin

2014-10-01

Interethnic differences in genetic polymorphism in genes encoding drug-metabolizing enzymes and transporters are one of the major factors that cause ethnic differences in drug response. This study aimed to investigate genetic polymorphisms in genes involved in drug metabolism, transport, and excretion among Korean, Japanese, and Chinese populations, the three major East Asian ethnic groups. The frequencies of 1936 variants representing 225 genes encoding drug-metabolizing enzymes and transporters were determined from 786 healthy participants (448 Korean, 208 Japanese, and 130 Chinese) using the Affymetrix Drug-Metabolizing Enzymes and Transporters Plus microarray. To compare allele or genotype frequencies in the high-dimensional data among the three East Asian ethnic groups, multiple testing, principal component analysis (PCA), and regularized multinomial logit model through least absolute shrinkage and selection operator were used. On microarray analysis, 1071 of 1936 variants (>50% of markers) were found to be monomorphic. In a large number of genetic variants, the fixation index and Pearson's correlation coefficient of minor allele frequencies were less than 0.034 and greater than 0.95, respectively, among the three ethnic groups. PCA identified 47 genetic variants with multiple testing, but was unable to discriminate ethnic groups by the first three components. Multinomial least absolute shrinkage and selection operator analysis identified 269 genetic variants that showed different frequencies among the three ethnic groups. However, none of those variants distinguished between the three ethnic groups during subsequent PCA. Korean, Japanese, and Chinese populations are not pharmacogenetically distant from one another, at least with regard to drug disposition, metabolism, and elimination.

Modularization of genetic elements promotes synthetic metabolic engineering.

PubMed

Qi, Hao; Li, Bing-Zhi; Zhang, Wen-Qian; Liu, Duo; Yuan, Ying-Jin

2015-11-15

In the context of emerging synthetic biology, metabolic engineering is moving to the next stage powered by new technologies. Systematical modularization of genetic elements makes it more convenient to engineer biological systems for chemical production or other desired purposes. In the past few years, progresses were made in engineering metabolic pathway using synthetic biology tools. Here, we spotlighted the topic of implementation of modularized genetic elements in metabolic engineering. First, we overviewed the principle developed for modularizing genetic elements and then discussed how the genetic modules advanced metabolic engineering studies. Next, we picked up some milestones of engineered metabolic pathway achieved in the past few years. Last, we discussed the rapid raised synthetic biology field of "building a genome" and the potential in metabolic engineering. Copyright © 2015 Elsevier Inc. All rights reserved.

γ-PGA Hydrolases of Phage Origin in Bacillus subtilis and Other Microbial Genomes.

PubMed

Mamberti, Stefania; Prati, Paola; Cremaschi, Paolo; Seppi, Claudio; Morelli, Carlo F; Galizzi, Alessandro; Fabbi, Massimo; Calvio, Cinzia

2015-01-01

Poly-γ-glutamate (γ-PGA) is an industrially interesting polymer secreted mainly by members of the class Bacilli which forms a shield able to protect bacteria from phagocytosis and phages. Few enzymes are known to degrade γ-PGA; among them is a phage-encoded γ-PGA hydrolase, PghP. The supposed role of PghP in phages is to ensure access to the surface of bacterial cells by dismantling the γ-PGA barrier. We identified four unannotated B. subtilis genes through similarity of their encoded products to PghP; in fact these genes reside in prophage elements of B. subtilis genome. The recombinant products of two of them demonstrate efficient polymer degradation, confirming that sequence similarity reflects functional homology. Genes encoding similar γ-PGA hydrolases were identified in phages specific for the order Bacillales and in numerous microbial genomes, not only belonging to that order. The distribution of the γ-PGA biosynthesis operon was also investigated with a bioinformatics approach; it was found that the list of organisms endowed with γ-PGA biosynthetic functions is larger than expected and includes several pathogenic species. Moreover in non-Bacillales bacteria the predicted γ-PGA hydrolase genes are preferentially found in species that do not have the genetic asset for polymer production. Our findings suggest that γ-PGA hydrolase genes might have spread across microbial genomes via horizontal exchanges rather than via phage infection. We hypothesize that, in natural habitats rich in γ-PGA supplied by producer organisms, the availability of hydrolases that release glutamate oligomers from γ-PGA might be a beneficial trait under positive selection.

Different Alleles of a Gene Encoding Leucoanthocyanidin Reductase (PaLAR3) Influence Resistance against the Fungus Heterobasidion parviporum in Picea abies1

PubMed Central

Ihrmark, Katarina

2016-01-01

Despite the fact that fungal diseases are a growing menace for conifers in modern silviculture, only a very limited number of molecular markers for pathogen resistance have been validated in conifer species. A previous genetic study indicated that the resistance of Norway spruce (Picea abies) to Heterobasidion annosum s.l., a pathogenic basidiomycete species complex, is linked to a quantitative trait loci that associates with differences in fungal growth in sapwood (FGS) that includes a gene, PaLAR3, which encodes a leucoanthocyanidin reductase. In this study, gene sequences showed the presence of two PaLAR3 allelic lineages in P. abies. Higher resistance was associated with the novel allele, which was found in low frequency in the four P. abies populations that we studied. Norway spruce plants carrying at least one copy of the novel allele showed a significant reduction in FGS after inoculation with Heterobasidion parviporum compared to their half-siblings carrying no copies, indicating dominance of this allele. The amount of (+) catechin, the enzymatic product of PaLAR3, was significantly higher in bark of trees homozygous for the novel allele. Although we observed that the in vitro activities of the enzymes encoded by the two alleles were similar, we could show that allele-specific transcript levels were significantly higher for the novel allele, indicating that regulation of gene expression is responsible for the observed effects in resistance, possibly caused by differences in cis-acting elements that we observe in the promoter region of the two alleles. PMID:27317690

An antisense RNA in a lytic cyanophage links psbA to a gene encoding a homing endonuclease.

PubMed

Millard, Andrew D; Gierga, Gregor; Clokie, Martha R J; Evans, David J; Hess, Wolfgang R; Scanlan, David J

2010-09-01

Cyanophage genomes frequently possess the psbA gene, encoding the D1 polypeptide of photosystem II. This protein is believed to maintain host photosynthetic capacity during infection and enhance phage fitness under high-light conditions. Although the first documented cyanophage-encoded psbA gene contained a group I intron, this feature has not been widely reported since, despite a plethora of new sequences becoming available. In this study, we show that in cyanophage S-PM2, this intron is spliced during the entire infection cycle. Furthermore, we report the widespread occurrence of psbA introns in marine metagenomic libraries, and with psbA often adjacent to a homing endonuclease (HE). Bioinformatic analysis of the intergenic region between psbA and the adjacent HE gene F-CphI in S-PM2 showed the presence of an antisense RNA (asRNA) connecting these two separate genetic elements. The asRNA is co-regulated with psbA and F-CphI, suggesting its involvement with their expression. Analysis of scaffolds from global ocean survey datasets shows this asRNA to be commonly associated with the 3' end of cyanophage psbA genes, implying that this potential mechanism of regulating marine 'viral' photosynthesis is evolutionarily conserved. Although antisense transcription is commonly found in eukaryotic and increasingly also in prokaryotic organisms, there has been no indication for asRNAs in lytic phages so far. We propose that this asRNA also provides a means of preventing the formation of mobile group I introns within cyanophage psbA genes.

Genes encoding a callose synthase and phytochrome A are adjacent to a MAP3Ka-like gene in Beta vulgaris USH20

USDA-ARS?s Scientific Manuscript database

MAP3Ka encodes a key conserved protein kinase responsible for orchestrating a rapid cascade of cellular events ultimately leading to localized cell death. Hypersensitive response, as it is termed, enables genetically-resistant plants to limit microbial invasion under the right environmental conditio...

Bifidobacterium animalis subsp. lactis ATCC 27673 Is a Genomically Unique Strain within Its Conserved Subspecies

PubMed Central

Loquasto, Joseph R.; Barrangou, Rodolphe; Dudley, Edward G.; Stahl, Buffy; Chen, Chun

2013-01-01

Many strains of Bifidobacterium animalis subsp. lactis are considered health-promoting probiotic microorganisms and are commonly formulated into fermented dairy foods. Analyses of previously sequenced genomes of B. animalis subsp. lactis have revealed little genetic diversity, suggesting that it is a monomorphic subspecies. However, during a multilocus sequence typing survey of Bifidobacterium, it was revealed that B. animalis subsp. lactis ATCC 27673 gave a profile distinct from that of the other strains of the subspecies. As part of an ongoing study designed to understand the genetic diversity of this subspecies, the genome of this strain was sequenced and compared to other sequenced genomes of B. animalis subsp. lactis and B. animalis subsp. animalis. The complete genome of ATCC 27673 was 1,963,012 bp, contained 1,616 genes and 4 rRNA operons, and had a G+C content of 61.55%. Comparative analyses revealed that the genome of ATCC 27673 contained six distinct genomic islands encoding 83 open reading frames not found in other strains of the same subspecies. In four islands, either phage or mobile genetic elements were identified. In island 6, a novel clustered regularly interspaced short palindromic repeat (CRISPR) locus which contained 81 unique spacers was identified. This type I-E CRISPR-cas system differs from the type I-C systems previously identified in this subspecies, representing the first identification of a different system in B. animalis subsp. lactis. This study revealed that ATCC 27673 is a strain of B. animalis subsp. lactis with novel genetic content and suggests that the lack of genetic variability observed is likely due to the repeated sequencing of a limited number of widely distributed commercial strains. PMID:23995933

Interdependence, Reflexivity, Fidelity, Impedance Matching, and the Evolution of Genetic Coding

PubMed Central

Carter, Charles W; Wills, Peter R

2018-01-01

Abstract Genetic coding is generally thought to have required ribozymes whose functions were taken over by polypeptide aminoacyl-tRNA synthetases (aaRS). Two discoveries about aaRS and their interactions with tRNA substrates now furnish a unifying rationale for the opposite conclusion: that the key processes of the Central Dogma of molecular biology emerged simultaneously and naturally from simple origins in a peptide•RNA partnership, eliminating the epistemological utility of a prior RNA world. First, the two aaRS classes likely arose from opposite strands of the same ancestral gene, implying a simple genetic alphabet. The resulting inversion symmetries in aaRS structural biology would have stabilized the initial and subsequent differentiation of coding specificities, rapidly promoting diversity in the proteome. Second, amino acid physical chemistry maps onto tRNA identity elements, establishing reflexive, nanoenvironmental sensing in protein aaRS. Bootstrapping of increasingly detailed coding is thus intrinsic to polypeptide aaRS, but impossible in an RNA world. These notions underline the following concepts that contradict gradual replacement of ribozymal aaRS by polypeptide aaRS: 1) aaRS enzymes must be interdependent; 2) reflexivity intrinsic to polypeptide aaRS production dynamics promotes bootstrapping; 3) takeover of RNA-catalyzed aminoacylation by enzymes will necessarily degrade specificity; and 4) the Central Dogma’s emergence is most probable when replication and translation error rates remain comparable. These characteristics are necessary and sufficient for the essentially de novo emergence of a coupled gene–replicase–translatase system of genetic coding that would have continuously preserved the functional meaning of genetically encoded protein genes whose phylogenetic relationships match those observed today. PMID:29077934

Quantitative trait loci associated with anthracnose resistance in sorghum

USDA-ARS?s Scientific Manuscript database

With an aim to develop a durable resistance to the fungal disease anthracnose, two unique genetic sources of resistance were selected to create genetic mapping populations to identify regions of the sorghum genome that encode anthracnose resistance. A series of quantitative trait loci were identifi...

Ovine Reference Materials and Assays for Prion Genetic Testing

USDA-ARS?s Scientific Manuscript database

Background: Genetic predisposition to scrapie in sheep is associated with variation in the peptide sequence of the ovine prion protein encoded by Prnp. Codon variants implicated in scrapie susceptibility or disease progression include those at amino acid positions 112, 136, 141, 154, and 171. Nin...

Molecular Genetics of Mitochondrial Disorders

ERIC Educational Resources Information Center

Wong, Lee-Jun C.

2010-01-01

Mitochondrial respiratory chain (RC) disorders (RCDs) are a group of genetically and clinically heterogeneous diseases because of the fact that protein components of the RC are encoded by both mitochondrial and nuclear genomes and are essential in all cells. In addition, the biogenesis, structure, and function of mitochondria, including DNA…

Thionin-D4E1 chimeric protein protects plants against bacterial infections

DOEpatents

Stover, Eddie W; Gupta, Goutam; Hao, Guixia

2017-08-08

The generation of a chimeric protein containing a first domain encoding either a pro-thionon or thionin, a second domain encoding D4E1 or pro-D4E1, and a third domain encoding a peptide linker located between the first domain and second domain is described. Either the first domain or the second domain is located at the amino terminal of the chimeric protein and the other domain (second domain or first domain, respectively) is located at the carboxyl terminal. The chimeric protein has antibacterial activity. Genetically altered plants and their progeny expressing a polynucleotide encoding the chimeric protein resist diseases caused by bacteria.

Molecular manipulations for enhancing luminescent bioreporters performance in the detection of toxic chemicals.

PubMed

Yagur-Kroll, Sharon; Belkin, Shimshon

2014-01-01

Microbial whole-cell bioreporters are genetically modified microorganisms that produce a quantifiable output in response to the presence of toxic chemicals or other stress factors. These bioreporters harbor a genetic fusion between a sensing element (usually a gene regulatory element responsive to the target) and a reporter element, the product of which may be quantitatively monitored either by its presence or by its activity. In this chapter we review genetic manipulations undertaken in order to improve bioluminescent bioreporter performance by increasing luminescent output, lowering the limit of detection, and shortening the response time. We describe molecular manipulations applied to all aspects of whole-cell bioreporters: the host strain, the expression system, the sensing element, and the reporter element. The molecular construction of whole-cell luminescent bioreporters, harboring fusions of gene promoter elements to reporter genes, has been around for over three decades; in most cases, these two genetic elements are combined "as is." This chapter outlines diverse molecular manipulations for enhancing the performance of such sensors.

