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Sample records for 28s ribosomal gene

  1. Phylogenetic Analysis of Ruminant Theileria spp. from China Based on 28S Ribosomal RNA Gene

    PubMed Central

    Gou, Huitian; Guan, Guiquan; Ma, Miling; Liu, Aihong; Liu, Zhijie; Xu, Zongke; Ren, Qiaoyun; Li, Youquan; Yang, Jifei; Chen, Ze

    2013-01-01

    Species identification using DNA sequences is the basis for DNA taxonomy. In this study, we sequenced the ribosomal large-subunit RNA gene sequences (3,037-3,061 bp) in length of 13 Chinese Theileria stocks that were infective to cattle and sheep. The complete 28S rRNA gene is relatively difficult to amplify and its conserved region is not important for phylogenetic study. Therefore, we selected the D2-D3 region from the complete 28S rRNA sequences for phylogenetic analysis. Our analyses of 28S rRNA gene sequences showed that the 28S rRNA was useful as a phylogenetic marker for analyzing the relationships among Theileria spp. in ruminants. In addition, the D2-D3 region was a short segment that could be used instead of the whole 28S rRNA sequence during the phylogenetic analysis of Theileria, and it may be an ideal DNA barcode. PMID:24327775

  2. DISCRIMINATION 28S RIBOSOMAL GENE OF TREMATODE CERCARIAE IN SNAILS FROM CHIANG MAI PROVINCE, THAILAND.

    PubMed

    Wongsawad, Chalobol; Wongsawad, Pheravut; Sukontason, Kom; Phalee, Anawat; Noikong-Phalee, Waraporn; Chai, Jong Yil

    2016-03-01

    Trematode cercariae are commonly found in many freshwater gastropods. These cercariae can serve to identify the occurrence of such trematodes as Centrocestus formosanus, Haplorchis taichui, Haplorchoides sp, and Stellantchasmus falcatus, which are important parasites in Chiang Mai Province, Thailand. As the species of these cercariae cannot be identified accurately based on morphology, this study employed sequencing of a fragment of 28S ribosomal DNA and phylogenetic analysis to identify the trematode cercariae found in freshwater gastropods in Chiang Mai Province. Eight types of trematode cercariae were identified, namely, distome cercaria (grouped with Philophthalmus spp clade), echinostome cercaria (grouped with Echinostoma spp clade), furcocercous cercaria (grouped with Posthodiplostomum sp/Alaria taxideae/Hysteromorpha triloba clade), monostome cercaria (grouped with Catatropis indicus clade), parapleurolophocercous cercaria (grouped with Haplorchoides sp clade), pleurolophocercous cercaria (grouped with Centrocestusformosanus clade), transversotrema cercaria (grouped with Transversotrema spp clade), and xiphidiocercaria (grouped with Prosthodendrium spp clade). These results provide important information that can be used for identifying these parasites in epidemiological surveys. PMID:27244956

  3. Integration of Bombyx mori R2 Sequences into the 28S Ribosomal RNA Genes of Drosophila melanogaster

    PubMed Central

    Eickbush, Danna G.; Luan, Dongmei D.; Eickbush, Thomas H.

    2000-01-01

    R2 non-long-terminal-repeat retrotransposable elements integrate into a precise location in the 28S rRNA genes of arthropods. The purified protein encoded by R2 can cleave the 28S gene target site and use the 3′ hydroxyl group generated by this cleavage to prime reverse transcription of its own RNA, a process called target-primed reverse transcription. An integration system is described here in which components from the R2 element of the silkmoth, Bombyx mori, are injected into the preblastoderm embryo of Drosophila melanogaster. Silkmoth R2 sequences were readily detected in the 28S rRNA genes of the surviving adults as well as in the genes of their progeny. The 3′ junctions of these insertions were similar to those seen in our in vitro assays, as well as those from endogenous R2 retrotransposition events. The 5′ junctions of the insertions originally contained major deletions of both R2 and 28S gene sequences, a problem overcome by the inclusion of upstream 28S gene sequences at the 5′ end of the injected RNA. The resulting 5′ junctions suggested a recombination event between the cDNA and the upstream target sequences. This in vivo integration system should help determine the mechanism of R2 retrotransposition and be useful as a delivery system to integrate defined DNA sequences into the rRNA genes of organisms. PMID:10594024

  4. Phylogenetic Relationships of Tribes Within Harpalinae (Coleoptera: Carabidae) as Inferred from 28S Ribosomal DNA and the Wingless Gene

    PubMed Central

    Ober, Karen A.; Maddison, David R.

    2008-01-01

    Harpalinae is a large, monophyletic subfamily of carabid ground beetles containing more than 19,000 species in approximately 40 tribes. The higher level phylogenetic relationships within harpalines were investigated based on nucleotide data from two nuclear genes, wingless and 28S rDNA. Phylogenetic analyses of combined data indicate that many harpaline tribes are monophyletic, however the reconstructed trees showed little support for deeper nodes. In addition, our results suggest that the Lebiomorph Assemblage (tribes Lebiini, Cyclosomini, Graphipterini, Perigonini, Odacanthini, Lachnophorini, Pentagonicini, Catapiesini and Calophaenini), which is united by a morphological synapomorphy, is not monophyletic, and the tribe Lebiini is paraphyletic with respect to members of Cyclosomini. Two unexpected clades of tribes were supported: the Zuphiitae, comprised of Anthiini, Zuphiini, Helluonini, Dryptini, Galeritini, and Physocrotaphini; and a clade comprised of Orthogoniini, Pseudomorphini, and Graphipterini. The data presented in this study represent a dense sample of taxa to examine the molecular phylogeny of Harpalinae and provide a useful framework to examine the origin and evolution of morphological and ecological diversity in this group. PMID:20302528

  5. Higher-level phylogeny of the Therevidae (Diptera: insecta) based on 28S ribosomal and elongation factor-1 alpha gene sequences.

    PubMed

    Yang, L; Wiegmann, B M; Yeates, D K; Irwin, M E

    2000-06-01

    Therevidae (stilleto flies) are a little-known family of asiloid brachyceran Diptera (Insecta). Separate and combined phylogenetic analyses of 1200 bases of the 28S ribosomal DNA and 1100 bases of elongation factor-1alpha were used to infer phylogenetic relationships within the family. The position of the enigmatic taxon Apsilocephala Kröber is evaluated in light of the molecular evidence. In all analyses, molecular data strongly support the monophyly of Therevidae, excluding Apsilocephala, and the division of Therevidae into two main clades corresponding to a previous classification of the family into the subfamilies Phycinae and Therevinae. Despite strong support for some relationships within these groups, relationships at the base of the two main clades are weakly supported. Short branch lengths for Australasian clades at the base of the Therevinae may represent a rapid radiation of therevids in Australia. PMID:10860652

  6. Phylogenetic relationships of the marine Haplosclerida (Phylum Porifera) employing ribosomal (28S rRNA) and mitochondrial (cox1, nad1) gene sequence data.

    PubMed

    Redmond, Niamh E; Raleigh, Jean; van Soest, Rob W M; Kelly, Michelle; Travers, Simon A A; Bradshaw, Brian; Vartia, Salla; Stephens, Kelly M; McCormack, Grace P

    2011-01-01

    The systematics of the poriferan Order Haplosclerida (Class Demospongiae) has been under scrutiny for a number of years without resolution. Molecular data suggests that the order needs revision at all taxonomic levels. Here, we provide a comprehensive view of the phylogenetic relationships of the marine Haplosclerida using many species from across the order, and three gene regions. Gene trees generated using 28S rRNA, nad1 and cox1 gene data, under maximum likelihood and Bayesian approaches, are highly congruent and suggest the presence of four clades. Clade A is comprised primarily of species of Haliclona and Callyspongia, and clade B is comprised of H. simulans and H. vansoesti (Family Chalinidae), Amphimedon queenslandica (Family Niphatidae) and Tabulocalyx (Family Phloeodictyidae), Clade C is comprised primarily of members of the Families Petrosiidae and Niphatidae, while Clade D is comprised of Aka species. The polyphletic nature of the suborders, families and genera described in other studies is also found here. PMID:21931685

  7. Phylogenetic Relationships of the Marine Haplosclerida (Phylum Porifera) Employing Ribosomal (28S rRNA) and Mitochondrial (cox1, nad1) Gene Sequence Data

    PubMed Central

    Redmond, Niamh E.; Raleigh, Jean; van Soest, Rob W. M.; Kelly, Michelle; Travers, Simon A. A.; Bradshaw, Brian; Vartia, Salla; Stephens, Kelly M.; McCormack, Grace P.