Effects of various types of stress on the metabolism of reserve carbohydrates in Saccharomyces cerevisiae: genetic evidence for a stress-induced recycling of glycogen and trehalose.

PubMed

Parrou, J L; Teste, M A; François, J

1997-06-01

It is well known that glycogen and trehalose accumulate in yeast under nutrient starvation or entering into the stationary phase of growth, and that high levels of trehalose are found in heat-shocked cells. However, effects of various types of stress on trehalose, and especially on glycogen, are poorly documented. Taking into account that almost all genes encoding the enzymes involved in the metabolism of these two reserve carbohydrates contain between one and several copies of the stress-responsive element (STRE), an investigation was made of the possibility of a link between the potential transcriptional induction of these genes and the accumulation of glycogen and trehalose under different stress conditions. Using transcriptional fusions, it was found that all these genes were induced in a similar fashion, although to various extents, by temperature, osmotic and oxidative stresses. Experiments performed with an msn2/msn4 double mutant proved that the transcriptional induction of the genes encoding glycogen synthase (GSY2) and trehalose-6-phosphate synthase (TPS1) was needed for the small increase in glycogen and trehalose upon exposure to a mild heat stress and salt shock. However, the extent of transcriptional activation of these genes upon stresses in wild-type strains was not correlated with a proportional rise in either glycogen or trehalose. The major explanation for this lack of correlation comes from the fact that genes encoding the enzymes of the biosynthetic and of the biodegradative pathways were almost equally induced. Hence, trehalose and glycogen accumulated to much higher levels in cells lacking neutral trehalose or glycogen phosphorylase exposed to stress conditions, which suggested that one of the major effects of stress in yeast is to induce a wasteful expenditure of energy by increasing the recycling of these molecules. We also found that transcriptional induction of STRE-controlled genes was abolished at temperatures above 40 degree C, while induction was still observed for a heat-shock-element regulated gene. Remarkably, trehalose accumulated to very high levels under this condition. This can be explained by a stimulation of trehalose synthase and inhibition of trehalose by high temperature.

Generative Representations for Automated Design of Robots

NASA Technical Reports Server (NTRS)

Homby, Gregory S.; Lipson, Hod; Pollack, Jordan B.

2007-01-01

A method of automated design of complex, modular robots involves an evolutionary process in which generative representations of designs are used. The term generative representations as used here signifies, loosely, representations that consist of or include algorithms, computer programs, and the like, wherein encoded designs can reuse elements of their encoding and thereby evolve toward greater complexity. Automated design of robots through synthetic evolutionary processes has already been demonstrated, but it is not clear whether genetically inspired search algorithms can yield designs that are sufficiently complex for practical engineering. The ultimate success of such algorithms as tools for automation of design depends on the scaling properties of representations of designs. A nongenerative representation (one in which each element of the encoded design is used at most once in translating to the design) scales linearly with the number of elements. Search algorithms that use nongenerative representations quickly become intractable (search times vary approximately exponentially with numbers of design elements), and thus are not amenable to scaling to complex designs. Generative representations are compact representations and were devised as means to circumvent the above-mentioned fundamental restriction on scalability. In the present method, a robot is defined by a compact programmatic form (its generative representation) and the evolutionary variation takes place on this form. The evolutionary process is an iterative one, wherein each cycle consists of the following steps: 1. Generative representations are generated in an evolutionary subprocess. 2. Each generative representation is a program that, when compiled, produces an assembly procedure. 3. In a computational simulation, a constructor executes an assembly procedure to generate a robot. 4. A physical-simulation program tests the performance of a simulated constructed robot, evaluating the performance according to a fitness criterion to yield a figure of merit that is fed back into the evolutionary subprocess of the next iteration. In comparison with prior approaches to automated evolutionary design of robots, the use of generative representations offers two advantages: First, a generative representation enables the reuse of components in regular and hierarchical ways and thereby serves a systematic means of creating more complex modules out of simpler ones. Second, the evolved generative representation may capture intrinsic properties of the design problem, so that variations in the representations move through the design space more effectively than do equivalent variations in a nongenerative representation. This method has been demonstrated by using it to design some robots that move, variously, by walking, rolling, or sliding. Some of the robots were built (see figure). Although these robots are very simple, in comparison with robots designed by humans, their structures are more regular, modular, hierarchical, and complex than are those of evolved designs of comparable functionality synthesized by use of nongenerative representations.

The VLSI design of a Reed-Solomon encoder using Berlekamps bit-serial multiplier algorithm

NASA Technical Reports Server (NTRS)

Truong, T. K.; Deutsch, L. J.; Reed, I. S.; Hsu, I. S.; Wang, K.; Yeh, C. S.

1982-01-01

Realization of a bit-serial multiplication algorithm for the encoding of Reed-Solomon (RS) codes on a single VLSI chip using NMOS technology is demonstrated to be feasible. A dual basis (255, 223) over a Galois field is used. The conventional RS encoder for long codes ofter requires look-up tables to perform the multiplication of two field elements. Berlekamp's algorithm requires only shifting and exclusive-OR operations.

Encoding and Retrieval Interference in Sentence Comprehension: Evidence from Agreement

PubMed Central

Villata, Sandra; Tabor, Whitney; Franck, Julie

2018-01-01

Long-distance verb-argument dependencies generally require the integration of a fronted argument when the verb is encountered for sentence interpretation. Under a parsing model that handles long-distance dependencies through a cue-based retrieval mechanism, retrieval is hampered when retrieval cues also resonate with non-target elements (retrieval interference). However, similarity-based interference may also stem from interference arising during the encoding of elements in memory (encoding interference), an effect that is not directly accountable for by a cue-based retrieval mechanism. Although encoding and retrieval interference are clearly distinct at the theoretical level, it is difficult to disentangle the two on empirical grounds, since encoding interference may also manifest at the retrieval region. We report two self-paced reading experiments aimed at teasing apart the role of each component in gender and number subject-verb agreement in Italian and English object relative clauses. In Italian, the verb does not agree in gender with the subject, thus providing no cue for retrieval. In English, although present tense verbs agree in number with the subject, past tense verbs do not, allowing us to test the role of number as a retrieval cue within the same language. Results from both experiments converge, showing similarity-based interference at encoding, and some evidence for an effect at retrieval. After having pointed out the non-negligible role of encoding in sentence comprehension, and noting that Lewis and Vasishth’s (2005) ACT-R model of sentence processing, the most fully developed cue-based retrieval approach to sentence processing does not predict encoding effects, we propose an augmentation of this model that predicts these effects. We then also propose a self-organizing sentence processing model (SOSP), which has the advantage of accounting for retrieval and encoding interference with a single mechanism. PMID:29403414

Evolved Populations of Shigella flexneri Phage Sf6 Acquire Large Deletions, Altered Genomic Architecture, and Faster Life Cycles.

PubMed

Dover, John A; Burmeister, Alita R; Molineux, Ian J; Parent, Kristin N

2016-09-19

Genomic architecture is the framework within which genes and regulatory elements evolve and where specific constructs may constrain or potentiate particular adaptations. One such construct is evident in phages that use a headful packaging strategy that results in progeny phage heads packaged with DNA until full rather than encapsidating a simple unit-length genome. Here, we investigate the evolution of the headful packaging phage Sf6 in response to barriers that impede efficient phage adsorption to the host cell. Ten replicate populations evolved faster Sf6 life cycles by parallel mutations found in a phage lysis gene and/or by large, 1.2- to 4.0-kb deletions that remove a mobile genetic IS911 element present in the ancestral phage genome. The fastest life cycles were found in phages that acquired both mutations. No mutations were found in genes encoding phage structural proteins, which were a priori expected from the experimental design that imposed a challenge for phage adsorption by using a Shigella flexneri host lacking receptors preferred by Sf6. We used DNA sequencing, molecular approaches, and physiological experiments on 82 clonal isolates taken from all 10 populations to reveal the genetic basis of the faster Sf6 life cycle. The majority of our isolates acquired deletions in the phage genome. Our results suggest that deletions are adaptive and can influence the duration of the phage life cycle while acting in conjunction with other lysis time-determining point mutations. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Origin and Evolution of European Community-Acquired Methicillin-Resistant Staphylococcus aureus

PubMed Central

Wirth, Thierry; Andersen, Paal S.; Skov, Robert L.; De Grassi, Anna; Simões, Patricia Martins; Tristan, Anne; Petersen, Andreas; Aziz, Maliha; Kiil, Kristoffer; Cirković, Ivana; Udo, Edet E.; del Campo, Rosa; Vuopio-Varkila, Jaana; Ahmad, Norazah; Tokajian, Sima; Peters, Georg; Schaumburg, Frieder; Olsson-Liljequist, Barbro; Givskov, Michael; Driebe, Elizabeth E.; Vigh, Henrik E.; Shittu, Adebayo; Ramdani-Bougessa, Nadjia; Rasigade, Jean-Philippe; Price, Lance B.; Vandenesch, Francois; Larsen, Anders R.; Laurent, Frederic

2014-01-01

ABSTRACT Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) was recognized in Europe and worldwide in the late 1990s. Within a decade, several genetically and geographically distinct CA-MRSA lineages carrying the small SCCmec type IV and V genetic elements and the Panton-Valentine leukocidin (PVL) emerged around the world. In Europe, the predominant CA-MRSA strain belongs to clonal complex 80 (CC80) and is resistant to kanamycin/amikacin and fusidic acid. CC80 was first reported in 1993 but was relatively rare until the late 1990s. It has since been identified throughout North Africa, the Middle East, and Europe, with recent sporadic reports in sub-Saharan Africa. While strongly associated with skin and soft tissue infections, it is rarely found among asymptomatic carriers. Methicillin-sensitive S. aureus (MSSA) CC80 strains are extremely rare except in sub-Saharan Africa. In the current study, we applied whole-genome sequencing to a global collection of both MSSA and MRSA CC80 isolates. Phylogenetic analyses strongly suggest that the European epidemic CA-MRSA lineage is derived from a PVL-positive MSSA ancestor from sub-Saharan Africa. Moreover, the tree topology suggests a single acquisition of both the SCCmec element and a plasmid encoding the fusidic acid resistance determinant. Four canonical SNPs distinguish the derived CA-MRSA lineage and include a nonsynonymous mutation in accessory gene regulator C (agrC). These changes were associated with a star-like expansion into Europe, the Middle East, and North Africa in the early 1990s, including multiple cases of cross-continent imports likely driven by human migrations. PMID:25161186

Mobile genetic elements and antibiotic resistance in mine soil amended with organic wastes.

PubMed

Garbisu, Carlos; Garaiyurrebaso, Olatz; Lanzén, Anders; Álvarez-Rodríguez, Itxaso; Arana, Lide; Blanco, Fernando; Smalla, Kornelia; Grohmann, Elisabeth; Alkorta, Itziar

2018-04-15

Metal resistance has been associated with antibiotic resistance due to co- or cross-resistance mechanisms. Here, metal contaminated mine soil treated with organic wastes was screened for the presence of mobile genetic elements (MGEs). The occurrence of conjugative IncP-1 and mobilizable IncQ plasmids, as well as of class 1 integrons, was confirmed by PCR and Southern blot hybridization, suggesting that bacteria from these soils have gene-mobilizing capacity with implications for the dissemination of resistance factors. Moreover, exogenous isolation of MGEs from the soil bacterial community was attempted under antibiotic selection pressure by using Escherichia coli as recipient. Seventeen putative transconjugants were identified based on increased antibiotic resistance. Metabolic traits and metal resistance of putative transconjugants were investigated, and whole genome sequencing was carried out for two of them. Most putative transconjugants displayed a multi-resistant phenotype for a broad spectrum of antibiotics. They also displayed changes regarding the ability to metabolise different carbon sources, RNA: DNA ratio, growth rate and biofilm formation. Genome sequencing of putative transconjugants failed to detect genes acquired by horizontal gene transfer, but instead revealed a number of nonsense mutations, including in ubiH, whose inactivation was linked to the observed resistance to aminoglycosides. Our results confirm that mine soils contain MGEs encoding antibiotic resistance. Moreover, they point out the role of spontaneous mutations in achieving low-level antibiotic resistance in a short time, which was associated with a trade-off in the capability to metabolise specific carbon sources. Copyright © 2017. Published by Elsevier B.V.

Genetic and Phenotypic Comparison of Facultative Methylotrophy between Methylobacterium extorquens Strains PA1 and AM1

PubMed Central

Nayak, Dipti D.; Marx, Christopher J.

2014-01-01

Methylobacterium extorquens AM1, a strain serendipitously isolated half a century ago, has become the best-characterized model system for the study of aerobic methylotrophy (the ability to grow on reduced single-carbon compounds). However, with 5 replicons and 174 insertion sequence (IS) elements in the genome as well as a long history of domestication in the laboratory, genetic and genomic analysis of M. extorquens AM1 face several challenges. On the contrary, a recently isolated strain - M. extorquens PA1- is closely related to M. extorquens AM1 (100% 16S rRNA identity) and contains a streamlined genome with a single replicon and only 20 IS elements. With the exception of the methylamine dehydrogenase encoding gene cluster (mau), genes known to be involved in methylotrophy are well conserved between M. extorquens AM1 and M. extorquens PA1. In this paper we report four primary findings regarding methylotrophy in PA1. First, with a few notable exceptions, the repertoire of methylotrophy genes between PA1 and AM1 is extremely similar. Second, PA1 grows faster with higher yields compared to AM1 on C1 and multi-C substrates in minimal media, but AM1 grows faster in rich medium. Third, deletion mutants in PA1 throughout methylotrophy modules have the same C1 growth phenotypes observed in AM1. Finally, the precision of our growth assays revealed several unexpected growth phenotypes for various knockout mutants that serve as leads for future work in understanding their basis and generality across Methylobacterium strains. PMID:25232997

The Systems Biology Markup Language (SBML) Level 3 Package: Layout, Version 1 Core.

PubMed

Gauges, Ralph; Rost, Ursula; Sahle, Sven; Wengler, Katja; Bergmann, Frank T

2015-06-01

Many software tools provide facilities for depicting reaction network diagrams in a visual form. Two aspects of such a visual diagram can be distinguished: the layout (i.e.: the positioning and connections) of the elements in the diagram, and the graphical form of the elements (for example, the glyphs used for symbols, the properties of the lines connecting them, and so on). For software tools that also read and write models in SBML (Systems Biology Markup Language) format, a common need is to store the network diagram together with the SBML representation of the model. This in turn raises the question of how to encode the layout and the rendering of these diagrams. The SBML Level 3 Version 1 Core specification does not provide a mechanism for explicitly encoding diagrams, but it does provide a mechanism for SBML packages to extend the Core specification and add additional syntactical constructs. The Layout package for SBML Level 3 adds the necessary features to SBML so that diagram layouts can be encoded in SBML files, and a companion package called SBML Rendering specifies how the graphical rendering of elements can be encoded. The SBML Layout package is based on the principle that reaction network diagrams should be described as representations of entities such as species and reactions (with direct links to the underlying SBML elements), and not as arbitrary drawings or graphs; for this reason, existing languages for the description of vector drawings (such as SVG) or general graphs (such as GraphML) cannot be used.

The Systems Biology Markup Language (SBML) Level 3 Package: Layout, Version 1 Core.

PubMed

Gauges, Ralph; Rost, Ursula; Sahle, Sven; Wengler, Katja; Bergmann, Frank Thomas

2015-09-04

Many software tools provide facilities for depicting reaction network diagrams in a visual form. Two aspects of such a visual diagram can be distinguished: the layout (i.e.: the positioning and connections) of the elements in the diagram, and the graphical form of the elements (for example, the glyphs used for symbols, the properties of the lines connecting them, and so on). For software tools that also read and write models in SBML (Systems Biology Markup Language) format, a common need is to store the network diagram together with the SBML representation of the model. This in turn raises the question of how to encode the layout and the rendering of these diagrams. The SBML Level 3 Version 1 Core specification does not provide a mechanism for explicitly encoding diagrams, but it does provide a mechanism for SBML packages to extend the Core specification and add additional syntactical constructs. The Layout package for SBML Level 3 adds the necessary features to SBML so that diagram layouts can be encoded in SBML files, and a companion package called SBML Rendering specifies how the graphical rendering of elements can be encoded. The SBML Layout package is based on the principle that reaction network diagrams should be described as representations of entities such as species and reactions (with direct links to the underlying SBML elements), and not as arbitrary drawings or graphs; for this reason, existing languages for the description of vector drawings (such as SVG) or general graphs (such as GraphML) cannot be used.

Finite element analysis and genetic algorithm optimization design for the actuator placement on a large adaptive structure

NASA Astrophysics Data System (ADS)

Sheng, Lizeng

The dissertation focuses on one of the major research needs in the area of adaptive/intelligent/smart structures, the development and application of finite element analysis and genetic algorithms for optimal design of large-scale adaptive structures. We first review some basic concepts in finite element method and genetic algorithms, along with the research on smart structures. Then we propose a solution methodology for solving a critical problem in the design of a next generation of large-scale adaptive structures---optimal placements of a large number of actuators to control thermal deformations. After briefly reviewing the three most frequently used general approaches to derive a finite element formulation, the dissertation presents techniques associated with general shell finite element analysis using flat triangular laminated composite elements. The element used here has three nodes and eighteen degrees of freedom and is obtained by combining a triangular membrane element and a triangular plate bending element. The element includes the coupling effect between membrane deformation and bending deformation. The membrane element is derived from the linear strain triangular element using Cook's transformation. The discrete Kirchhoff triangular (DKT) element is used as the plate bending element. For completeness, a complete derivation of the DKT is presented. Geometrically nonlinear finite element formulation is derived for the analysis of adaptive structures under the combined thermal and electrical loads. Next, we solve the optimization problems of placing a large number of piezoelectric actuators to control thermal distortions in a large mirror in the presence of four different thermal loads. We then extend this to a multi-objective optimization problem of determining only one set of piezoelectric actuator locations that can be used to control the deformation in the same mirror under the action of any one of the four thermal loads. A series of genetic algorithms, GA Version 1, 2 and 3, were developed to find the optimal locations of piezoelectric actuators from the order of 1021 ˜ 1056 candidate placements. Introducing a variable population approach, we improve the flexibility of selection operation in genetic algorithms. Incorporating mutation and hill climbing into micro-genetic algorithms, we are able to develop a more efficient genetic algorithm. Through extensive numerical experiments, we find that the design search space for the optimal placements of a large number of actuators is highly multi-modal and that the most distinct nature of genetic algorithms is their robustness. They give results that are random but with only a slight variability. The genetic algorithms can be used to get adequate solution using a limited number of evaluations. To get the highest quality solution, multiple runs including different random seed generators are necessary. The investigation time can be significantly reduced using a very coarse grain parallel computing. Overall, the methodology of using finite element analysis and genetic algorithm optimization provides a robust solution approach for the challenging problem of optimal placements of a large number of actuators in the design of next generation of adaptive structures.

Convergent Evolution of Ribonuclease H in LTR Retrotransposons and Retroviruses

PubMed Central

Ustyantsev, Kirill; Novikova, Olga; Blinov, Alexander; Smyshlyaev, Georgy

2015-01-01

Ty3/Gypsy long terminals repeat (LTR) retrotransposons are structurally and phylogenetically close to retroviruses. Two notable structural differences between these groups of genetic elements are 1) the presence in retroviruses of an additional envelope gene, env, which mediates infection, and 2) a specific dual ribonuclease H (RNH) domain encoded by the retroviral pol gene. However, similar to retroviruses, many Ty3/Gypsy LTR retrotransposons harbor additional env-like genes, promoting concepts of the infective mode of these retrotransposons. Here, we provide a further line of evidence of similarity between retroviruses and some Ty3/Gypsy LTR retrotransposons. We identify that, together with their additional genes, plant Ty3/Gypsy LTR retrotransposons of the Tat group have a second RNH, as do retroviruses. Most importantly, we show that the resulting dual RNHs of Tat LTR retrotransposons and retroviruses emerged independently, providing strong evidence for their convergent evolution. The convergent resemblance of Tat LTR retrotransposons and retroviruses may indicate similar selection pressures acting on these diverse groups of elements and reveal potential evolutionary constraints on their structure. We speculate that dual RNH is required to accelerate retrotransposon evolution through increased rates of strand transfer events and subsequent recombination events. PMID:25605791

Toward mapping the biology of the genome.

PubMed

Chanock, Stephen

2012-09-01

This issue of Genome Research presents new results, methods, and tools from The ENCODE Project (ENCyclopedia of DNA Elements), which collectively represents an important step in moving beyond a parts list of the genome and promises to shape the future of genomic research. This collection sheds light on basic biological questions and frames the current debate over the optimization of tools and methodological challenges necessary to compare and interpret large complex data sets focused on how the genome is organized and regulated. In a number of instances, the authors have highlighted the strengths and limitations of current computational and technical approaches, providing the community with useful standards, which should stimulate development of new tools. In many ways, these papers will ripple through the scientific community, as those in pursuit of understanding the "regulatory genome" will heavily traverse the maps and tools. Similarly, the work should have a substantive impact on how genetic variation contributes to specific diseases and traits by providing a compendium of functional elements for follow-up study. The success of these papers should not only be measured by the scope of the scientific insights and tools but also by their ability to attract new talent to mine existing and future data.

What Do We Know About NOD-Like Receptors in Plant Immunity?

PubMed

Zhang, Xiaoxiao; Dodds, Peter N; Bernoux, Maud

2017-08-04

The first plant disease resistance (R) genes were identified and cloned more than two decades ago. Since then, many more R genes have been identified and characterized in numerous plant pathosystems. Most of these encode members of the large family of intracellular NLRs (NOD-like receptors), which also includes animal immune receptors. New discoveries in this expanding field of research provide new elements for our understanding of plant NLR function. But what do we know about plant NLR function today? Genetic, structural, and functional analyses have uncovered a number of commonalities and differences in pathogen recognition strategies as well as how NLRs are regulated and activate defense signaling, but many unknowns remain. This review gives an update on the latest discoveries and breakthroughs in this field, with an emphasis on structural findings and some comparison to animal NLRs, which can provide additional insights and paradigms in plant NLR function.

Helicos BioSciences.

PubMed

Milos, Patrice

2008-04-01

Helicos BioSciences Corporation is a life sciences company developing revolutionary new single molecule sequencing technology to provide the path to the US$1000 genome. True Single Molecule Sequencing (tSMS) will drive advancements in pharmacogenomics that can enable a better understanding of an individual's susceptibility to disease, develop more effective disease diagnoses and differentiate response to disease therapies. During 2007, genome-wide disease-association studies, the encylopedia of DNA elements (ENCODE) and the published genome sequence of two individuals have revealed human genome variation far more extensive than originally believed. These also demonstrated that common variations explain only a fraction of the genetic basis of disease. Therefore, the capability to understand an individual genome is critical in setting the foundation for the next great revolution in healthcare. Helicos is committed to this vision and will provide cost-effective genome sequencing and comprehensive analysis of the transcribed genome that can unlock the era of personalized healthcare.

Metaproteomics reveals abundant transposase expression in mutualistic endosymbionts

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

Kleiner, Manuel; Young, Jacque C; Shah, Manesh B

2013-01-01

Transposases, enzymes that catalyze the movement of mobile genetic elements, are the most abundant genes in nature. While many bacteria encode an abundance of transposases in their genomes, the current paradigm is that transposase gene expression is tightly regulated and generally low due to its severe mutagenic effects. In the current study, we detected the highest number of transposase proteins ever reported in bacteria, in symbionts of the gutless marine worm Olavius algarvensis using metaproteomics. At least 26 different transposases from 12 different families were detected and genomic and proteomic analyses suggest many of these are active. This high expressionmore » of transposases indicates that the mechanisms for their tight regulation have been disabled or destroyed. Based on recent studies on other symbionts and pathogens that showed high transposase transcription, we speculate that abundant transposase expression might be common in symbionts and pathogens.« less

Memory Retrieval in Mice and Men

PubMed Central

Ben-Yakov, Aya; Dudai, Yadin; Mayford, Mark R.

2015-01-01

Retrieval, the use of learned information, was until recently mostly terra incognita in the neurobiology of memory, owing to shortage of research methods with the spatiotemporal resolution required to identify and dissect fast reactivation or reconstruction of complex memories in the mammalian brain. The development of novel paradigms, model systems, and new tools in molecular genetics, electrophysiology, optogenetics, in situ microscopy, and functional imaging, have contributed markedly in recent years to our ability to investigate brain mechanisms of retrieval. We review selected developments in the study of explicit retrieval in the rodent and human brain. The picture that emerges is that retrieval involves coordinated fast interplay of sparse and distributed corticohippocampal and neocortical networks that may permit permutational binding of representational elements to yield specific representations. These representations are driven largely by the activity patterns shaped during encoding, but are malleable, subject to the influence of time and interaction of the existing memory with novel information. PMID:26438596

Brain-wide neuronal dynamics during motor adaptation in zebrafish.

PubMed

Ahrens, Misha B; Li, Jennifer M; Orger, Michael B; Robson, Drew N; Schier, Alexander F; Engert, Florian; Portugues, Ruben

2012-05-09

A fundamental question in neuroscience is how entire neural circuits generate behaviour and adapt it to changes in sensory feedback. Here we use two-photon calcium imaging to record the activity of large populations of neurons at the cellular level, throughout the brain of larval zebrafish expressing a genetically encoded calcium sensor, while the paralysed animals interact fictively with a virtual environment and rapidly adapt their motor output to changes in visual feedback. We decompose the network dynamics involved in adaptive locomotion into four types of neuronal response properties, and provide anatomical maps of the corresponding sites. A subset of these signals occurred during behavioural adjustments and are candidates for the functional elements that drive motor learning. Lesions to the inferior olive indicate a specific functional role for olivocerebellar circuitry in adaptive locomotion. This study enables the analysis of brain-wide dynamics at single-cell resolution during behaviour.

Brain-wide neuronal dynamics during motor adaptation in zebrafish

PubMed Central

Ahrens, Misha B; Li, Jennifer M; Orger, Michael B; Robson, Drew N; Schier, Alexander F; Engert, Florian; Portugues, Ruben

2013-01-01

A fundamental question in neuroscience is how entire neural circuits generate behavior and adapt it to changes in sensory feedback. Here we use two-photon calcium imaging to record activity of large populations of neurons at the cellular level throughout the brain of larval zebrafish expressing a genetically-encoded calcium sensor, while the paralyzed animals interact fictively with a virtual environment and rapidly adapt their motor output to changes in visual feedback. We decompose the network dynamics involved in adaptive locomotion into four types of neural response properties, and provide anatomical maps of the corresponding sites. A subset of these signals occurred during behavioral adjustments and are candidates for the functional elements that drive motor learning. Lesions to the inferior olive indicate a specific functional role for olivocerebellar circuitry in adaptive locomotion. This study enables the analysis of brain-wide dynamics at single-cell resolution during behavior. PMID:22622571

EcoFlex: A Multifunctional MoClo Kit for E. coli Synthetic Biology.

PubMed

Lai, Hung-En; Moore, Simon; Polizzi, Karen; Freemont, Paul

2018-01-01

Development of advanced synthetic biology tools is always in demand since they act as a platform technology to enable rapid prototyping of biological constructs in a high-throughput manner. EcoFlex is a modular cloning (MoClo) kit for Escherichia coli and is based on the Golden Gate principles, whereby Type IIS restriction enzymes (BsaI, BsmBI, BpiI) are used to construct modular genetic elements (biological parts) in a bottom-up approach. Here, we describe a collection of plasmids that stores various biological parts including promoters, RBSs, terminators, ORFs, and destination vectors, each encoding compatible overhangs allowing hierarchical assembly into single transcription units or a full-length polycistronic operon or biosynthetic pathway. A secondary module cloning site is also available for pathway optimization, in order to limit library size if necessary. Here, we show the utility of EcoFlex using the violacein biosynthesis pathway as an example.