    2011-01-01

    The systematics of the poriferan Order Haplosclerida (Class Demospongiae) has been under scrutiny for a number of years without resolution. Molecular data suggests that the order needs revision at all taxonomic levels. Here, we provide a comprehensive view of the phylogenetic relationships of the marine Haplosclerida using many species from across the order, and three gene regions. Gene trees generated using 28S rRNA, nad1 and cox1 gene data, under maximum likelihood and Bayesian approaches, are highly congruent and suggest the presence of four clades. Clade A is comprised primarily of species of Haliclona and Callyspongia, and clade B is comprised of H. simulans and H. vansoesti (Family Chalinidae), Amphimedon queenslandica (Family Niphatidae) and Tabulocalyx (Family Phloeodictyidae), Clade C is comprised primarily of members of the Families Petrosiidae and Niphatidae, while Clade D is comprised of Aka species. The polyphletic nature of the suborders, families and genera described in other studies is also found here. PMID:21931685

  8. PCR Primers for Metazoan Nuclear 18S and 28S Ribosomal DNA Sequences

    PubMed Central

    Machida, Ryuji J.; Knowlton, Nancy

    2012-01-01

    Background Metagenetic analyses, which amplify and sequence target marker DNA regions from environmental samples, are increasingly employed to assess the biodiversity of communities of small organisms. Using this approach, our understanding of microbial diversity has expanded greatly. In contrast, only a few studies using this approach to characterize metazoan diversity have been reported, despite the fact that many metazoan species are small and difficult to identify or are undescribed. One of the reasons for this discrepancy is the availability of universal primers for the target taxa. In microbial studies, analysis of the 16S ribosomal DNA is standard. In contrast, the best gene for metazoan metagenetics is less clear. In the present study, we have designed primers that amplify the nuclear 18S and 28S ribosomal DNA sequences of most metazoan species with the goal of providing effective approaches for metagenetic analyses of metazoan diversity in environmental samples, with a particular emphasis on marine biodiversity. Methodology/Principal Findings Conserved regions suitable for designing PCR primers were identified using 14,503 and 1,072 metazoan sequences of the nuclear 18S and 28S rDNA regions, respectively. The sequence similarity of both these newly designed and the previously reported primers to the target regions of these primers were compared for each phylum to determine the expected amplification efficacy. The nucleotide diversity of the flanking regions of the primers was also estimated for genera or higher taxonomic groups of 11 phyla to determine the variable regions within the genes. Conclusions/Significance The identified nuclear ribosomal DNA primers (five primer pairs for 18S and eleven for 28S) and the results of the nucleotide diversity analyses provide options for primer combinations for metazoan metagenetic analyses. Additionally, advantages and disadvantages of not only the 18S and 28S ribosomal DNA, but also other marker regions as targets

  9. Intragenomic sequence variation at the ITS1 - ITS2 region and at the 18S and 28S nuclear ribosomal DNA genes of the New Zealand mud snail, Potamopyrgus antipodarum (Hydrobiidae: mollusca)

    USGS Publications Warehouse

    Hoy, Marshal S.; Rodriguez, Rusty J.

    2013-01-01

    Molecular genetic analysis was conducted on two populations of the invasive non-native New Zealand mud snail (Potamopyrgus antipodarum), one from a freshwater ecosystem in Devil's Lake (Oregon, USA) and the other from an ecosystem of higher salinity in the Columbia River estuary (Hammond Harbor, Oregon, USA). To elucidate potential genetic differences between the two populations, three segments of nuclear ribosomal DNA (rDNA), the ITS1-ITS2 regions and the 18S and 28S rDNA genes were cloned and sequenced. Variant sequences within each individual were found in all three rDNA segments. Folding models were utilized for secondary structure analysis and results indicated that there were many sequences which contained structure-altering polymorphisms, which suggests they could be nonfunctional pseudogenes. In addition, analysis of molecular variance (AMOVA) was used for hierarchical analysis of genetic variance to estimate variation within and among populations and within individuals. AMOVA revealed significant variation in the ITS region between the populations and among clones within individuals, while in the 5.8S rDNA significant variation was revealed among individuals within the two populations. High levels of intragenomic variation were found in the ITS regions, which are known to be highly variable in many organisms. More interestingly, intragenomic variation was also found in the 18S and 28S rDNA, which has rarely been observed in animals and is so far unreported in Mollusca. We postulate that in these P. antipodarum populations the effects of concerted evolution are diminished due to the fact that not all of the rDNA genes in their polyploid genome should be essential for sustaining cellular function. This could lead to a lessening of selection pressures, allowing mutations to accumulate in some copies, changing them into variant sequences.                   

  10. Molecular Identification of Sibling Species of Sclerodermus (Hymenoptera: Bethylidae) That Parasitize Buprestid and Cerambycid Beetles by Using Partial Sequences of Mitochondrial DNA Cytochrome Oxidase Subunit 1 and 28S Ribosomal RNA Gene

    PubMed Central

    Jiang, Yuan; Yang, Zhongqi; Wang, Xiaoyi; Hou, Yuxia

    2015-01-01

    The species belonging to Sclerodermus (Hymenoptera: Bethylidae) are currently the most important insect natural enemies of wood borer pests, mainly buprestid and cerambycid beetles, in China. However, some sibling species of this genus are very difficult to distinguish because of their similar morphological features. To address this issue, we conducted phylogenetic and genetic analyses of cytochrome oxidase subunit I (COI) and 28S RNA gene sequences from eight species of Sclerodermus reared from different wood borer pests. The eight sibling species were as follows: S. guani Xiao et Wu, S. sichuanensis Xiao, S. pupariae Yang et Yao, and Sclerodermus spp. (Nos. 1–5). A 594-bp fragment of COI and 750-bp fragment of 28S were subsequently sequenced. For COI, the G-C content was found to be low in all the species, averaging to about 30.0%. Sequence divergences (Kimura-2-parameter distances) between congeneric species averaged to 4.5%, and intraspecific divergences averaged to about 0.09%. Further, the maximum sequence divergences between congeneric species and Sclerodermus sp. (No. 5) averaged to about 16.5%. All 136 samples analyzed were included in six reciprocally monophyletic clades in the COI neighbor-joining (NJ) tree. The NJ tree inferred from the 28S rRNA sequence yielded almost identical results, but the samples from S. guani, S. sichuanensis, S. pupariae, and Sclerodermus spp. (Nos. 1–4) clustered together and only Sclerodermus sp. (No. 5) clustered separately. Our findings indicate that the standard barcode region of COI can be efficiently used to distinguish morphologically similar Sclerodermus species. Further, we speculate that Sclerodermus sp. (No. 5) might be a new species of Sclerodermus. PMID:25782000

  11. The sequence of 28S ribosomal RNA varies within and between human cell lines.

    PubMed Central

    Leffers, H; Andersen, A H

    1993-01-01

    The primary structure of 28S ribosomal RNA constitutes a conserved core which is similar among most 23S-like rRNAs and expansion segments which occur at specific positions in the sequence. The expansion segments account for most of the size difference between prokaryotic (archaeal and eubacterial) and eukaryotic rRNAs and they exhibit a sequence variation which is unique among rRNAs. We have investigated the sequence variation of one of the expansion segments, V8, by sequencing a total of 111 V8 segments from 9 different human cell lines and tissues and have found 35 different variants. The variation occur mainly at two 'hot spots' which are separated by 170 nucleotides in the primary sequence but are neighbours in the secondary structure. The sequence of V8 segments varies both within and between human cell lines and tissues. The implications for the evolution of the eukaryotic 28S rRNA are discussed together with possible functions of the expansion segments. We also present a secondary structure model for the V8 segment based on comparative sequence analysis and chemical and enzymatic foot printing. Images PMID:8464736

  12. Reconstruction of phylogenetic relationships in dermatomycete genus Trichophyton Malmsten 1848 based on ribosomal internal transcribed spacer region, partial 28S rRNA and beta-tubulin genes sequences.

    PubMed

    Pchelin, Ivan M; Zlatogursky, Vasily V; Rudneva, Mariya V; Chilina, Galina A; Rezaei-Matehkolaei, Ali; Lavnikevich, Dmitry M; Vasilyeva, Natalya V; Taraskina, Anastasia E

    2016-09-01

    Trichophyton spp. are important causative agents of superficial mycoses. The phylogeny of the genus and accurate strain identification, based on the ribosomal ITS region sequencing, are still under development. The present work is aimed at (i) inferring the genus phylogeny from partial ITS, LSU and BT2 sequences (ii) description of ribosomal ITS region polymorphism in 15 strains of Trichophyton interdigitale. We performed DNA sequence-based species identification and phylogenetic analysis on 48 strains belonging to the genus Trichophyton. Phylogenetic relationships were inferred by maximum likelihood and Bayesian methods on concatenated ITS, LSU and BT2 sequences. Ribosomal ITS region polymorphisms were assessed directly on the alignment. By phylogenetic reconstruction, we reveal major anthropophilic and zoophilic species clusters in the genus Trichophyton. We describe several sequences of the ITS region of T. interdigitale, which do not fit in the traditional polymorphism scheme and propose emendations in this scheme for discrimination between ITS sequence types in T. interdigitale. The new polymorphism scheme will allow inclusion of a wider spectrum of isolates while retaining its explanatory power. This scheme was also found to be partially congruent with NTS typing technique. PMID:27071492

  13. Nucleotide sequence neighbouring a late modified guanylic residue within the 28S ribosomal RNA of several eukaryotic cells.

    PubMed Central

    Eladari, M E; Hampe, A; Galibert, F

    1977-01-01

    The nucleotide sequence of a particular T1 oligonucleotide found in 41S and 28S RNAs of several cellular cell lines (human, mouse, rat and chicken fibroblast) but absent in 45S ribosomal RNA has been deduced. Its primary structure : A-U-U*-G*-psi-U-C-A-C-C-C-A-C-U-A-A-U-A-Gp shows the presence of a modified G residue which explains the existence of this oligonucleotide in the T1 fingerprint of 41S RNA and 28S. Its absence on the 45S RNA T1 fingerprint is accounted for by a late modification. Images PMID:561392

  14. Proteomic analysis of the mammalian mitochondrial ribosome. Identification of protein components in the 28 S small subunit.

    PubMed

    Suzuki, T; Terasaki, M; Takemoto-Hori, C; Hanada, T; Ueda, T; Wada, A; Watanabe, K

    2001-08-31

    The mammalian mitochondrial ribosome (mitoribosome) has a highly protein-rich composition with a small sedimentation coefficient of 55 S, consisting of 39 S large and 28 S small subunits. In the previous study, we analyzed 39 S large subunit proteins from bovine mitoribosome (Suzuki, T., Terasaki, M., Takemoto-Hori, C., Hanada, T., Ueda, T., Wada, A., and Watanabe, K. (2001) J. Biol. Chem. 276, 21724-21736). The results suggested structural compensation for the rRNA deficit through proteins of increased molecular mass in the mitoribosome. We report here the identification of 28 S small subunit proteins. Each protein was separated by radical-free high-reducing two-dimensional polyacrylamide gel electrophoresis and analyzed by liquid chromatography/mass spectrometry/mass spectrometry using electrospray ionization/ion trap mass spectrometer to identify cDNA sequence by expressed sequence tag data base searches in silico. Twenty one proteins from the small subunit were identified, including 11 new proteins along with their complete cDNA sequences from human and mouse. In addition to these proteins, three new proteins were also identified in the 55 S mitoribosome. We have clearly identified a mitochondrial homologue of S12, which is a key regulatory protein of translation fidelity and a candidate for the autosomal dominant deafness gene, DFNA4. The apoptosis-related protein DAP3 was found to be a component of the small subunit, indicating a new function for the mitoribosome in programmed cell death. In summary, we have mapped a total of 55 proteins from the 55 S mitoribosome on the two-dimensional polyacrylamide gels. PMID:11402041

  15. Studies on the low molecular weight RNA associated with 28S ribosomal RNA from Crotalus durissus terrificus liver.

    PubMed Central

    Giorgini, J F; De Lucca, F L

    1976-01-01

    A low molecular weight RNA was released from the purified rattlesnake 28 S RNA by brief heat treatment as well as by treatment with 80% dimethylsulfoxide or formamide. The sedimentation coeficient of this low molecular weight RNA was found to be 5.5 S, corresponding to a nucleotide number of 140 and a molecular weight of 46 000. It was also observed that 5.5S RNA is present in equimolar ratio to 5 S rRNA. Heat treatment of the purified 60 S ribosomal subunit also released the 5.5 S RNA. The possibility that this low molecular weight RNA is located on the surface of the large ribosomal subunit is discussed. PMID:1250695

  16. Nearly complete 28S rRNA gene sequences confirm new hypotheses of sponge evolution.

    PubMed

    Thacker, Robert W; Hill, April L; Hill, Malcolm S; Redmond, Niamh E; Collins, Allen G; Morrow, Christine C; Spicer, Lori; Carmack, Cheryl A; Zappe, Megan E; Pohlmann, Deborah; Hall, Chelsea; Diaz, Maria C; Bangalore, Purushotham V

    2013-09-01

    The highly collaborative research sponsored by the NSF-funded Assembling the Porifera Tree of Life (PorToL) project is providing insights into some of the most difficult questions in metazoan systematics. Our understanding of phylogenetic relationships within the phylum Porifera has changed considerably with increased taxon sampling and data from additional molecular markers. PorToL researchers have falsified earlier phylogenetic hypotheses, discovered novel phylogenetic alliances, found phylogenetic homes for enigmatic taxa, and provided a more precise understanding of the evolution of skeletal features, secondary metabolites, body organization, and symbioses. Some of these exciting new discoveries are shared in the papers that form this issue of Integrative and Comparative Biology. Our analyses of over 300 nearly complete 28S ribosomal subunit gene sequences provide specific case studies that illustrate how our dataset confirms new hypotheses of sponge evolution. We recovered monophyletic clades for all 4 classes of sponges, as well as the 4 major clades of Demospongiae (Keratosa, Myxospongiae, Haploscleromorpha, and Heteroscleromorpha), but our phylogeny differs in several aspects from traditional classifications. In most major clades of sponges, families within orders appear to be paraphyletic. Although additional sampling of genes and taxa are needed to establish whether this pattern results from a lack of phylogenetic resolution or from a paraphyletic classification system, many of our results are congruent with those obtained from 18S ribosomal subunit gene sequences and complete mitochondrial genomes. These data provide further support for a revision of the traditional classification of sponges. PMID:23748742

  17. Nearly Complete 28S rRNA Gene Sequences Confirm New Hypotheses of Sponge Evolution

    PubMed Central

    Thacker, Robert W.; Hill, April L.; Hill, Malcolm S.; Redmond, Niamh E.; Collins, Allen G.; Morrow, Christine C.; Spicer, Lori; Carmack, Cheryl A.; Zappe, Megan E.; Pohlmann, Deborah; Hall, Chelsea; Diaz, Maria C.; Bangalore, Purushotham V.

    2013-01-01

    The highly collaborative research sponsored by the NSF-funded Assembling the Porifera Tree of Life (PorToL) project is providing insights into some of the most difficult questions in metazoan systematics. Our understanding of phylogenetic relationships within the phylum Porifera has changed considerably with increased taxon sampling and data from additional molecular markers. PorToL researchers have falsified earlier phylogenetic hypotheses, discovered novel phylogenetic alliances, found phylogenetic homes for enigmatic taxa, and provided a more precise understanding of the evolution of skeletal features, secondary metabolites, body organization, and symbioses. Some of these exciting new discoveries are shared in the papers that form this issue of Integrative and Comparative Biology. Our analyses of over 300 nearly complete 28S ribosomal subunit gene sequences provide specific case studies that illustrate how our dataset confirms new hypotheses of sponge evolution. We recovered monophyletic clades for all 4 classes of sponges, as well as the 4 major clades of Demospongiae (Keratosa, Myxospongiae, Haploscleromorpha, and Heteroscleromorpha), but our phylogeny differs in several aspects from traditional classifications. In most major clades of sponges, families within orders appear to be paraphyletic. Although additional sampling of genes and taxa are needed to establish whether this pattern results from a lack of phylogenetic resolution or from a paraphyletic classification system, many of our results are congruent with those obtained from 18S ribosomal subunit gene sequences and complete mitochondrial genomes. These data provide further support for a revision of the traditional classification of sponges. PMID:23748742

  18. Identification of Dermatophyte Species by 28S Ribosomal DNA Sequencing with a Commercial Kit

    PubMed Central

    Ninet, Béatrice; Jan, Isabelle; Bontems, Olympia; Léchenne, Barbara; Jousson, Olivier; Panizzon, Renato; Lew, Daniel; Monod, Michel

    2003-01-01

    We have shown that dermatophyte species can be easily identified on the basis of a DNA sequence encoding a part of the large-subunit (LSU) rRNA (28S rRNA) by using the MicroSeq D2 LSU rRNA Fungal Sequencing Kit. Two taxa causing distinct dermatophytoses were clearly distinguished among isolates of the Trichophyton mentagrophytes species complex. PMID:12574293

  19. 28S ribosomal RNA sequences separate five prominent Lygus (Hemiptera: Miridae) pest species into three species clusters

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A segment of the nuclear 28S rRNA gene was compared among six species of Lygus (L. hesperus, L. keltoni, L. borealis, L. elisus, L. lineolaris, L. vanduzeei). The DNA sequences separate into three main groups. The LL group contains L. lineolaris and L. vanduzeei. Group LBLE is comprised of L. elisus...