A high-quality human reference panel reveals the complexity and distribution of genomic structural variants.

PubMed

Hehir-Kwa, Jayne Y; Marschall, Tobias; Kloosterman, Wigard P; Francioli, Laurent C; Baaijens, Jasmijn A; Dijkstra, Louis J; Abdellaoui, Abdel; Koval, Vyacheslav; Thung, Djie Tjwan; Wardenaar, René; Renkens, Ivo; Coe, Bradley P; Deelen, Patrick; de Ligt, Joep; Lameijer, Eric-Wubbo; van Dijk, Freerk; Hormozdiari, Fereydoun; Uitterlinden, André G; van Duijn, Cornelia M; Eichler, Evan E; de Bakker, Paul I W; Swertz, Morris A; Wijmenga, Cisca; van Ommen, Gert-Jan B; Slagboom, P Eline; Boomsma, Dorret I; Schönhuth, Alexander; Ye, Kai; Guryev, Victor

2016-10-06

Structural variation (SV) represents a major source of differences between individual human genomes and has been linked to disease phenotypes. However, the majority of studies provide neither a global view of the full spectrum of these variants nor integrate them into reference panels of genetic variation. Here, we analyse whole genome sequencing data of 769 individuals from 250 Dutch families, and provide a haplotype-resolved map of 1.9 million genome variants across 9 different variant classes, including novel forms of complex indels, and retrotransposition-mediated insertions of mobile elements and processed RNAs. A large proportion are previously under reported variants sized between 21 and 100 bp. We detect 4 megabases of novel sequence, encoding 11 new transcripts. Finally, we show 191 known, trait-associated SNPs to be in strong linkage disequilibrium with SVs and demonstrate that our panel facilitates accurate imputation of SVs in unrelated individuals.

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

Hood, Iris V.; Berger, James M.

Replisome assembly requires the loading of replicative hexameric helicases onto origins by AAA+ ATPases. How loader activity is appropriately controlled remains unclear. Here, we use structural and biochemical analyses to establish how an antimicrobial phage protein interferes with the function of theStaphylococcus aureusreplicative helicase loader, DnaI. The viral protein binds to the loader’s AAA+ ATPase domain, allowing binding of the host replicative helicase but impeding loader self-assembly and ATPase activity. Close inspection of the complex highlights an unexpected locus for the binding of an interdomain linker element in DnaI/DnaC-family proteins. We find that the inhibitor protein is genetically coupled tomore » a phage-encoded homolog of the bacterial helicase loader, which we show binds to the host helicase but not to the inhibitor itself. These findings establish a new approach by which viruses can hijack host replication processes and explain how loader activity is internally regulated to prevent aberrant auto-association.« less

Neutral details associated with emotional events are encoded: evidence from a cued recall paradigm.

PubMed

Mickley Steinmetz, Katherine R; Knight, Aubrey G; Kensinger, Elizabeth A

2016-11-01

Enhanced emotional memory often comes at the cost of memory for surrounding background information. Narrowed-encoding theories suggest that this is due to narrowed attention for emotional information at encoding, leading to impaired encoding of background information. Recent work has suggested that an encoding-based theory may be insufficient. Here, we examined whether cued recall-instead of previously used recognition memory tasks-would reveal evidence that non-emotional information associated with emotional information was effectively encoded. Participants encoded positive, negative, or neutral objects on neutral backgrounds. At retrieval, they were given either the item or the background as a memory cue and were asked to recall the associated scene element. Counter to narrowed-encoding theories, emotional items were more likely than neutral items to trigger recall of the associated background. This finding suggests that there is a memory trace of this contextual information and that emotional cues may facilitate retrieval of this information.

Tick-Borne Transmission of Two Genetically Distinct Anaplasma marginale Strains following Superinfection of the Mammalian Reservoir Host

USDA-ARS?s Scientific Manuscript database

Strain superinfection affects the dynamics of epidemiological spread of pathogens through a host population. Superinfection has recently been shown to occur for genetically distinct strains of the tick-borne pathogen Anaplasma marginale that encode distinctly different surface protein variants. Supe...

An Efficient Method for Electroporation of Small Interfering RNAs into ENCODE Project Tier 1 GM12878 and K562 Cell Lines.

PubMed

Muller, Ryan Y; Hammond, Ming C; Rio, Donald C; Lee, Yeon J

2015-12-01

The Encyclopedia of DNA Elements (ENCODE) Project aims to identify all functional sequence elements in the human genome sequence by use of high-throughput DNA/cDNA sequencing approaches. To aid the standardization, comparison, and integration of data sets produced from different technologies and platforms, the ENCODE Consortium selected several standard human cell lines to be used by the ENCODE Projects. The Tier 1 ENCODE cell lines include GM12878, K562, and H1 human embryonic stem cell lines. GM12878 is a lymphoblastoid cell line, transformed with the Epstein-Barr virus, that was selected by the International HapMap Project for whole genome and transcriptome sequencing by use of the Illumina platform. K562 is an immortalized myelogenous leukemia cell line. The GM12878 cell line is attractive for the ENCODE Projects, as it offers potential synergy with the International HapMap Project. Despite the vast amount of sequencing data available on the GM12878 cell line through the ENCODE Project, including transcriptome, chromatin immunoprecipitation-sequencing for histone marks, and transcription factors, no small interfering siRNA-mediated knockdown studies have been performed in the GM12878 cell line, as cationic lipid-mediated transfection methods are inefficient for lymphoid cell lines. Here, we present an efficient and reproducible method for transfection of a variety of siRNAs into the GM12878 and K562 cell lines, which subsequently results in targeted protein depletion.

Genotyping and phenotyping of CYP2D6 and CYP3A isoenzymes in patients with alcohol use disorder: correlation with haloperidol plasma concentration.

PubMed

Sychev, Dmitry A; Zastrozhin, Mikhail S; Miroshnichenko, Igor I; Baymeeva, Natalia V; Smirnov, Valery V; Grishina, Elena A; Ryzhikova, Kristina A; Mirzaev, Karin B; Markov, Dmitry D; Skryabin, Valentin Y; Snalina, Nataliya E; Nosikova, Polina G; Savchenko, Ludmila M; Bryun, Evgeny A

2017-09-26

Haloperidol is used for the treatment of alcohol use disorders in patients with signs of alcohol-related psychosis. Haloperidol therapy poses a high risk of adverse drug reactions (ADR). Contradictory data, which include the effects of genetic polymorphisms in genes encoding the elements of haloperidol biotransformation system on haloperidol metabolism rate and plasma drug concentration ratio, are described in patients with different genotypes. The primary objective of this study was to investigate the effects of CYP2D6 and CYP3A5 genetic polymorphisms on haloperidol equilibrium concentration in patients with alcohol use disorder. The study included 69 male patients with alcohol use disorder. Genotyping was performed using the allele-specific real-time PCR. CYP2D6 and CYP3A were phenotyped with HPLC-MS using the concentration of endogenous substrate of the enzyme and its urinary metabolites [6-hydroxy-1,2,3,4-tetrahydro-β-carboline(6-HO-THBC) to pinoline ratio for CYP2D6 and 6-β-hydroxycortisol to cortisol ratio for CYP3A]. The equilibrium plasma concentration was determined using LC-MS-MS. Results indicated that both C/D indexes and equilibrium concentration levels depend on CYP2D6 genetic polymorphism, but only in patients receiving haloperidol intramuscular injections [0.26 (0.09; 0.48) vs. 0.54 (0.44; 0.74), p=0.037]. The study demonstrates that CYP2D6 genetic polymorphism (1846G>A) can affect haloperidol concentration levels in patients with alcohol use disorder.

An Integrated Encyclopedia of DNA Elements in the Human Genome

PubMed Central

2012-01-01

Summary The human genome encodes the blueprint of life, but the function of the vast majority of its nearly three billion bases is unknown. The Encyclopedia of DNA Elements (ENCODE) project has systematically mapped regions of transcription, transcription factor association, chromatin structure, and histone modification. These data enabled us to assign biochemical functions for 80% of the genome, in particular outside of the well-studied protein-coding regions. Many discovered candidate regulatory elements are physically associated with one another and with expressed genes, providing new insights into the mechanisms of gene regulation. The newly identified elements also show a statistical correspondence to sequence variants linked to human disease, and can thereby guide interpretation of this variation. Overall the project provides new insights into the organization and regulation of our genes and genome, and an expansive resource of functional annotations for biomedical research. PMID:22955616

Genetic and molecular studies of a composite chromosomal element (Tn3705) containing a Tn916-modified structure (Tn3704) in Streptococcus anginosus F22.

PubMed

Clermont, D; Horaud, T

1994-01-01

The plasmid-free Streptococcus anginosus F22 contained a conjugative element, Tn3705, encoding resistance to erythromycin (Emr) and tetracycline-minocycline (Tcr-Mnr). We mapped a chromosomal region (> 52 kb) of F22, corresponding to the internal part of Tn3705. Molecular analysis of Tn3705 revealed it to be a composite structure: it included in its central part a transposon designated Tn3704 (20.3 kb +/- 0.5 kb), which had a modified structure in comparison with that of Tn916 and on which the Emr Tcr-Mn4 markers were localized. Tn3705 inserted from F22 into the chromosome of various streptococcal transconjugants as well as that of Enterococcus faecalis transconjugants without changing its structure. In contrast, from the chromosome of an E. faecalis::Tn3705 transconjugant only Tn3704 inserted, at various sites, into another E. faecalis chromosome. Sugar fermentations occurred after the insertion of Tn3704 into the chromosome of an asaccharolytic E. faecalis strain. Transposition of only Tn3704 from the chromosome of E. faecalis::Tn3705 onto pIP964, an E. faecalis hemolysin plasmid, yielded two different pIP964 derivatives. The size of the entire element Tn3705 was estimated to be about 70.0 kb by pulsed-field electrophoresis.

Recessive mutations in the INS gene result in neonatal diabetes through reduced insulin biosynthesis

PubMed Central

Garin, Intza; Edghill, Emma L.; Akerman, Ildem; Rubio-Cabezas, Oscar; Rica, Itxaso; Locke, Jonathan M.; Maestro, Miguel Angel; Alshaikh, Adnan; Bundak, Ruveyde; del Castillo, Gabriel; Deeb, Asma; Deiss, Dorothee; Fernandez, Juan M.; Godbole, Koumudi; Hussain, Khalid; O’Connell, Michele; Klupa, Thomasz; Kolouskova, Stanislava; Mohsin, Fauzia; Perlman, Kusiel; Sumnik, Zdenek; Rial, Jose M.; Ugarte, Estibaliz; Vasanthi, Thiruvengadam; Johnstone, Karen; Flanagan, Sarah E.; Martínez, Rosa; Castaño, Carlos; Patch, Ann-Marie; Fernández-Rebollo, Eduardo; Raile, Klemens; Morgan, Noel; Harries, Lorna W.; Castaño, Luis; Ellard, Sian; Ferrer, Jorge; de Nanclares, Guiomar Perez; Hattersley, Andrew T.

2010-01-01

Heterozygous coding mutations in the INS gene that encodes preproinsulin were recently shown to be an important cause of permanent neonatal diabetes. These dominantly acting mutations prevent normal folding of proinsulin, which leads to beta-cell death through endoplasmic reticulum stress and apoptosis. We now report 10 different recessive INS mutations in 15 probands with neonatal diabetes. Functional studies showed that recessive mutations resulted in diabetes because of decreased insulin biosynthesis through distinct mechanisms, including gene deletion, lack of the translation initiation signal, and altered mRNA stability because of the disruption of a polyadenylation signal. A subset of recessive mutations caused abnormal INS transcription, including the deletion of the C1 and E1 cis regulatory elements, or three different single base-pair substitutions in a CC dinucleotide sequence located between E1 and A1 elements. In keeping with an earlier and more severe beta-cell defect, patients with recessive INS mutations had a lower birth weight (−3.2 SD score vs. −2.0 SD score) and were diagnosed earlier (median 1 week vs. 10 weeks) compared to those with dominant INS mutations. Mutations in the insulin gene can therefore result in neonatal diabetes as a result of two contrasting pathogenic mechanisms. Moreover, the recessively inherited mutations provide a genetic demonstration of the essential role of multiple sequence elements that regulate the biosynthesis of insulin in man. PMID:20133622

International Metadata Initiatives: Lessons in Bibliographic Control.

ERIC Educational Resources Information Center

Caplan, Priscilla

This paper looks at a subset of metadata schemes, including the Text Encoding Initiative (TEI) header, the Encoded Archival Description (EAD), the Dublin Core Metadata Element Set (DCMES), and the Visual Resources Association (VRA) Core Categories for visual resources. It examines why they developed as they did, major point of difference from…

Analysis of a MULE-cyanide hydratase gene fusion in Verticillium dahliae

USDA-ARS?s Scientific Manuscript database

The genome of the phytopathogenic fungus Verticillium dahliae encodes numerous Class II “cut-and-paste” transposable elements, including those of a small group of MULE transposons. We have previously identified a fusion event between a MULE transposon sequence and sequence encoding a cyanide hydrata...