  20. 28S ribosomal RNA sequences separate five prominent Lygus (Hemiptera: Miridae) pest species into three species clu

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A segment of the 28S rRNA gene was compared among six species of Lygus (L. hesperus, L. keltoni, L. borealis, L. elisus, L. lineolaris, L. vanduzeii). The DNA sequences separate into three main groups. The LL group contains L. lineolaris and L. vanduzeii. Group LBLE is comprised of L. elisus and mos...

  1. The SBP2 protein central to selenoprotein synthesis contacts the human ribosome at expansion segment 7L of the 28S rRNA.

    PubMed

    Kossinova, Olga; Malygin, Alexey; Krol, Alain; Karpova, Galina

    2014-07-01

    SBP2 is a pivotal protein component in selenoprotein synthesis. It binds the SECIS stem-loop in the 3' UTR of selenoprotein mRNA and interacts with both the specialized translation elongation factor and the ribosome at the 60S subunit. In this work, our goal was to identify the binding partners of SBP2 on the ribosome. Cross-linking experiments with bifunctional reagents demonstrated that the SBP2-binding site on the human ribosome is mainly formed by the 28S rRNA. Direct hydroxyl radical probing of the entire 28S rRNA revealed that SBP2 bound to 80S ribosomes or 60S subunits protects helix ES7L-E in expansion segment 7 of the 28S rRNA. Diepoxybutane cross-linking confirmed the interaction of SBP2 with helix ES7L-E. Additionally, binding of SBP2 to the ribosome led to increased reactivity toward chemical probes of a few bases in ES7L-E and in the universally conserved helix H89, indicative of conformational changes in the 28S rRNA in response to SBP2 binding. This study revealed for the first time that SBP2 makes direct contacts with a discrete region of the human 28S rRNA. PMID:24850884

  2. The Strepsiptera problem: phylogeny of the holometabolous insect orders inferred from 18S and 28S ribosomal DNA sequences and morphology.

    PubMed

    Whiting, M F; Carpenter, J C; Wheeler, Q D; Wheeler, W C

    1997-03-01

    Phylogenetic relationships among the holometabolous insect orders were inferred from cladistic analysis of nucleotide sequences of 18S ribosomal DNA (rDNA) (85 exemplars) and 28S rDNA (52 exemplars) and morphological characters. Exemplar outgroup taxa were Collembola (1 sequence), Archaeognatha (1), Ephemerida (1), Odonata (2), Plecoptera (2), Blattodea (1), Mantodea (1), Dermaptera (1), Orthoptera (1), Phasmatodea (1), Embioptera (1), Psocoptera (1), Phthiraptera (1), Hemiptera (4), and Thysanoptera (1). Exemplar ingroup taxa were Coleoptera: Archostemata (1), Adephaga (2), and Polyphaga (7); Megaloptera (1); Raphidioptera (1); Neuroptera (sensu stricto = Planipennia): Mantispoidea (2), Hemerobioidea (2), and Myrmeleontoidea (2); Hymenoptera: Symphyta (4) and Apocrita (19); Trichoptera: Hydropsychoidea (1) and Limnephiloidea (2); Lepidoptera: Ditrysia (3); Siphonaptera: Pulicoidea (1) and Ceratophylloidea (2); Mecoptera: Meropeidae (1), Boreidae (1), Panorpidae (1), and Bittacidae (2); Diptera: Nematocera (1), Brachycera (2), and Cyclorrhapha (1); and Strepsiptera: Corioxenidae (1), Myrmecolacidae (1), Elenchidae (1), and Stylopidae (3). We analyzed approximately 1 kilobase of 18S rDNA, starting 398 nucleotides downstream of the 5' end, and approximately 400 bp of 28S rDNA in expansion segment D3. Multiple alignment of the 18S and 28S sequences resulted in 1,116 nucleotide positions with 24 insert regions and 398 positions with 14 insert regions, respectively. All Strepsiptera and Neuroptera have large insert regions in 18S and 28S. The secondary structure of 18S insert 23 is composed of long stems that are GC rich in the basal Strepsiptera and AT rich in the more derived Strepsiptera. A matrix of 176 morphological characters was analyzed for holometabolous orders. Incongruence length difference tests indicate that the 28S + morphological data sets are incongruent but that 28S + 18S, 18S + morphology, and 28S + 18S + morphology fail to reject the hypothesis of

  3. Fungal community analysis in the deep-sea sediments of the Pacific Ocean assessed by comparison of ITS, 18S and 28S ribosomal DNA regions

    NASA Astrophysics Data System (ADS)

    Xu, Wei; Luo, Zhu-Hua; Guo, Shuangshuang; Pang, Ka-Lai

    2016-03-01

    We investigated the diversity of fungal communities in 6 different deep-sea sediment samples of the Pacific Ocean based on three different types of clone libraries, including internal transcribed spacer (ITS), 18S rDNA, and 28S rDNA regions. A total of 1978 clones were generated from 18 environmental clone libraries, resulting in 140 fungal operational taxonomic units (OTUs), including 18 OTUs from ITS, 44 OTUs from 18S rDNA, and 78 OTUs from 28S rDNA gene primer sets. The majority of the recovered sequences belonged to diverse phylotypes of the Ascomycota and Basidiomycota. Additionally, our study revealed a total of 46 novel fungal phylotypes, which showed low similarities (<97%) with available fungal sequences in the GenBank, including a novel Zygomycete lineage, suggesting possible new fungal taxa occurring in the deep-sea sediments. The results suggested that 28S rDNA is an efficient target gene to describe fungal community in deep-sea environment.

  4. Identification of Scopulariopsis species by partial 28S rRNA gene sequence analysis.

    PubMed

    Jagielski, Tomasz; Kosim, Kinga; Skóra, Magdalena; Macura, Anna Barbara; Bielecki, Jacek

    2013-01-01

    The genus Scopulariopsis contains over 30 species of mitosporic moulds, which although usually saprophytic may also act as opportunistic pathogens in humans. They have mainly been associated with onychomycosis, and only sporadically reported as a cause of deep tissue infections or systemic disease. Identification of Scopulariopsis species still largely relies on phenotype-based methods. There is a need for a molecular diagnostic approach, that would allow to reliably discriminate between different Scopulariopsis species. The aim of this study was to apply sequence analysis of partial 28S rRNA gene for species identification of Scopulariopsis clinical isolates. Although the method employed did reveal some genetic polymorphism among Scopulariopsis isolates tested, it was not enough for species delineation. For this to be achieved, other genetic loci, within and beyond the rDNA operon, need to be investigated. PMID:24459837

  5. [Phylogeny of protostome moulting animals (Ecdysozoa) inferred from 18 and 28S rRNA gene sequences].

    PubMed

    Petrov, N B; Vladychenskaia, N S

    2005-01-01

    Reliability of reconstruction of phylogenetic relationships within a group of protostome moulting animals was evaluated by means of comparison of 18 and 28S rRNA gene sequences sets both taken separately and combined. Reliability of reconstructions was evaluated by values of the bootstrap support of major phylogenetic tree nodes and by degree of congruence of phylogenetic trees inferred by various methods. By both criteria, phylogenetic trees reconstructed from the combined 18 and 28S rRNA gene sequences were better than those inferred from 18 and 28S sequences taken separately. Results obtained are consistent with phylogenetic hypothesis separating protostome animals into two major clades, moulting Ecdysozoa (Priapulida + Kinorhyncha, Nematoda + Nematomorpha, Onychophora + Tardigrada, Myriapoda + Chelicerata, Crustacea + Hexapoda) and unmoulting Lophotrochozoa (Plathelminthes, Nemertini, Annelida, Mollusca, Echiura, Sipuncula). Clade Cephalorhyncha does not include nematomorphs (Nematomorpha). Conclusion was taken that it is necessary to use combined 18 and 28S data in phylogenetic studies. PMID:16083008

  6. Fungal community structure in disease suppressive soils assessed by 28S LSU gene sequencing.

    PubMed

    Penton, C Ryan; Gupta, V V S R; Tiedje, James M; Neate, Stephen M; Ophel-Keller, Kathy; Gillings, Michael; Harvey, Paul; Pham, Amanda; Roget, David K