The complete genome sequence of the acarbose producer Actinoplanes sp. SE50/110

PubMed Central

2012-01-01

Background Actinoplanes sp. SE50/110 is known as the wild type producer of the alpha-glucosidase inhibitor acarbose, a potent drug used worldwide in the treatment of type-2 diabetes mellitus. As the incidence of diabetes is rapidly rising worldwide, an ever increasing demand for diabetes drugs, such as acarbose, needs to be anticipated. Consequently, derived Actinoplanes strains with increased acarbose yields are being used in large scale industrial batch fermentation since 1990 and were continuously optimized by conventional mutagenesis and screening experiments. This strategy reached its limits and is generally superseded by modern genetic engineering approaches. As a prerequisite for targeted genetic modifications, the complete genome sequence of the organism has to be known. Results Here, we present the complete genome sequence of Actinoplanes sp. SE50/110 [GenBank:CP003170], the first publicly available genome of the genus Actinoplanes, comprising various producers of pharmaceutically and economically important secondary metabolites. The genome features a high mean G + C content of 71.32% and consists of one circular chromosome with a size of 9,239,851 bp hosting 8,270 predicted protein coding sequences. Phylogenetic analysis of the core genome revealed a rather distant relation to other sequenced species of the family Micromonosporaceae whereas Actinoplanes utahensis was found to be the closest species based on 16S rRNA gene sequence comparison. Besides the already published acarbose biosynthetic gene cluster sequence, several new non-ribosomal peptide synthetase-, polyketide synthase- and hybrid-clusters were identified on the Actinoplanes genome. Another key feature of the genome represents the discovery of a functional actinomycete integrative and conjugative element. Conclusions The complete genome sequence of Actinoplanes sp. SE50/110 marks an important step towards the rational genetic optimization of the acarbose production. In this regard, the identified actinomycete integrative and conjugative element could play a central role by providing the basis for the development of a genetic transformation system for Actinoplanes sp. SE50/110 and other Actinoplanes spp. Furthermore, the identified non-ribosomal peptide synthetase- and polyketide synthase-clusters potentially encode new antibiotics and/or other bioactive compounds, which might be of pharmacologic interest. PMID:22443545

The complete genome sequence of the acarbose producer Actinoplanes sp. SE50/110.

PubMed

Schwientek, Patrick; Szczepanowski, Rafael; Rückert, Christian; Kalinowski, Jörn; Klein, Andreas; Selber, Klaus; Wehmeier, Udo F; Stoye, Jens; Pühler, Alfred

2012-03-23

Actinoplanes sp. SE50/110 is known as the wild type producer of the alpha-glucosidase inhibitor acarbose, a potent drug used worldwide in the treatment of type-2 diabetes mellitus. As the incidence of diabetes is rapidly rising worldwide, an ever increasing demand for diabetes drugs, such as acarbose, needs to be anticipated. Consequently, derived Actinoplanes strains with increased acarbose yields are being used in large scale industrial batch fermentation since 1990 and were continuously optimized by conventional mutagenesis and screening experiments. This strategy reached its limits and is generally superseded by modern genetic engineering approaches. As a prerequisite for targeted genetic modifications, the complete genome sequence of the organism has to be known. Here, we present the complete genome sequence of Actinoplanes sp. SE50/110 [GenBank:CP003170], the first publicly available genome of the genus Actinoplanes, comprising various producers of pharmaceutically and economically important secondary metabolites. The genome features a high mean G + C content of 71.32% and consists of one circular chromosome with a size of 9,239,851 bp hosting 8,270 predicted protein coding sequences. Phylogenetic analysis of the core genome revealed a rather distant relation to other sequenced species of the family Micromonosporaceae whereas Actinoplanes utahensis was found to be the closest species based on 16S rRNA gene sequence comparison. Besides the already published acarbose biosynthetic gene cluster sequence, several new non-ribosomal peptide synthetase-, polyketide synthase- and hybrid-clusters were identified on the Actinoplanes genome. Another key feature of the genome represents the discovery of a functional actinomycete integrative and conjugative element. The complete genome sequence of Actinoplanes sp. SE50/110 marks an important step towards the rational genetic optimization of the acarbose production. In this regard, the identified actinomycete integrative and conjugative element could play a central role by providing the basis for the development of a genetic transformation system for Actinoplanes sp. SE50/110 and other Actinoplanes spp. Furthermore, the identified non-ribosomal peptide synthetase- and polyketide synthase-clusters potentially encode new antibiotics and/or other bioactive compounds, which might be of pharmacologic interest.

Identification, Characterization, and Application of the Replicon Region of the Halophilic Temperate Sphaerolipovirus SNJ1.

PubMed

Wang, Yuchen; Sima, Linshan; Lv, Jie; Huang, Suiyuan; Liu, Ying; Wang, Jiao; Krupovic, Mart; Chen, Xiangdong

2016-07-15

The temperate haloarchaeal virus SNJ1 displays lytic and lysogenic life cycles. During the lysogenic cycle, the virus resides in its host, Natrinema sp. strain J7-1, in the form of an extrachromosomal circular plasmid, pHH205. In this study, a 3.9-kb region containing seven predicted genes organized in two operons was identified as the minimal replicon of SNJ1. Only RepA, encoded by open reading frame 11-12 (ORF11-12), was found to be essential for replication, and its expression increased during the lytic cycle. Sequence analysis suggested that RepA is a distant homolog of HUH endonucleases, a superfamily that includes rolling-circle replication initiation proteins from various viruses and plasmids. In addition to RepA, two genetic elements located within both termini of the 3.9-kb replicon were also required for SNJ1 replication. SNJ1 genome and SNJ1 replicon-based shuttle vectors were present at 1 to 3 copies per chromosome. However, the deletion of ORF4 significantly increased the SNJ1 copy number, suggesting that the product of ORF4 is a negative regulator of SNJ1 abundance. Shuttle vectors based on the SNJ1 replicon were constructed and validated for stable expression of heterologous proteins, both in J7 derivatives and in Natrinema pallidum JCM 8980(T), suggesting their broad applicability as genetic tools for Natrinema species. Archaeal viruses exhibit striking morphological diversity and unique gene content. In this study, the minimal replicon of the temperate haloarchaeal virus SNJ1 was identified. A number of ORFs and genetic elements controlling virus genome replication, maintenance, and copy number were characterized. In addition, based on the replicon, a novel expression shuttle vector has been constructed and validated for protein expression and purification in Natrinema sp. CJ7 and Natrinema pallidum JCM 8980(T) This study not only provided mechanistic and functional insights into SNJ1 replication but also led to the development of useful genetic tools to investigate SNJ1 and other viruses infecting Natrinema species as well as their hosts. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Transmission of ESBL-producing Enterobacteriaceae and their mobile genetic elements—identification of sources by whole genome sequencing: study protocol for an observational study in Switzerland

PubMed Central

Stadler, Tanja; Meinel, Dominik; Aguilar-Bultet, Lisandra; Huisman, Jana S; Schindler, Ruth; Egli, Adrian; Seth-Smith, Helena M B; Eichenberger, Lucas; Brodmann, Peter; Hübner, Philipp; Bagutti, Claudia; Tschudin-Sutter, Sarah

2018-01-01

Introduction Extended-spectrum beta-lactamases (ESBL)-producing Enterobacteriaceae were first described in relation with hospital-acquired infections. In the 2000s, the epidemiology of ESBL-producing organisms changed as especially ESBL-producing Escherichia coli was increasingly described as an important cause of community-acquired infections, supporting the hypothesis that in more recent years ESBL-producing Enterobacteriaceae have probably been imported into hospitals rather than vice versa. Transmission of ESBL-producing Enterobacteriaceae is complicated by ESBL genes being encoded on self-transmissible plasmids, which can be exchanged among the same and different bacterial species. The aim of this research project is to quantify hospital-wide transmission of ESBL-producing Enterobacteriaceae on both the level of bacterial species and the mobile genetic elements and to determine if hospital-acquired infections caused by ESBL producers are related to strains and mobile genetic elements predominantly circulating in the community or in the healthcare setting. This distinction is critical in prevention since the former emphasises the urgent need to establish or reinforce antibiotic stewardship programmes, and the latter would call for more rigorous infection control. Methods and analysis This protocol presents an observational study that will be performed at the University Hospital Basel and in the city of Basel, Switzerland. ESBL-producing Enterobacteriaceae will be collected from any specimens obtained by routine clinical practice or by active screening in both inpatient and outpatient settings, as well as from wastewater samples and foodstuffs, both collected monthly over a 12-month period for analyses by whole genome sequencing. Bacterial chromosomal, plasmid and ESBL-gene sequences will be compared within the cohort to determine genetic relatedness and migration between humans and their environment. Ethics and dissemination This study has been approved by the local ethics committee (Ethikkommission Nordwest-und Zentralschweiz) as a quality control project (Project-ID 2017–00100). The results of this study will be published in peer-reviewed medical journals, communicated to participants, the general public and all relevant stakeholders. PMID:29455172

pHlash: A New Genetically Encoded and Ratiometric Luminescence Sensor of Intracellular pH

PubMed Central

Robertson, J. Brian; Johnson, Carl Hirschie

2012-01-01

We report the development of a genetically encodable and ratiometic pH probe named “pHlash” that utilizes Bioluminescence Resonance Energy Transfer (BRET) rather than fluorescence excitation. The pHlash sensor–composed of a donor luciferase that is genetically fused to a Venus fluorophore–exhibits pH dependence of its spectral emission in vitro. When expressed in either yeast or mammalian cells, pHlash reports basal pH and cytosolic acidification in vivo. Its spectral ratio response is H+ specific; neither Ca++, Mg++, Na+, nor K+ changes the spectral form of its luminescence emission. Moreover, it can be used to image pH in single cells. This is the first BRET-based sensor of H+ ions, and it should allow the approximation of pH in cytosolic and organellar compartments in applications where current pH probes are inadequate. PMID:22905204

Optogenetics: a new enlightenment age for zebrafish neurobiology.

PubMed

Del Bene, Filippo; Wyart, Claire

2012-03-01

Zebrafish became a model of choice for neurobiology because of the transparency of its brain and because of its amenability to genetic manipulation. In particular, at early stages of development the intact larva is an ideal system to apply optical techniques for deep imaging in the nervous system, as well as genetically encoded tools for targeting subsets of neurons and monitoring and manipulating their activity. For these applications,new genetically encoded optical tools, fluorescent sensors, and light-gated channels have been generated,creating the field of "optogenetics." It is now possible to monitor and control neuronal activity with minimal perturbation and unprecedented spatio-temporal resolution.We describe here the main achievements that have occurred in the last decade in imaging and manipulating neuronal activity in intact zebrafish larvae. We provide also examples of functional dissection of neuronal circuits achieved with the applications of these techniques in the visual and locomotor systems.

pHlash: a new genetically encoded and ratiometric luminescence sensor of intracellular pH.

PubMed

Zhang, Yunfei; Xie, Qiguang; Robertson, J Brian; Johnson, Carl Hirschie

2012-01-01

We report the development of a genetically encodable and ratiometic pH probe named "pHlash" that utilizes Bioluminescence Resonance Energy Transfer (BRET) rather than fluorescence excitation. The pHlash sensor-composed of a donor luciferase that is genetically fused to a Venus fluorophore-exhibits pH dependence of its spectral emission in vitro. When expressed in either yeast or mammalian cells, pHlash reports basal pH and cytosolic acidification in vivo. Its spectral ratio response is H(+) specific; neither Ca(++), Mg(++), Na(+), nor K(+) changes the spectral form of its luminescence emission. Moreover, it can be used to image pH in single cells. This is the first BRET-based sensor of H(+) ions, and it should allow the approximation of pH in cytosolic and organellar compartments in applications where current pH probes are inadequate.

In silico Evolutionary Developmental Neurobiology and the Origin of Natural Language

NASA Astrophysics Data System (ADS)

Szathmáry, Eörs; Szathmáry, Zoltán; Ittzés, Péter; Orbaán, Geroő; Zachár, István; Huszár, Ferenc; Fedor, Anna; Varga, Máté; Számadó, Szabolcs

It is justified to assume that part of our genetic endowment contributes to our language skills, yet it is impossible to tell at this moment exactly how genes affect the language faculty. We complement experimental biological studies by an in silico approach in that we simulate the evolution of neuronal networks under selection for language-related skills. At the heart of this project is the Evolutionary Neurogenetic Algorithm (ENGA) that is deliberately biomimetic. The design of the system was inspired by important biological phenomena such as brain ontogenesis, neuron morphologies, and indirect genetic encoding. Neuronal networks were selected and were allowed to reproduce as a function of their performance in the given task. The selected neuronal networks in all scenarios were able to solve the communication problem they had to face. The most striking feature of the model is that it works with highly indirect genetic encoding--just as brains do.

Ligand interaction scan: a general method for engineering ligand-sensitive protein alleles.

PubMed

Erster, Oran; Eisenstein, Miriam; Liscovitch, Mordechai

2007-05-01

The ligand interaction scan (LIScan) method is a general procedure for engineering small molecule ligand-regulated forms of a protein that is complementary to other 'reverse' genetic and chemical-genetic methods for drug-target validation. It involves insertional mutagenesis by a chemical-genetic 'switch', comprising a genetically encoded peptide module that binds with high affinity to a small-molecule ligand. We demonstrated the method with TEM-1 beta-lactamase, using a tetracysteine hexapeptide insert and a biarsenical fluorescein ligand (FlAsH).

Genetically Engineered Cyanobacteria

NASA Technical Reports Server (NTRS)

Zhou, Ruanbao (Inventor); Gibbons, William (Inventor)

2015-01-01

The disclosed embodiments provide cyanobacteria spp. that have been genetically engineered to have increased production of carbon-based products of interest. These genetically engineered hosts efficiently convert carbon dioxide and light into carbon-based products of interest such as long chained hydrocarbons. Several constructs containing polynucleotides encoding enzymes active in the metabolic pathways of cyanobacteria are disclosed. In many instances, the cyanobacteria strains have been further genetically modified to optimize production of the carbon-based products of interest. The optimization includes both up-regulation and down-regulation of particular genes.