    2014-01-01

    Natural biological suppression of soil-borne diseases is a function of the activity and composition of soil microbial communities. Soil microbe and phytopathogen interactions can occur prior to crop sowing and/or in the rhizosphere, subsequently influencing both plant growth and productivity. Research on suppressive microbial communities has concentrated on bacteria although fungi can also influence soil-borne disease. Fungi were analyzed in co-located soils 'suppressive' or 'non-suppressive' for disease caused by Rhizoctonia solani AG 8 at two sites in South Australia using 454 pyrosequencing targeting the fungal 28S LSU rRNA gene. DNA was extracted from a minimum of 125 g of soil per replicate to reduce the micro-scale community variability, and from soil samples taken at sowing and from the rhizosphere at 7 weeks to cover the peak Rhizoctonia infection period. A total of ∼ 994,000 reads were classified into 917 genera covering 54% of the RDP Fungal Classifier database, a high diversity for an alkaline, low organic matter soil. Statistical analyses and community ordinations revealed significant differences in fungal community composition between suppressive and non-suppressive soil and between soil type/location. The majority of differences associated with suppressive soils were attributed to less than 40 genera including a number of endophytic species with plant pathogen suppression potentials and mycoparasites such as Xylaria spp. Non-suppressive soils were dominated by Alternaria, Gibberella and Penicillum. Pyrosequencing generated a detailed description of fungal community structure and identified candidate taxa that may influence pathogen-plant interactions in stable disease suppression. PMID:24699870

  7. Fungal Community Structure in Disease Suppressive Soils Assessed by 28S LSU Gene Sequencing

    PubMed Central

    Penton, C. Ryan; Gupta, V. V. S. R.; Tiedje, James M.; Neate, Stephen M.; Ophel-Keller, Kathy; Gillings, Michael; Harvey, Paul; Pham, Amanda; Roget, David K.

    2014-01-01

    Natural biological suppression of soil-borne diseases is a function of the activity and composition of soil microbial communities. Soil microbe and phytopathogen interactions can occur prior to crop sowing and/or in the rhizosphere, subsequently influencing both plant growth and productivity. Research on suppressive microbial communities has concentrated on bacteria although fungi can also influence soil-borne disease. Fungi were analyzed in co-located soils ‘suppressive’ or ‘non-suppressive’ for disease caused by Rhizoctonia solani AG 8 at two sites in South Australia using 454 pyrosequencing targeting the fungal 28S LSU rRNA gene. DNA was extracted from a minimum of 125 g of soil per replicate to reduce the micro-scale community variability, and from soil samples taken at sowing and from the rhizosphere at 7 weeks to cover the peak Rhizoctonia infection period. A total of ∼994,000 reads were classified into 917 genera covering 54% of the RDP Fungal Classifier database, a high diversity for an alkaline, low organic matter soil. Statistical analyses and community ordinations revealed significant differences in fungal community composition between suppressive and non-suppressive soil and between soil type/location. The majority of differences associated with suppressive soils were attributed to less than 40 genera including a number of endophytic species with plant pathogen suppression potentials and mycoparasites such as Xylaria spp. Non-suppressive soils were dominated by Alternaria, Gibberella and Penicillum. Pyrosequencing generated a detailed description of fungal community structure and identified candidate taxa that may influence pathogen-plant interactions in stable disease suppression. PMID:24699870

  8. Mammalian mitochondrial ribosomal small subunit (MRPS) genes: A putative role in human disease.

    PubMed

    Gopisetty, Gopal; Thangarajan, Rajkumar

    2016-09-01

    Mitochondria are prominently understood as power houses producing ATP the primary energy currency of the cell. However, mitochondria are also known to play an important role in apoptosis and autophagy, and mitochondrial dysregulation can lead to pathological outcomes. Mitochondria are known to contain 1500 proteins of which only 13 are coded by mitochondrial DNA and the rest are coded by nuclear genes. Protein synthesis in mitochondria involves mitochondrial ribosomes which are 55-60S particles and are composed of small 28S and large 39S subunits. A feature of mammalian mitoribosome which differentiate it from bacterial ribosomes is the increased protein content. The human mitochondrial ribosomal protein (MRP) gene family comprises of 30 genes which code for mitochondrial ribosomal small subunit and 50 genes for the large subunit. The present review focuses on the mitochondrial ribosomal small subunit genes (MRPS), presents an overview of the literature and data gleaned from publicly available gene and protein expression databases. The survey revealed aberrations in MRPS gene expression patterns in varied human diseases indicating a putative role in their etiology. PMID:27170550

  9. Introns regulate the production of ribosomal proteins by modulating splicing of duplicated ribosomal protein genes.

    PubMed

    Petibon, Cyrielle; Parenteau, Julie; Catala, Mathieu; Elela, Sherif Abou

    2016-05-01

    Most budding yeast introns exist in the many duplicated ribosomal protein genes (RPGs) and it has been posited that they remain there to modulate the expression of RPGs and cell growth in response to stress. However, the mechanism by which introns regulate the expression of RPGs and their impact on the synthesis of ribosomal proteins remain unclear. In this study, we show that introns determine the ratio of ribosomal protein isoforms through asymmetric paralog-specific regulation of splicing. Exchanging the introns and 3' untranslated regions of the duplicated RPS9 genes altered the splicing efficiency and changed the ratio of the ribosomal protein isoforms. Mutational analysis of the RPS9 genes indicated that splicing is regulated by variations in the intron structure and the 3' untranslated region. Together these data suggest that preferential splicing of duplicated RPGs provides a means for adjusting the ratio of different ribosomal protein isoforms, while maintaining the overall expression level of each ribosomal protein. PMID:26945043

  10. Contrasting evolutionary patterns of 28S and ITS rRNA genes reveal high intragenomic variation in Cephalenchus (Nematoda): Implications for species delimitation.

    PubMed

    Pereira, Tiago José; Baldwin, James Gordon

    2016-05-01

    Concerted evolution is often assumed to be the evolutionary force driving multi-family genes, including those from ribosomal DNA (rDNA) repeat, to complete homogenization within a species, although cases of non-concerted evolution have been also documented. In this study, sequence variation of 28S and ITS ribosomal RNA (rRNA) genes in the genus Cephalenchus is assessed at three different levels, intragenomic, intraspecific, and interspecific. The findings suggest that not all Cephalenchus species undergo concerted evolution. High levels of intraspecific polymorphism, mostly due to intragenomic variation, are found in Cephalenchus sp1 (BRA-01). Secondary structure analyses of both rRNA genes and across different species show a similar substitution pattern, including mostly compensatory (CBC) and semi-compensatory (SBC) base changes, thus suggesting the functionality of these rRNA copies despite the variation found in some species. This view is also supported by low sequence variation in the 5.8S gene in relation to the flanking ITS-1 and ITS-2 as well as by the existence of conserved motifs in the former gene. It is suggested that potential cross-fertilization in some Cephalenchus species, based on inspection of female reproductive system, might contribute to both intragenomic and intraspecific polymorphism of their rRNA genes. These results reinforce the potential implications of intragenomic and intraspecific genetic diversity on species delimitation, especially in biodiversity studies based solely on metagenetic approaches. Knowledge of sequence variation will be crucial for accurate species diversity estimation using molecular methods. PMID:26926945

  11. Phylogenetic utility of ribosomal genes for reconstructing the phylogeny of five Chinese satyrine tribes (Lepidoptera, Nymphalidae)

    PubMed Central

    Yang, Mingsheng; Zhang, Yalin

    2015-01-01

    Abstract Satyrinae is one of twelve subfamilies of the butterfly family Nymphalidae, which currently includes nine tribes. However, phylogenetic relationships among them remain largely unresolved, though different researches have been conducted based on both morphological and molecular data. However, ribosomal genes have never been used in tribe level phylogenetic analyses of Satyrinae. In this study we investigate for the first time the phylogenetic relationships among the tribes Elymniini, Amathusiini, Zetherini and Melanitini which are indicated to be a monophyletic group, and the Satyrini, using two ribosomal genes (28s rDNA and 16s rDNA) and four protein-coding genes (EF-1α, COI, COII and Cytb). We mainly aim to assess the phylogenetic informativeness of the ribosomal genes as well as clarify the relationships among different tribes. Our results show the two ribosomal genes generally have the same high phylogenetic informativeness compared with EF-1α; and we infer the 28s rDNA would show better informativeness if the 28s rDNA sequence data for each sampling taxon are obtained in this study. The placement of the monotypic genus Callarge Leech in Zetherini is confirmed for the first time based on molecular evidence. In addition, our maximum likelihood (ML) and Bayesian inference (BI) trees consistently show that the involved Satyrinae including the Amathusiini is monophyletic with high support values. Although the relationships among the five tribes are identical among ML and BI analyses and are mostly strongly-supported in BI analysis, those in ML analysis are lowly- or moderately- supported. Therefore, the relationships among the related five tribes recovered herein need further verification based on more sampling taxa. PMID:25878526

  12. The Genes for Cytoplasmic Ribosomal Ribonucleic Acid in Higher Plants

    PubMed Central

    Scott, N. Steele; Ingle, J.