Is junk DNA bunk? A critique of ENCODE

PubMed Central

Doolittle, W. Ford

2013-01-01

Do data from the Encyclopedia Of DNA Elements (ENCODE) project render the notion of junk DNA obsolete? Here, I review older arguments for junk grounded in the C-value paradox and propose a thought experiment to challenge ENCODE’s ontology. Specifically, what would we expect for the number of functional elements (as ENCODE defines them) in genomes much larger than our own genome? If the number were to stay more or less constant, it would seem sensible to consider the rest of the DNA of larger genomes to be junk or, at least, assign it a different sort of role (structural rather than informational). If, however, the number of functional elements were to rise significantly with C-value then, (i) organisms with genomes larger than our genome are more complex phenotypically than we are, (ii) ENCODE’s definition of functional element identifies many sites that would not be considered functional or phenotype-determining by standard uses in biology, or (iii) the same phenotypic functions are often determined in a more diffuse fashion in larger-genomed organisms. Good cases can be made for propositions ii and iii. A larger theoretical framework, embracing informational and structural roles for DNA, neutral as well as adaptive causes of complexity, and selection as a multilevel phenomenon, is needed. PMID:23479647

Localization and physical mapping of genes encoding the A+U-rich element RNA-binding protein AUF1 to human chromosomes 4 and X

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

Wagner, B.J.; Long, L.; Pettenati, M.J.

Messenger RNAs encoding many oncoproteins and cytokines are relatively unstable. Their instability, which ensures appropriate levels and timing of expression, is controlled in part by proteins that bind to A + U-rich instability elements (AREs) present in the 3{prime}-untranslated regions of the mRNAs. cDNAs encoding the AUF1 family of ARE-binding proteins were cloned from human and murine cDNA libraries. In the present study monochromosomal somatic cell hybrids were used to localize two AUF1 loci to human chromosomes 4 and X. In situ hybridization analyses using P1 clones as probes identified the 4q21.1-q21.2 and Xq12 regions as the locations of themore » AUF1 genes. 10 refs., 2 figs.« less

Identifying Genetic Sources of Phenotypic Heterogeneity in Orofacial Clefts by Targeted Sequencing.

PubMed

Carlson, Jenna C; Taub, Margaret A; Feingold, Eleanor; Beaty, Terri H; Murray, Jeffrey C; Marazita, Mary L; Leslie, Elizabeth J

2017-07-17

Orofacial clefts (OFCs), including nonsyndromic cleft lip with or without cleft palate (NSCL/P), are common birth defects. NSCL/P is highly heterogeneous with multiple phenotypic presentations. Two common subtypes of NSCL/P are cleft lip (CL) and cleft lip with cleft palate (CLP) which have different population prevalence. Similarly, NSCL/P can be divided into bilateral and unilateral clefts, with unilateral being the most common. Individuals with unilateral NSCL/P are more likely to be affected on the left side of the upper lip, but right side affection also occurs. Moreover, NSCL/P is twice as common in males as in females. The goal of this study is to discover genetic variants that have different effects in case subgroups. We conducted both common variant and rare variant analyses in 1034 individuals of Asian ancestry with NSCL/P, examining four sources of heterogeneity within CL/P: cleft type, sex, laterality, and side. We identified several regions associated with subtype differentiation: cleft type differences in 8q24 (p = 1.00 × 10 -4 ), laterality differences in IRF6, a gene previously implicated with wound healing (p = 2.166 × 10 -4 ), sex differences and side of unilateral CL differences in FGFR2 (p = 3.00 × 10 -4 ; p = 6.00 × 10 -4 ), and sex differences in VAX1 (p < 1.00 × 10 -4 ) among others. Many of the regions associated with phenotypic modification were either adjacent to or overlapping functional elements based on ENCODE chromatin marks and published craniofacial enhancers. We have identified multiple common and rare variants as potential phenotypic modifiers of NSCL/P, and suggest plausible elements responsible for phenotypic heterogeneity, further elucidating the complex genetic architecture of OFCs. Birth Defects Research 109:1030-1038, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Genetic diversity of genes encoding OKP and LEN beta-lactamases produced by clinical Klebsiella pneumoniae strains in Portugal.

PubMed

Mendonça, Nuno; Ferreira, Eugénia; Caniça, Manuela

2009-03-01

Of the 308 clinical Klebsiella pneumoniae strains collected in 21 Portuguese health institutions, 11 encoded for LEN and 9 for OKP enzymes; of these, 15 were new enzymes. Ninety-one percent of LEN and all OKP producer strains were resistant to amoxicillin. We demonstrate that these beta-lactamase were highly diverse.

Mutations of the X-linked genes encoding neuroligins NLGN3 and NLGN4 are associated with autism.

PubMed

Jamain, Stéphane; Quach, Hélène; Betancur, Catalina; Råstam, Maria; Colineaux, Catherine; Gillberg, I Carina; Soderstrom, Henrik; Giros, Bruno; Leboyer, Marion; Gillberg, Christopher; Bourgeron, Thomas

2003-05-01

Many studies have supported a genetic etiology for autism. Here we report mutations in two X-linked genes encoding neuroligins NLGN3 and NLGN4 in siblings with autism-spectrum disorders. These mutations affect cell-adhesion molecules localized at the synapse and suggest that a defect of synaptogenesis may predispose to autism.

Hydrogenase polypeptide and methods of use

DOEpatents

Adams, Michael W.W.; Hopkins, Robert C.; Jenney, JR, Francis E.; Sun, Junsong

2016-02-02

Provided herein are polypeptides having hydrogenase activity. The polypeptide may be multimeric, and may have hydrogenase activity of at least 0.05 micromoles H.sub.2 produced min.sup.-1 mg protein.sup.-1. Also provided herein are polynucleotides encoding the polypeptides, genetically modified microbes that include polynucleotides encoding one or more subunits of the multimeric polypeptide, and methods for making and using the polypeptides.

Origin and evolution of SINEs in eukaryotic genomes.

PubMed

Kramerov, D A; Vassetzky, N S

2011-12-01

Short interspersed elements (SINEs) are one of the two most prolific mobile genomic elements in most of the higher eukaryotes. Although their biology is still not thoroughly understood, unusual life cycle of these simple elements amplified as genomic parasites makes their evolution unique in many ways. In contrast to most genetic elements including other transposons, SINEs emerged de novo many times in evolution from available molecules (for example, tRNA). The involvement of reverse transcription in their amplification cycle, huge number of genomic copies and modular structure allow variation mechanisms in SINEs uncommon or rare in other genetic elements (module exchange between SINE families, dimerization, and so on.). Overall, SINE evolution includes their emergence, progressive optimization and counteraction to the cell's defense against mobile genetic elements.

Method and apparatus for optical encoding with compressible imaging

NASA Technical Reports Server (NTRS)

Leviton, Douglas B. (Inventor)

2006-01-01

The present invention presents an optical encoder with increased conversion rates. Improvement in the conversion rate is a result of combining changes in the pattern recognition encoder's scale pattern with an image sensor readout technique which takes full advantage of those changes, and lends itself to operation by modern, high-speed, ultra-compact microprocessors and digital signal processors (DSP) or field programmable gate array (FPGA) logic elements which can process encoder scale images at the highest speeds. Through these improvements, all three components of conversion time (reciprocal conversion rate)--namely exposure time, image readout time, and image processing time--are minimized.

Whole-exome sequencing reveals genetic variants associated with chronic kidney disease characterized by tubulointerstitial damages in North Central Region, Sri Lanka.

PubMed

Nanayakkara, Shanika; Senevirathna, S T M L D; Parahitiyawa, Nipuna B; Abeysekera, Tilak; Chandrajith, Rohana; Ratnatunga, Neelakanthi; Hitomi, Toshiaki; Kobayashi, Hatasu; Harada, Kouji H; Koizumi, Akio

2015-09-01

The familial clustering observed in chronic kidney disease of uncertain etiology (CKDu) characterized by tubulointerstitial damages in the North Central Region of Sri Lanka strongly suggests the involvement of genetic factors in its pathogenesis. The objective of the present study is to use whole-exome sequencing to identify the genetic variants associated with CKDu. Whole-exome sequencing of eight CKDu cases and eight controls was performed, followed by direct sequencing of candidate loci in 301 CKDu cases and 276 controls. Association study revealed rs34970857 (c.658G > A/p.V220M) located in the KCNA10 gene encoding a voltage-gated K channel as the most promising SNP with the highest odds ratio of 1.74. Four rare variants were identified in gene encoding Laminin beta2 (LAMB2) which is known to cause congenital nephrotic syndrome. Three out of four variants in LAMB2 were novel variants found exclusively in cases. Genetic investigations provide strong evidence on the presence of genetic susceptibility for CKDu. Possibility of presence of several rare variants associated with CKDu in this population is also suggested.

Acral peeling skin syndrome resulting from a homozygous nonsense mutation in the CSTA gene encoding cystatin A.

PubMed

Krunic, Aleksandar L; Stone, Kristina L; Simpson, Michael A; McGrath, John A

2013-01-01

Acral peeling skin syndrome (APSS) is a clinically and genetically heterogeneous disorder. We used whole-exome sequencing to identify the molecular basis of APSS in a consanguineous Jordanian-American pedigree. We identified a homozygous nonsense mutation (p.Lys22X) in the CSTA gene, encoding cystatin A, that was confirmed using Sanger sequencing. Cystatin A is a protease inhibitor found in the cornified cell envelope, and loss-of-function mutations have previously been reported in two cases of exfoliative ichthyosis. Our study expands the molecular pathology of APSS and demonstrates the value of next-generation sequencing in the genetic characterization of inherited skin diseases. © 2013 Wiley Periodicals, Inc.

Quantitative and Comparative Profiling of Protease Substrates through a Genetically Encoded Multifunctional Photocrosslinker.

PubMed

He, Dan; Xie, Xiao; Yang, Fan; Zhang, Heng; Su, Haomiao; Ge, Yun; Song, Haiping; Chen, Peng R

2017-11-13

A genetically encoded, multifunctional photocrosslinker was developed for quantitative and comparative proteomics. By bearing a bioorthogonal handle and a releasable linker in addition to its photoaffinity warhead, this probe enables the enrichment of transient and low-abundance prey proteins after intracellular photocrosslinking and prey-bait separation, which can be subject to stable isotope dimethyl labeling and mass spectrometry analysis. This quantitative strategy (termed isoCAPP) allowed a comparative proteomic approach to be adopted to identify the proteolytic substrates of an E. coli protease-chaperone dual machinery DegP. Two newly identified substrates were subsequently confirmed by proteolysis experiments. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

De novo mutations in the gene encoding the synaptic scaffolding protein SHANK3 in patients ascertained for schizophrenia

PubMed Central

Gauthier, Julie; Champagne, Nathalie; Lafrenière, Ronald G.; Xiong, Lan; Spiegelman, Dan; Brustein, Edna; Lapointe, Mathieu; Peng, Huashan; Côté, Mélanie; Noreau, Anne; Hamdan, Fadi F.; Addington, Anjené M.; Rapoport, Judith L.; DeLisi, Lynn E.; Krebs, Marie-Odile; Joober, Ridha; Fathalli, Ferid; Mouaffak, Fayçal; Haghighi, Ali P.; Néri, Christian; Dubé, Marie-Pierre; Samuels, Mark E.; Marineau, Claude; Stone, Eric A.; Awadalla, Philip; Barker, Philip A.; Carbonetto, Salvatore; Drapeau, Pierre; Rouleau, Guy A.

2010-01-01

Schizophrenia likely results from poorly understood genetic and environmental factors. We studied the gene encoding the synaptic protein SHANK3 in 285 controls and 185 schizophrenia patients with unaffected parents. Two de novo mutations (R1117X and R536W) were identified in two families, one being found in three affected brothers, suggesting germline mosaicism. Zebrafish and rat hippocampal neuron assays revealed behavior and differentiation defects resulting from the R1117X mutant. As mutations in SHANK3 were previously reported in autism, the occurrence of SHANK3 mutations in subjects with a schizophrenia phenotype suggests a molecular genetic link between these two neurodevelopmental disorders. PMID:20385823

Catching the engram: strategies to examine the memory trace.

PubMed

Sakaguchi, Masanori; Hayashi, Yasunori

2012-09-21

Memories are stored within neuronal ensembles in the brain. Modern genetic techniques can be used to not only visualize specific neuronal ensembles that encode memories (e.g., fear, craving) but also to selectively manipulate those neurons. These techniques are now being expanded for the study of various types of memory. In this review, we will summarize the genetic methods used to visualize and manipulate neurons involved in the representation of memory engrams. The methods will help clarify how memory is encoded, stored and processed in the brain. Furthermore, these approaches may contribute to our understanding of the pathological mechanisms associated with human memory disorders and, ultimately, may aid the development of therapeutic strategies to ameliorate these diseases.

An Oral DNA Vaccine Encoding Endoglin Eradicates Breast Tumors by Blocking Their Blood Supply

DTIC Science & Technology

2006-05-01

W81XWH-04-1-0489 TITLE: An Oral DNA Vaccine Encoding Endoglin Eradicates Breast Tumors by Blocking Their Blood Supply PRINCIPAL...Encoding Endoglin Eradicates Breast Tumors by Blocking Their Blood Supply 5b. GRANT NUMBER W81XWH-04-1-0489 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR...blocking renewal of blood vessel growth in the tumor bed, have been proposed as suitable antitumor strategies. Endoglin (CD105) is a suitable

BAIAP2 is related to emotional modulation of human memory strength.

PubMed

Luksys, Gediminas; Ackermann, Sandra; Coynel, David; Fastenrath, Matthias; Gschwind, Leo; Heck, Angela; Rasch, Bjoern; Spalek, Klara; Vogler, Christian; Papassotiropoulos, Andreas; de Quervain, Dominique

2014-01-01

Memory performance is the result of many distinct mental processes, such as memory encoding, forgetting, and modulation of memory strength by emotional arousal. These processes, which are subserved by partly distinct molecular profiles, are not always amenable to direct observation. Therefore, computational models can be used to make inferences about specific mental processes and to study their genetic underpinnings. Here we combined a computational model-based analysis of memory-related processes with high density genetic information derived from a genome-wide study in healthy young adults. After identifying the best-fitting model for a verbal memory task and estimating the best-fitting individual cognitive parameters, we found a common variant in the gene encoding the brain-specific angiogenesis inhibitor 1-associated protein 2 (BAIAP2) that was related to the model parameter reflecting modulation of verbal memory strength by negative valence. We also observed an association between the same genetic variant and a similar emotional modulation phenotype in a different population performing a picture memory task. Furthermore, using functional neuroimaging we found robust genotype-dependent differences in activity of the parahippocampal cortex that were specifically related to successful memory encoding of negative versus neutral information. Finally, we analyzed cortical gene expression data of 193 deceased subjects and detected significant BAIAP2 genotype-dependent differences in BAIAP2 mRNA levels. Our findings suggest that model-based dissociation of specific cognitive parameters can improve the understanding of genetic underpinnings of human learning and memory.

Pharmacogenetics and human molecular genetics of opiate and cocaine addictions and their treatments.

PubMed

Kreek, Mary Jeanne; Bart, Gavin; Lilly, Charles; LaForge, K Steven; Nielsen, David A

2005-03-01

Opiate and cocaine addictions are major social and medical problems that impose a significant burden on society. Despite the size and scope of these problems, there are few effective treatments for these addictions. Methadone maintenance is an effective and most widely used treatment for opiate addiction, allowing normalization of many physiological abnormalities caused by chronic use of short-acting opiates. There are no pharmacological treatments for cocaine addiction. Epidemiological, linkage, and association studies have demonstrated a significant contribution of genetic factors to the addictive diseases. This article reviews the molecular genetics and pharmacogenetics of opiate and cocaine addictions, focusing primarily on genes of the opioid and monoaminergic systems that have been associated with or have evidence for linkage to opiate or cocaine addiction. This evidence has been marshalled either through identification of variant alleles that lead to functional alterations of gene products, altered gene expression, or findings of linkage or association studies. Studies of polymorphisms in the mu opioid receptor gene, which encodes the receptor target of some endogenous opioids, heroin, morphine, and synthetic opioids, have contributed substantially to knowledge of genetic influences on opiate and cocaine addiction. Other genes of the endogenous opioid and monoaminergic systems, particularly genes encoding dopamine beta-hydroxylase, and the dopamine, serotonin, and norepinephrine transporters have also been implicated. Variants in genes encoding proteins involved in metabolism or biotransformation of drugs of abuse and also of treatment agents are reviewed.

Identification of a peroxisome proliferator-responsive element upstream of the gene encoding rat peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase.

PubMed Central

Zhang, B; Marcus, S L; Sajjadi, F G; Alvares, K; Reddy, J K; Subramani, S; Rachubinski, R A; Capone, J P

1992-01-01

Ciprofibrate, a hypolipidemic drug that acts as a peroxisome proliferator, induces the transcription of genes encoding peroxisomal beta-oxidation enzymes. To identify cis-acting promoter elements involved in this induction, 5.8 kilobase pairs of promoter sequence from the gene encoding rat peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (EC 4.2.1.17/EC 1.1.1.35) was inserted upstream of a luciferase reporter gene. Transfection of this expression vector into rat hepatoma H4IIEC3 cells in the presence of ciprofibrate resulted in a 5- to 10-fold, cell type-specific increase in luciferase activity as compared to cells transfected in the absence of drug. A peroxisome proliferator-responsive element (PPRE) was localized to a 196-nucleotide region centered at position -2943 from the transcription start site. This PPRE conferred ciprofibrate responsiveness on a heterologous promoter and functioned independently of orientation or position. Gel retardation analysis with nuclear extracts demonstrated that ciprofibrate-treated or untreated H4IIEC3 cells, but not HeLa cells or monkey kidney cells, contained sequence-specific DNA binding factors that interact with the PPRE. These results have implications for understanding the mechanisms of coordinated transcriptional induction of genes encoding peroxisomal proteins by hypolipidemic agents and other peroxisome proliferators. Images PMID:1502166

Lanthanide-Containing Polymer Microspheres by Multiple-Stage Dispersion Polymerization for Highly Multiplexed Bioassays

PubMed Central

Abdelrahman, Ahmed I.; Dai, Sheng; Thickett, Stuart C.; Ornatsky, Olga; Bandura, Dmitry; Baranov, Vladimir; Winnik, Mitchell A.

2009-01-01

We describe the synthesis and characterization of metal-encoded polystyrene microspheres by multiple-stage dispersion polymerization with diameters on the order of 2 µm and a very narrow size distribution. Different lanthanides were loaded into these microspheres through the addition of a mixture of LnCl3 salts and excess acrylic acid or acetoacetylethyl methacrylate (AAEM) dissolved in ethanol to the reaction after about 10% conversion of styrene, i.e., well after the particle nucleation stage was complete. Individual microspheres contain ca. 106 – 108 chelated lanthanide ions, of either a single element or a mixture of elements. These microspheres were characterized one-by-one utilizing a novel mass cytometer with an inductively coupled plasma (ICP) ionization source and time-of-flight (TOF) mass spectrometry detection. Microspheres containing a range of different metals at different levels of concentration were synthesized to meet the requirements of binary encoding and enumeration encoding protocols. With four different metals at five levels of concentration, we could achieve a variability of 624, and the strategy we report should allow one to obtain much larger variability. To demonstrate the usefulness of element-encoded beads for highly multiplexed immunoassays, we carried out a proof-of-principle model bioassay involving conjugation of mouse IgG to the surface of La and Tm containing particles, and its detection by an anti-mouse IgG bearing a metal-chelating polymer with Pr. PMID:19807075

Lanthanide-containing polymer microspheres by multiple-stage dispersion polymerization for highly multiplexed bioassays.

PubMed

Abdelrahman, Ahmed I; Dai, Sheng; Thickett, Stuart C; Ornatsky, Olga; Bandura, Dmitry; Baranov, Vladimir; Winnik, Mitchell A

2009-10-28

We describe the synthesis and characterization of metal-encoded polystyrene microspheres by multiple-stage dispersion polymerization with diameters on the order of 2 mum and a very narrow size distribution. Different lanthanides were loaded into these microspheres through the addition of a mixture of lanthanide salts (LnCl(3)) and excess acrylic acid (AA) or acetoacetylethyl methacrylate (AAEM) dissolved in ethanol to the reaction after about 10% conversion of styrene, that is, well after the particle nucleation stage was complete. Individual microspheres contain ca. 10(6)-10(8) chelated lanthanide ions, of either a single element or a mixture of elements. These microspheres were characterized one-by-one utilizing a novel mass cytometer with an inductively coupled plasma (ICP) ionization source and time-of-flight (TOF) mass spectrometry detection. Microspheres containing a range of different metals at different levels of concentration were synthesized to meet the requirements of binary encoding and enumeration encoding protocols. With four different metals at five levels of concentration, we could achieve a variability of 624, and the strategy we report should allow one to obtain much larger variability. To demonstrate the usefulness of element-encoded beads for highly multiplexed immunoassays, we carried out a proof-of-principle model bioassay involving conjugation of mouse IgG to the surface of La and Tm containing particles and its detection by an antimouse IgG bearing a metal-chelating polymer with Pr.

Parasite genetics and the immune host: recombination between antigenic types of Eimeria maxima as an entrée to the identification of protective antigens.

PubMed

Blake, Damer P; Hesketh, Patricia; Archer, Andrew; Carroll, Fionnadh; Smith, Adrian L; Shirley, Martin W

2004-11-01

The genomes of protozoan parasites encode thousands of gene products and identification of the subset that stimulates a protective immune response is a daunting task. Most screens for vaccine candidates identify molecules by capacity to induce immune responses rather than protection. This paper describes the core findings of a strategy developed with the coccidial parasite Eimeria maxima to rationally identify loci within its genome that encode immunoprotective antigens. Our strategy uses a novel combination of parasite genetics, DNA fingerprinting, drug-resistance and strain-specific immunity and centres on two strains of E. maxima that each induce a lethal strain-specific protective immune response in the host and show a differential response to anti-Eimeria chemotherapy. Through classical mating studies with these strains we have demonstrated that loci encoding molecules stimulating strain-specific protective immunity or resistance to the anti-coccidial drug robenidine segregate independently. Furthermore, passage of populations of recombinant parasites in the face of killing in the immune host was accompanied by the elimination of some polymorphic DNA markers defining the parent strain used to immunise the host. Consideration of the numbers of parasites recombinant for the two traits implicates very few antigen-encoding loci. Our data provide a potential strategy to identify putative antigen-encoding loci in other parasites.

Genetic diversity among major endemic strains of Leptospira interrogans in China

PubMed Central

He, Ping; Sheng, Yue-Ying; Shi, Yao-Zhou; Jiang, Xiu-Gao; Qin, Jin-Hong; Zhang, Zhi-Ming; Zhao, Guo-Ping; Guo, Xiao-Kui

2007-01-01

Background Leptospirosis is a world-widely distributed zoonosis. Humans become infected via exposure to pathogenic Leptospira spp. from contaminated water or soil. The availability of genomic sequences of Leptospira interrogans serovar Lai and serovar Copenhageni opened up opportunities to identify genetic diversity among different pathogenic strains of L. interrogans representing various kinds of serotypes (serogroups and serovars). Results Comparative genomic hybridization (CGH) analysis was used to compare the gene content of L. interrogans serovar Lai strain Lai with that of other 10 L. interrogans strains prevailed in China and one identified from Brazil using a microarray spotted with 3,528 protein coding sequences (CDSs) of strain Lai. The cutoff ratio of sample/reference (S/R) hybridization for detecting the absence of genes from one tested strain was set by comparing the ratio of S/R hybridization and the in silico sequence similarities of strain Lai and serovar Copenhageni strain Fiocruz L1-130. Among the 11 strains tested, 275 CDSs were found absent from at least one strain. The common backbone of the L. interrogans genome was estimated to contain about 2,917 CDSs. The genes encoding fundamental cellular functions such as translation, energy production and conversion were conserved. While strain-specific genes include those that encode proteins related to either cell surface structures or carbohydrate transport and metabolism. We also found two genomic islands (GIs) in strain Lai containing genes divergently absent in other strains. Because genes encoding proteins with potential pathogenic functions are located within GIs, these elements might contribute to the variations in disease manifestation. Differences in genes involved in O-antigen biosynthesis were also identified for strains belonging to different serogroups, which offers an opportunity for future development of genomic typing tools for serological classification. Conclusion CGH analyses for pathogenic leptospiral strains prevailed in China against the L. interrogans serovar Lai strain Lai CDS-spotted microarrays revealed 2,917 common backbone CDSs and strain specific genes encoding proteins mainly related to cell surface structures and carbohydrated transport/metabolism. Of the 275 CDSs considered absent from at least one of the L. interrogans strains tested, most of them were clustered in the rfb gene cluster and two putative genomic islands (GI A and B) in strain Lai. The strain-specific genes detected via this work will provide a knowledge base for further investigating the pathogenesis of L interrogans and/or for the development of effective vaccines and/or diagnostic tools. PMID:17603913

Principles of metadata organization at the ENCODE data coordination center

PubMed Central

Hong, Eurie L.; Sloan, Cricket A.; Chan, Esther T.; Davidson, Jean M.; Malladi, Venkat S.; Strattan, J. Seth; Hitz, Benjamin C.; Gabdank, Idan; Narayanan, Aditi K.; Ho, Marcus; Lee, Brian T.; Rowe, Laurence D.; Dreszer, Timothy R.; Roe, Greg R.; Podduturi, Nikhil R.; Tanaka, Forrest; Hilton, Jason A.; Cherry, J. Michael

2016-01-01

The Encyclopedia of DNA Elements (ENCODE) Data Coordinating Center (DCC) is responsible for organizing, describing and providing access to the diverse data generated by the ENCODE project. The description of these data, known as metadata, includes the biological sample used as input, the protocols and assays performed on these samples, the data files generated from the results and the computational methods used to analyze the data. Here, we outline the principles and philosophy used to define the ENCODE metadata in order to create a metadata standard that can be applied to diverse assays and multiple genomic projects. In addition, we present how the data are validated and used by the ENCODE DCC in creating the ENCODE Portal (https://www.encodeproject.org/). Database URL: www.encodeproject.org PMID:26980513

Parallel and competitive processes in hierarchical analysis: perceptual grouping and encoding of closure.

PubMed

Han, S; Humphreys, G W; Chen, L

1999-10-01

The role of perceptual grouping and the encoding of closure of local elements in the processing of hierarchical patterns was studied. Experiments 1 and 2 showed a global advantage over the local level for 2 tasks involving the discrimination of orientation and closure, but there was a local advantage for the closure discrimination task relative to the orientation discrimination task. Experiment 3 showed a local precedence effect for the closure discrimination task when local element grouping was weakened by embedding the stimuli from Experiment 1 in a background made up of cross patterns. Experiments 4A and 4B found that dissimilarity of closure between the local elements of hierarchical stimuli and the background figures could facilitate the grouping of closed local elements and enhanced the perception of global structure. Experiment 5 showed that the advantage for detecting the closure of local elements in hierarchical analysis also held under divided- and selective-attention conditions. Results are consistent with the idea that grouping between local elements takes place in parallel and competes with the computation of closure of local elements in determining the selection between global and local levels of hierarchical patterns for response.

Pathogenicity Island Cross Talk Mediated by Recombination Directionality Factors Facilitates Excision from the Chromosome.