    1973-01-01

    The genes for cytoplasmic ribosomal RNA are partially resolved from the bulk of the DNA by CsCl equilibrium centrifugation. Although in some plants the buoyant density of the ribosomal RNA genes is as expected from the base composition of ribosomal RNA, others show a large discrepancy which cannot be due to the presence of low G-C spacer-DNA. The cross-hybridization observed with 1.3 and 0.7 × 106 molecular weight ribosomal RNAs and DNA, which varies greatly with different plant species, is not due to contamination of the ribosomal RNAs, and is specific for the ribosomal DNA of each species, probably largely restricted to those sequences coding for the two stable ribosomal RNAs. The double reciprocal plot may be used for the extrapolation of saturation values only with caution, because in these cases such plots are not linear over the whole of the hybridization reaction. PMID:16658392

  13. The ribosomal gene spacer region in archaebacteria

    NASA Technical Reports Server (NTRS)

    Achenbach-Richter, L.; Woese, C. R.

    1988-01-01

    Sequences for the spacer regions that separate the 16S and 23S ribosomal RNA genes have been determined for four more (strategically placed) archaebacteria. These confirm the general rule that methanogens and extreme halophiles have spacers that contain a single tRNAala gene, while tRNA genes are not found in the spacer region of the true extreme thermophiles. The present study also shows that the spacer regions from the sulfate reducing Archaeglobus and the extreme thermophile Thermococcus (both of which cluster phylogenetically with the methanogens and extreme halophiles) contain each a tRNAala gene. Thus, not only all methanogens and extreme halophiles show this characteristic, but all organisms on the "methanogen branch" of the archaebacterial tree appear to do so. The finding of a tRNA gene in the spacer region of the extreme thermophile Thermococcus celer is the first known phenotypic property that links this organism with its phylogenetic counterparts, the methanogens, rather than with its phenotypic counterparts, the sulfur-dependent extreme thermophiles.

  14. Secondary structure and phylogenetic utility of the ribosomal large subunit (28S) in monogeneans of the genus Thaparocleidus and Bifurcohaptor (Monogenea: Dactylogyridae).

    PubMed

    Chaudhary, Anshu; Singh, Hridaya Shanker

    2013-04-01

    Present communication deals with secondary structure of 28S rDNA of two already known species of monogeneans viz., Bifurcohaptor indicus and Thaparocleidus parvulus parasitizing gill filaments of a freshwater fish, Mystus vittatus for phylogenetic inference. Secondary structure data are best used as accessory taxonomic characters as their phylogenetic resolving power and confidence in validity. Secondary structure of the 28S rDNA transcript could provide information for identifying homologous nucleotide characters, useful for cladistic inference of relationships. Such structure data could be used as taxonomic character. The study supports that species-level sequence variability renders 28S sequence as a unique window for examining the behavior of fast evolving, non-coding DNA sequences. Apart from this it also confirms that molecular similarity present in various species could be host-induced. PMID:24431545

  15. Molecular mechanisms of ribosomal protein gene coregulation.

    PubMed

    Reja, Rohit; Vinayachandran, Vinesh; Ghosh, Sujana; Pugh, B Franklin

    2015-09-15

    The 137 ribosomal protein genes (RPGs) of Saccharomyces provide a model for gene coregulation. We examined the positional and functional organization of their regulators (Rap1 [repressor activator protein 1], Fhl1, Ifh1, Sfp1, and Hmo1), the transcription machinery (TFIIB, TFIID, and RNA polymerase II), and chromatin at near-base-pair resolution using ChIP-exo, as RPGs are coordinately reprogrammed. Where Hmo1 is enriched, Fhl1, Ifh1, Sfp1, and Hmo1 cross-linked broadly to promoter DNA in an RPG-specific manner and demarcated by general minor groove widening. Importantly, Hmo1 extended 20-50 base pairs (bp) downstream from Fhl1. Upon RPG repression, Fhl1 remained in place. Hmo1 dissociated, which was coupled to an upstream shift of the +1 nucleosome, as reflected by the Hmo1 extension and core promoter region. Fhl1 and Hmo1 may create two regulatable and positionally distinct barriers, against which chromatin remodelers position the +1 nucleosome into either an activating or a repressive state. Consistent with in vitro studies, we found that specific TFIID subunits, in addition to cross-linking at the core promoter, made precise cross-links at Rap1 sites, which we interpret to reflect native Rap1-TFIID interactions. Our findings suggest how sequence-specific DNA binding regulates nucleosome positioning and transcription complex assembly >300 bp away and how coregulation coevolved with coding sequences. PMID:26385964

  16. The Hymenopteran Tree of Life: Evidence from Protein-Coding Genes and Objectively Aligned Ribosomal Data

    PubMed Central

    Klopfstein, Seraina; Vilhelmsen, Lars; Heraty, John M.; Sharkey, Michael; Ronquist, Fredrik

    2013-01-01

    Previous molecular analyses of higher hymenopteran relationships have largely been based on subjectively aligned ribosomal sequences (18S and 28S). Here, we reanalyze the 18S and 28S data (unaligned about 4.4 kb) using an objective and a semi-objective alignment approach, based on MAFFT and BAli-Phy, respectively. Furthermore, we present the first analyses of a substantial protein-coding data set (4.6 kb from one mitochondrial and four nuclear genes). Our results indicate that previous studies may have suffered from inflated support values due to subjective alignment of the ribosomal sequences, but apparently not from significant biases. The protein data provide independent confirmation of several earlier results, including the monophyly of non-xyelid hymenopterans, Pamphilioidea + Unicalcarida, Unicalcarida, Vespina, Apocrita, Proctotrupomorpha and core Proctotrupomorpha. The protein data confirm that Aculeata are nested within a paraphyletic Evaniomorpha, but cast doubt on the monophyly of Evanioidea. Combining the available morphological, ribosomal and protein-coding data, we examine the total-evidence signal as well as congruence and conflict among the three data sources. Despite an emerging consensus on many higher-level hymenopteran relationships, several problems remain unresolved or contentious, including rooting of the hymenopteran tree, relationships of the woodwasps, placement of Stephanoidea and Ceraphronoidea, and the sister group of Aculeata. PMID:23936325

  17. Introns regulate the production of ribosomal proteins by modulating splicing of duplicated ribosomal protein genes

    PubMed Central

    Petibon, Cyrielle; Parenteau, Julie; Catala, Mathieu; Elela, Sherif Abou

    2016-01-01

    Most budding yeast introns exist in the many duplicated ribosomal protein genes (RPGs) and it has been posited that they remain there to modulate the expression of RPGs and cell growth in response to stress. However, the mechanism by which introns regulate the expression of RPGs and their impact on the synthesis of ribosomal proteins remain unclear. In this study, we show that introns determine the ratio of ribosomal protein isoforms through asymmetric paralog-specific regulation of splicing. Exchanging the introns and 3′ untranslated regions of the duplicated RPS9 genes altered the splicing efficiency and changed the ratio of the ribosomal protein isoforms. Mutational analysis of the RPS9 genes indicated that splicing is regulated by variations in the intron structure and the 3′ untranslated region. Together these data suggest that preferential splicing of duplicated RPGs provides a means for adjusting the ratio of different ribosomal protein isoforms, while maintaining the overall expression level of each ribosomal protein. PMID:26945043

  18. A combination of morphology and 28S rRNA gene sequences provide grouping and ranking criteria to merge eight into three Ambispora species (Ambisporaceae, Glomeromycota).

    PubMed

    Bills, Robert J; Morton, Joseph B

    2015-08-01

    Ambispora, the only genus in Ambisporaceae and one of three deeply rooted families in Archaeosporales, Glomeromycetes, is amended. Analysis of the morphology of specimens from types and living cultures and 28S ribosomal DNA (rDNA; LSU) sequences resulted in two major changes that redefined Ambispora to include only species with the potential for spore dimorphism (acaulosporoid and glomoid). First, species described as producing only glomoid spores (Ambispora leptoticha, Ambispora fecundispora, and Ambispora callosa), only acaulosporoid spores (Ambispora jimgerdemannii), or both spore morphotypes (Ambispora appendicula) were synonymized with a redefined dimorphic species, A. leptoticha. LSU sequences and more conserved SSU gene data indicated little divergence between genotypes formerly classified as separate species. Second, Ambispora fennica was synonymized with Ambispora gerdemannii based on morphological and LSU sequence variation equivalent to that measured in the sister clade A. leptoticha. With this analysis, Ambispora was reduced to three species: A. leptoticha, A. gerdemannii, and Ambispora granatensis. Morphological and molecular characters were given equal treatment in this study, as each data set informed and clarified grouping and ranking decisions. The two inner layers of the acaulosporoid spore wall were the only structural characters uniquely defining each of these three species; all other characters were shared. Phenotypes of glomoid spores were indistinguishable between species, and thus were informative only at the genus level. Distinct subclade structure of the LSU gene tree suggests fixation of discrete variants typical of clonal reproduction and possible retention of polymorphisms in rDNA repeats, so that not all discrete genetic variants are indicative of speciation. PMID:25638691

  19. Relationship between organization and function of ribosomal genes in Drosophila melanogaster

    SciTech Connect

    Karpen, G.H.