PubMed

Carpenter, Megan R; Rozovsky, Sharon; Boyd, E Fidelma

2015-12-14

Pathogenicity islands (PAIs) are mobile integrated genetic elements (MIGEs) that contain a diverse range of virulence factors and are essential in the evolution of pathogenic bacteria. PAIs are widespread among bacteria and integrate into the host genome, commonly at a tRNA locus, via integrase-mediated site-specific recombination. The excision of PAIs is the first step in the horizontal transfer of these elements and is not well understood. In this study, we examined the role of recombination directionality factors (RDFs) and their relationship with integrases in the excision of two PAIs essential for Vibrio cholerae host colonization: Vibrio pathogenicity island 1 (VPI-1) and VPI-2. VPI-1 does not contain an RDF, which allowed us to answer the question of whether RDFs are an absolute requirement for excision. We found that an RDF was required for efficient excision of VPI-2 but not VPI-1 and that RDFs can induce excision of both islands. Expression data revealed that the RDFs act as transcriptional repressors to both VPI-1- and VPI-2-encoded integrases. We demonstrated that the RDFs Vibrio excision factor A (VefA) and VefB bind at the attachment sites (overlapping the int promoter region) of VPI-1 and VPI-2, thus supporting this mode of integrase repression. In addition, V. cholerae RDFs are promiscuous due to their dual functions of promoting excision of both VPI-1 and VPI-2 and acting as negative transcriptional regulators of the integrases. This is the first demonstration of cross talk between PAIs mediated via RDFs which reveals the complex interactions that occur between separately acquired MIGEs. Deciphering the mechanisms of pathogenicity island excision is necessary for understanding the evolution and spread of these elements to their nonpathogenic counterparts. Such mechanistic insight would assist in predicting the mobility of uncharacterized genetic elements. This study identified extensive RDF-mediated cross talk between two nonhomologous VPIs and demonstrated the dual functionality of RDF proteins: (i) inducing PAI excision and (ii) acting as transcriptional regulators. Findings from this study may be implicated in determining the mobilome contribution of other bacteria with multiple MIGEs. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Fault detection and bypass in a sequence information signal processor

NASA Technical Reports Server (NTRS)

Peterson, John C. (Inventor); Chow, Edward T. (Inventor)

1992-01-01

The invention comprises a plurality of scan registers, each such register respectively associated with a processor element; an on-chip comparator, encoder and fault bypass register. Each scan register generates a unitary signal the logic state of which depends on the correctness of the input from the previous processor in the systolic array. These unitary signals are input to a common comparator which generates an output indicating whether or not an error has occurred. These unitary signals are also input to an encoder which identifies the location of any fault detected so that an appropriate multiplexer can be switched to bypass the faulty processor element. Input scan data can be readily programmed to fully exercise all of the processor elements so that no fault can remain undetected.

The Translational Apparatus of Plastids and Its Role in Plant Development

PubMed Central

Tiller, Nadine; Bock, Ralph

2014-01-01

Chloroplasts (plastids) possess a genome and their own machinery to express it. Translation in plastids occurs on bacterial-type 70S ribosomes utilizing a set of tRNAs that is entirely encoded in the plastid genome. In recent years, the components of the chloroplast translational apparatus have been intensely studied by proteomic approaches and by reverse genetics in the model systems tobacco (plastid-encoded components) and Arabidopsis (nucleus-encoded components). This work has provided important new insights into the structure, function, and biogenesis of chloroplast ribosomes, and also has shed fresh light on the molecular mechanisms of the translation process in plastids. In addition, mutants affected in plastid translation have yielded strong genetic evidence for chloroplast genes and gene products influencing plant development at various levels, presumably via retrograde signaling pathway(s). In this review, we describe recent progress with the functional analysis of components of the chloroplast translational machinery and discuss the currently available evidence that supports a significant impact of plastid translational activity on plant anatomy and morphology. PMID:24589494

Persistence of antigen is required to maintain transplantation tolerance induced by genetic modification of bone marrow stem cells.

PubMed

Tian, C; Bagley, J; Iacomini, J

2006-09-01

Genetic modification of hematopoietic stem cells (HSCs) resulting in a state of molecular chimerism can be used to induce donor-specific tolerance to allografts. However, the requirements for maintaining tolerance in molecular chimeras remain unknown. Here, we examined whether long-term expression of a retrovirally encoded alloantigen in hematopoietic cells is required to maintain donor-specific tolerance in molecular chimeras. To this end, mice were reconstituted with syngeneic bone marrow transduced with retroviruses carrying the gene encoding the allogeneic MHC class I molecule Kb. Following induction of molecular chimerism, mice were depleted of cells expressing Kb by administration of the anti-Kb monoclonal antibody Y-3. Mice that were effectively depleted of cells expressing the retrovirally encoded MHC class I antigen rejected Kb disparate skin allografts. In contrast, control molecular chimeras accepted Kb disparate skin allografts indefinitely. These data suggest maintenance of tolerance in molecular chimeras requires long-term expression of retrovirally transduced alloantigen on the progeny of retrovirally transduced HSCs.

The role of Epstein–Barr virus in epithelial malignancies

PubMed Central

Tsao, Sai-Wah; Tsang, Chi Man; To, Ka-Fai; Lo, Kwok-Wai

2015-01-01

The close association of Epstein–Barr virus (EBV) infection with non-keratinizing nasopharyngeal carcinomas and a subset of gastric carcinomas suggests that EBV infection is a crucial event in these cancers. The difficulties encountered in infecting and transforming primary epithelial cells in experimental systems suggest that the role of EBV in epithelial malignancies is complex and multifactorial in nature. Genetic alterations in the premalignant epithelium may support the establishment of latent EBV infection, which is believed to be an initiation event. Oncogenic properties have been reported in multiple EBV latent genes. The BamH1 A rightwards transcripts (BARTs) and the BART-encoded microRNAs (miR-BARTs) are highly expressed in EBV-associated epithelial malignancies and may induce malignant transformation. However, enhanced proliferation may not be the crucial function of EBV infection in epithelial malignancies, at least in the early stages of cancer development. EBV-encoded gene products may confer anti-apoptotic properties and promote the survival of infected premalignant epithelial cells harbouring genetic alterations. Multiple EBV-encoded microRNAs have been reported to have immune evasion functions. Genetic alterations in host cells, as well as inflammatory stroma, could modulate the expression of EBV genes and alter the growth properties of infected premalignant epithelial cells, encouraging their selection during carcinogenesis. PMID:25251730

FRET-based genetically-encoded sensors for quantitative monitoring of metabolites.

PubMed

Mohsin, Mohd; Ahmad, Altaf; Iqbal, Muhammad

2015-10-01

Neighboring cells in the same tissue can exist in different states of dynamic activities. After genomics, proteomics and metabolomics, fluxomics is now equally important for generating accurate quantitative information on the cellular and sub-cellular dynamics of ions and metabolite, which is critical for functional understanding of organisms. Various spectrometry techniques are used for monitoring ions and metabolites, although their temporal and spatial resolutions are limited. Discovery of the fluorescent proteins and their variants has revolutionized cell biology. Therefore, novel tools and methods targeting sub-cellular compartments need to be deployed in specific cells and targeted to sub-cellular compartments in order to quantify the target-molecule dynamics directly. We require tools that can measure cellular activities and protein dynamics with sub-cellular resolution. Biosensors based on fluorescence resonance energy transfer (FRET) are genetically encoded and hence can specifically target sub-cellular organelles by fusion to proteins or targetted sequences. Since last decade, FRET-based genetically encoded sensors for molecules involved in energy production, reactive oxygen species and secondary messengers have helped to unravel key aspects of cellular physiology. This review, describing the design and principles of sensors, presents a database of sensors for different analytes/processes, and illustrate examples of application in quantitative live cell imaging.

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

PubMed Central

Naganathan, Saranga; Grunbeck, Amy; Tian, He; Huber, Thomas; Sakmar, Thomas P.

2013-01-01

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes. PMID:24056801

Identification, variation and transcription of pneumococcal repeat sequences

PubMed Central

2011-01-01

Background Small interspersed repeats are commonly found in many bacterial chromosomes. Two families of repeats (BOX and RUP) have previously been identified in the genome of Streptococcus pneumoniae, a nasopharyngeal commensal and respiratory pathogen of humans. However, little is known about the role they play in pneumococcal genetics. Results Analysis of the genome of S. pneumoniae ATCC 700669 revealed the presence of a third repeat family, which we have named SPRITE. All three repeats are present at a reduced density in the genome of the closely related species S. mitis. However, they are almost entirely absent from all other streptococci, although a set of elements related to the pneumococcal BOX repeat was identified in the zoonotic pathogen S. suis. In conjunction with information regarding their distribution within the pneumococcal chromosome, this suggests that it is unlikely that these repeats are specialised sequences performing a particular role for the host, but rather that they constitute parasitic elements. However, comparing insertion sites between pneumococcal sequences indicates that they appear to transpose at a much lower rate than IS elements. Some large BOX elements in S. pneumoniae were found to encode open reading frames on both strands of the genome, whilst another was found to form a composite RNA structure with two T box riboswitches. In multiple cases, such BOX elements were demonstrated as being expressed using directional RNA-seq and RT-PCR. Conclusions BOX, RUP and SPRITE repeats appear to have proliferated extensively throughout the pneumococcal chromosome during the species' past, but novel insertions are currently occurring at a relatively slow rate. Through their extensive secondary structures, they seem likely to affect the expression of genes with which they are co-transcribed. Software for annotation of these repeats is freely available from ftp://ftp.sanger.ac.uk/pub/pathogens/strep_repeats/. PMID:21333003

A Mariner Transposon-Based Signature-Tagged Mutagenesis System for the Analysis of Oral Infection by Listeria monocytogenes

PubMed Central

Cummins, Joanne; Casey, Pat G.; Joyce, Susan A.; Gahan, Cormac G. M.

2013-01-01

Listeria monocytogenes is a Gram-positive foodborne pathogen and the causative agent of listerosis a disease that manifests predominately as meningitis in the non-pregnant individual or infection of the fetus and spontaneous abortion in pregnant women. Common-source outbreaks of foodborne listeriosis are associated with significant morbidity and mortality. However, relatively little is known concerning the mechanisms that govern infection via the oral route. In order to aid functional genetic analysis of the gastrointestinal phase of infection we designed a novel signature-tagged mutagenesis (STM) system based upon the invasive L. monocytogenes 4b serotype H7858 strain. To overcome the limitations of gastrointestinal infection by L. monocytogenes in the mouse model we created a H7858 strain that is genetically optimised for oral infection in mice. Furthermore our STM system was based upon a mariner transposon to favour numerous and random transposition events throughout the L. monocytogenes genome. Use of the STM bank to investigate oral infection by L. monocytogenes identified 21 insertion mutants that demonstrated significantly reduced potential for infection in our model. The sites of transposon insertion included lmOh7858_0671 (encoding an internalin homologous to Lmo0610), lmOh7858_0898 (encoding a putative surface-expressed LPXTG protein homologous to Lmo0842), lmOh7858_2579 (encoding the HupDGC hemin transport system) and lmOh7858_0399 (encoding a putative fructose specific phosphotransferase system). We propose that this represents an optimised STM system for functional genetic analysis of foodborne/oral infection by L. monocytogenes. PMID:24069416

Isozyme variation in lychee (Litchi chinensis Sonn.)

USDA-ARS?s Scientific Manuscript database

A genetic diversity analysis involving 49 lychee (Litchi chinensis SOM.) accessions using eight enzyme systems encoding 12 loci (Zdh-I, Zdh-2, Mdh-2, Per-l, Pgi-2, Pgm-1, Pgm-2, Sk& Tpi-1, Tpi-2, Ugpp-1, and Ugpp-2) revealed moderate to high levels of genetic variability. Cluster analysis of the iso...

Epigenetics: A Fascinating Field with Profound Research, Clinical, & Public Health Implications

ERIC Educational Resources Information Center

Stein, Richard A.; Davis, Devra Lee

2012-01-01

Epigenetics is emerging as one of the most dynamic and vibrant biomedical areas. Multiple lines of evidence confirm that inherited genetic changes alone cannot fully explain all phenotypic characteristics of live organisms, and additional factors, which are not encoded in the DNA sequence, are involved. The contribution of non-genetic factors is…

Characterization of bla(CMY)-encoding plasmids among Salmonella isolated in the United States in 2007.

PubMed

Folster, Jason P; Pecic, Gary; McCullough, Andre; Rickert, Regan; Whichard, Jean M

2011-12-01

Salmonella enterica is one of the most common bacterial causes of foodborne illness, and nontyphoidal Salmonella is estimated to cause ∼1.2 million illnesses in the United States each year. Plasmids are mobile genetic elements that play a critical role in the dissemination of antimicrobial resistance determinants. AmpC-type CMY β-lactamases (bla(CMY)) confer resistance to extended-spectrum cephalosporins and β-lactam/β-lactamase inhibitor combinations and are commonly plasmid-encoded. A variety of plasmids have been shown to encode CMY β-lactamases and certain plasmids may be associated with particular Salmonella serotypes or environmental sources. In this study, we characterized bla(CMY) β-lactamase-encoding plasmids among Salmonella isolates. Isolates of Salmonella from specimens collected from humans in 2007 were submitted to the Centers for Disease Control and Prevention National Antimicrobial Resistance Monitoring System laboratory for susceptibility testing. Three percent (65/2161) of Salmonella isolates displayed resistance to ceftriaxone (minimum inhibitory concentration [MIC] ≥4 mg/L) and amoxicillin/clavulanic acid (MIC ≥32 mg/L), a combination associated with the presence of a bla(CMY) mechanism of resistance. Sixty-four (98.5%) isolates were polymerase chain reaction-positive for bla(CMY) genes. Transformation and conjugation studies showed that 95% (61/64) of the bla(CMY) genes were plasmid-encoded. Most of the bla(CMY)-positive isolates were serotype Typhimurium, Newport, Heidelberg, and Agona. Forty-three plasmids were replicon type IncA/C, 15 IncI1, 2 contained multiple replicon loci, and 1 was untypeable. IncI1 plasmids conferred only the bla(CMY)-associated resistance phenotype, whereas IncA/C plasmids conferred additional multi-drug resistance (MDR) phenotypes to drugs such as chloramphenicol, sulfisoxazole, and tetracycline. Most of the IncI1 plasmids (12/15) were sequence type 12 by plasmid multi-locus sequence typing. CMY β-lactamase-encoding plasmids among human isolates of Salmonella in the United States tended to be large MDR IncA/C plasmids or single resistance determinant IncI1 plasmids. In general, IncI1 plasmids were identified among serotypes commonly associated with poultry, whereas IncA/C plasmids were more likely to be identified among cattle/beef-associated serotypes.