    1987-01-01

    In most eukaryotic organisms, the genes that encode the 18S and 28S ribosomal RNAs (rDNA genes) are tandemly repeated, and are located in constitutive heterochromatin and/or centromeric or telomeric regions. P-element mediated transformation was used to investigate the relationship between rDNA organization and function in Drosophila melanogaster. Tritiated-uridine incorporation under heat shock conditions and in situ hybridization to rRNA were used to demonstrate that a single rDNA gene inserted into euchromatin can be transcribed at a high rate, in polytene nuclei. P-element-mediated transformation of a single Drosophila rDNA gene was also utilized to investigate the ability of ribosomal DNA to organize a nucleolus. Cytological approaches demonstrated that structures resembling the endogenous nucleoli were preferentially associated with four different sites of rDNA insertion, in polytene nuclei. These mini-nucleoli also contained components specific to the nucleolus, as shown by in situ hybridization to rRNA and indirect immunofluorescence with an antibody that binds to Drosophila nucleoli. The transformed genes were able to partially rescue mutant phenotypes due to a deficiency of rDNA, indicating that the mini-nucleoli were functional.

  20. Phylogenetic Analysis of the Spider Mite Sub-Family Tetranychinae (Acari: Tetranychidae) Based on the Mitochondrial COI Gene and the 18S and the 5′ End of the 28S rRNA Genes Indicates That Several Genera Are Polyphyletic

    PubMed Central

    Matsuda, Tomoko; Morishita, Maiko; Hinomoto, Norihide; Gotoh, Tetsuo

    2014-01-01

    The spider mite sub-family Tetranychinae includes many agricultural pests. The internal transcribed spacer (ITS) region of nuclear ribosomal RNA genes and the cytochrome c oxidase subunit I (COI) gene of mitochondrial DNA have been used for species identification and phylogenetic reconstruction within the sub-family Tetranychinae, although they have not always been successful. The 18S and 28S rRNA genes should be more suitable for resolving higher levels of phylogeny, such as tribes or genera of Tetranychinae because these genes evolve more slowly and are made up of conserved regions and divergent domains. Therefore, we used both the 18S (1,825–1,901 bp) and 28S (the 5′ end of 646–743 bp) rRNA genes to infer phylogenetic relationships within the sub-family Tetranychinae with a focus on the tribe Tetranychini. Then, we compared the phylogenetic tree of the 18S and 28S genes with that of the mitochondrial COI gene (618 bp). As observed in previous studies, our phylogeny based on the COI gene was not resolved because of the low bootstrap values for most nodes of the tree. On the other hand, our phylogenetic tree of the 18S and 28S genes revealed several well-supported clades within the sub-family Tetranychinae. The 18S and 28S phylogenetic trees suggest that the tribes Bryobiini, Petrobiini and Eurytetranychini are monophyletic and that the tribe Tetranychini is polyphyletic. At the genus level, six genera for which more than two species were sampled appear to be monophyletic, while four genera (Oligonychus, Tetranychus, Schizotetranychus and Eotetranychus) appear to be polyphyletic. The topology presented here does not fully agree with the current morphology-based taxonomy, so that the diagnostic morphological characters of Tetranychinae need to be reconsidered. PMID:25289639

  1. Lophotrochozoa internal phylogeny: new insights from an up-to-date analysis of nuclear ribosomal genes

    PubMed Central

    Paps, Jordi; Baguñà, Jaume; Riutort, Marta

    2009-01-01

    Resolving the relationships among animal phyla is a key biological problem that remains to be solved. Morphology is unable to determine the relationships among most phyla and although molecular data have unveiled a new evolutionary scenario, they have their own limitations. Nuclear ribosomal genes (18S and 28S rDNA) have been used effectively for many years. However, they are considered of limited use for resolving deep divergences such as the origin of the bilaterians, due to certain drawbacks such as the long-branch attraction (LBA) problem. Here, we attempt to overcome these pitfalls by combining several methods suggested in previous studies and routinely used in contemporary standard phylogenetic analyses but that have not yet been applied to any bilaterian phylogeny based on these genes. The methods used include maximum likelihood and Bayesian inference, the application of models with rate heterogeneity across sites, wide taxon sampling and compartmentalized analyses for each problematic clade. The results obtained show that the combination of the above-mentioned methodologies minimizes the LBA effect, and a new Lophotrochozoa phylogeny emerges. Also, the Acoela and Nemertodermatida are confirmed with maximum support as the first branching bilaterians. Ribosomal RNA genes are thus a reliable source for the study of deep divergences in the metazoan tree, provided that the data are treated carefully. PMID:19129141

  2. Differential Expression of Ribosomal Genes in Brain and Blood of Alzheimer's Disease Patients.

    PubMed

    Rasmussen, Lucas; de Labio, Roger W; Viani, Gustavo A; Chen, Elizabeth; Villares, Joao; Bertolucci, Paulo-Henrique; Minett, Thais S; Turecki, Gustavo; Cecyre, Danielle; Drigo, Sandra A; Smith, Marilia C; Payao, Spencer L M

    2015-01-01

    Changes in rRNA and rDNA expression have been associated with cellular and organism aging and have been linked to Alzheimer's disease (AD) pathogenesis. In this study, we investigated the mRNA expression of ribosomal genes (28S/18S) and β-amyloid precursor protein (APP) in different post mortem brain tissue regions (the entorhinal and auditory cortices and the hippocampus) of AD patients and elderly control subjects and also evaluated the extent of expression in peripheral blood from young, healthy, elderly, and Alzheimer's disease patients in order to investigate whether these individuals experienced the effects of aging. The comparative threshold cycle (CT) method via Real Time Polymerase Chain Reaction and the Polymerase Chain Reaction- Restriction Fragment Length Polymorphism (PCR-RFLP) were used to analyze gene expression and the Apolipoprotein E (APOE) genotype, respectively. When the brain areas were analyzed collectively, we observed a significant decrease in APP expression and a significant increase in levels of mRNA of 18S and 28S in Alzheimer's disease patients compared to healthy elderly individuals. Furthermore, there was a significant upregulation of 28SrRNA in the entorhinal cortex and hippocampus, but not in the auditory cortex of patients with AD. On the other hand, tests of blood samples verified a decreased expression of 28S rRNA in patients with AD. These results support the hypothesis that changes in rRNA are present in AD patients, are tissue-specific, and seem to occur independently and differently in each tissue. However, the next challenge is to discover the mechanisms responsible for the differences in expression observed in the blood and the brain in both healthy elderly individuals and Alzheimer's disease patients, as well as the impact of these genes on AD pathogenesis. PMID:26502820

  3. Epigeneitc silencing of ribosomal RNA genes by Mybbp1a

    PubMed Central

    2012-01-01

    Background Transcription of the ribosomal RNA gene repeats by Pol I occurs in the nucleolus and is a fundamental step in ribosome biogenesis and protein translation. Due to tight coordination between ribosome biogenesis and cell proliferation, transcription of rRNA and stable maintenance of rDNA clusters are thought to be under intricate control by intercalated mechanisms, particularly at the epigenetic level. Methods and Results Here we identify the nucleolar protein Myb-binding protein 1a (Mybbp1a) as a novel negative regulator of rRNA expression. Suppression of rDNA transcription by Mybbp1a was linked to promoter regulation as illustrated by its binding to the chromatin around the hypermethylated, inactive rDNA gene promoters. Our data further showed that downregulation of Mybbp1a abrogated the local DNA methylation levels and histone marks associated with gene silencing, and altered the promoter occupancy of various factors such UBF and HDACs, consequently leading to elevated rRNA expression. Mechanistically, we propose that Mybbp1a maintains rDNA repeats in a silenced state while in association with the negative epigenetic modifiers HDAC1/2. Conclusions Results from our present work reveal a previously unrecognized co-repressor role of Mybbp1a in rRNA expression. They are further consistent with the scenario that Mybbp1a is an integral constituent of the rDNA epigenetic regulation that underlies the balanced state of rDNA clusters. PMID:22686419

  4. Phylogenetic Analysis Using the 28S rRNA Gene Reveals That the Genus Paracreptotrema (Digenea: Allocreadiidae) Is Not Monophyletic; Description of Two New Genera and One New Species.

    PubMed

    de León, Gerardo Pérez-Ponce; Pinacho-Pinacho, Carlos D; Mendoza-Garfias, Berenit; Choudhury, Anindo; García-Varela, Martín

    2016-02-01

    This study investigates the systematics of Paracreptotrema Choudhury, Pérez-Ponce de León, Brooks and Daverdin, 2006 using morphological data (stained whole mounts and scanning electron microscopy) and partial sequences of the 28S ribosomal rRNA gene, obtained from freshly collected material. In total, 484 specimens representing 4 species, i.e., Paracreptotrema blancoi (157), Paracreptotrema profundulusi (12), Paracreptotrema rosenthali (8), and Paracreptotrema blancoi sensu Salgado-Maldonado et al. (2011) (307) were collected. Existing museum depositions were also studied. The 28S rRNA gene sequences of these Paracreptotrema spp. were aligned, along with sequences from 22 other allocreadiids and 4 other non-allocreadiid xiphidiatan species. Bayesian inference and maximum likelihood analyses indicated a paraphyletic Paracreptotrema split into 3 clades: 1 comprising P. blancoi and P. rosenthali that was sister to a clade formed by 3 other species of allocreadiids (species of Wallinia, Creptotrematina, and Auriculostoma) typically found in characid fishes, a second clade formed solely by Paracreptotrema heterandriae as the sister taxon of the aforementioned species, and a third by P. profundulusi and specimens erroneously identified as P. blancoi. Two new taxa were erected to reflect these results: Paracreptotrematoides for Paracreptotrema heterandriae, and Pseudoparacreptotrema for Paracreptotrema profundulusi and P. macroacetabulata (the species erroneously identified as P. blancoi from profundulids across Middle America). Closer consideration of the morphology corroborates these findings. The revised systematics also indicated that Paracreptotrema spp. are found in poeciliids, whereas Pseudoparacreptotrema spp. parasitize profundulids. The study demonstrates the value of an integrative taxonomy approach to address the apparently complicated systematics of the allocreadiids. PMID:26561039

  5. Development and evaluation of a 28S rRNA gene-based nested PCR assay for P. falciparum and P. vivax

    PubMed Central

    Pakalapati, Deepak; Garg, Shilpi; Middha, Sheetal; Acharya, Jyoti; Subudhi, Amit K; Boopathi, Arunachalam P; Saxena, Vishal; Kochar, Sanjay K; Kochar, Dhanpat K; Das, Ashis

    2013-01-01

    The 28S rRNA gene was amplified and sequenced from P. falciparum and P. vivax isolates collected from northwest India. Based upon the sequence diversity of the Plasmodium 28SrRNA gene in comparison with its human counterpart, various nested polymerase chain reaction (PCR) primers were designed from the 3R region of the 28SrRNA gene and evaluated on field isolates. This is the first report demonstrating the utility of this gene for species-specific diagnosis of malaria for these two species, prevalent in India. The initial evaluation on 363 clinical isolates indicated that, in comparison with microscopy, which showed sensitivity and specificity of 85.39% and 100% respectively, the sensitivity and specificity of the nested PCR assay was found to be 99.08% and 100% respectively. This assay was also successful in detecting mixed infections that are undetected by microscopy. Our results demonstrate the utility of the 28S rRNA gene as a diagnostic target for the detection of the major plasmodial species infecting humans. PMID:23816509

  6. D2 Region of the 28S RNA Gene: A Too-Conserved Fragment for Inferences on Phylogeny of South American Triatomines.

    PubMed

    Guerra, Ana Letícia; Alevi, Kaio Cesar Chaboli; Banho, Cecília Artico; de Oliveira, Jader; da Rosa, João Aristeu; Vilela de Azeredo-Oliveira, Maria Tercília

    2016-09-01

    The brasiliensis complex is composed of five triatomine species, and different approaches suggest that Triatoma lenti and Triatoma petrochiae may be the new members. Therefore, this study sought to analyze the phylogenetic relationships within this complex by means of the D2 region of the 28S RNA gene, and to analyze the degree of polymorphism and phylogenetic significance of this gene for South American triatomines. Phylogenetic analysis by using sequence fragments of the D2 domain did not allow to perform phylogenetic inferences on species within the brasiliensis complex, because the gene alignment composed of a matrix with 37 specimens exhibited only two variable sites along the 567 base pairs used. Furthermore, if all South American species are included, only four variable sites were detected, reflecting the high degree of gene conservation. Therefore, we do not recommend the use of this gene for phylogenetic reconstruction for this group of Chagas disease vectors. PMID:27382073

  7. Characterization of recombinant bacteriophages containing mosquito ribosomal RNA genes

    SciTech Connect

    Park, Y.J.

    1988-01-01

    A family of nine recombinant bacteriophages containing rRNA genes from cultured cells of the mosquito, Aedes albopictus, has been isolated by screening two different genomic DNA libraries - Charon 30 and EMBL 3 using {sup 32}P-labeled 18S and 28S rRNA as probes. These nine recombinant bacteriophages were characterized by restriction mapping, Southern blotting, and S1 nuclease analysis. The 18S rRNA coding region contains an evolutionarily conserved EcoRI site near the 3{prime}-end, and measures 1800 bp. The 28S rRNA genes were divided into {alpha} and {beta} coding regions measuring 1750 bp and 2000 bp, respectively. The gap between these two regions measures about 340 bp. No insertion sequences were found in the rRNA coding regions. The entire rDNA repeat unit had a minimum length of 15.6 kb, including a nontranscribed spacer region. The non-transcribed spacer region of cloned A. albopictus rDNA contained a common series of seven PvuI sites within a 1250 bp region upstream of the 18S rRNA coding region, and a proportion of this region also showed heterogeneity both in the length and in the restriction sites.

  8. Pulmonate phylogeny based on 28S rRNA gene sequences: a framework for discussing habitat transitions and character transformation.

    PubMed

    Holznagel, W E; Colgan, D J; Lydeard, C

    2010-12-01

    Pulmonate snails occupy a wide range of marine, estuarine, freshwater and terrestrial environments. Non-terrestrial forms are supposed to be basal in pulmonate evolution but the group's phylogeny is not well resolved either morphologically or on the basis of available DNA sequence data. The lack of a robust phylogeny makes it difficult to understand character polarization and habitat transformation in pulmonates. We have investigated pulmonate relationships using 27 new sequences of 28S rRNA from pulmonates and outgroups, augmented with data from GenBank. The complete alignments comprised about 3.8kb. Maximum parsimony, maximum likelihood and Bayesian analyses of alignments generated under different assumptions are reported. Complete alignments appear to have a degree of substitution saturation so where there is conflict between hypothesised relationships more weight is given to analyses where regions of random similarity are excluded and which are not affected by this complication. Monophyly of the five main pulmonate groups was robustly supported in almost all analyses. The marine group Amphiboloidea and the freshwater Glacidorbidae are the most basal. The remaining pulmonates (Siphonariidae, Hygrophila and Eupulmonata) form a moderately-supported monophyletic group in all analyses bar one probably affected by saturation of substitutions. Siphonariidae, a predominantly marine and intertidal family, and Eupulmonata (mainly terrestrial with marine, estuarine and freshwater species) form a strongly supported clade that is the sister group to Hygrophila (freshwater). Multiple colonizations of freshwater and terrestrial habitats by pulmonate snails are suggested. No analyses strongly support the possibility of habitat reversions. The colonizations of freshwater by Hygrophila and of land by Stylommatophora were apparently phylogenetically independent although it cannot yet be excluded that there were transient terrestrial phases in the history of the former group or

  9. Genomic architecture and inheritance of human ribosomal RNA gene clusters

    PubMed Central

    Stults, Dawn M.; Killen, Michael W.; Pierce, Heather H.; Pierce, Andrew J.

    2008-01-01

    The finishing of the Human Genome Project largely completed the detailing of human euchromatic sequences; however, the most highly repetitive regions of the genome still could not be assembled. The 12 gene clusters producing the structural RNA components of the ribosome are critically important for cellular viability, yet fall into this unassembled region of the Human Genome Project. To determine the extent of human variation in ribosomal RNA gene content (rDNA) and patterns of rDNA cluster inheritance, we have determined the physical lengths of the rDNA clusters in peripheral blood white cells of healthy human volunteers. The cluster lengths exhibit striking variability between and within human individuals, ranging from 50 kb to >6 Mb, manifest essentially complete heterozygosity, and provide each person with their own unique rDNA electrophoretic karyotype. Analysis of these rDNA fingerprints in multigenerational human families demonstrates that the rDNA clusters are subject to meiotic rearrangement at a frequency >10% per cluster, per meiosis. With this high intrinsic recombinational instability, the rDNA clusters may serve as a unique paradigm of potential human genomic plasticity. PMID:18025267

  10. 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

  11. Autogenous Regulation of Splicing of the Transcript of a Yeast Ribosomal Protein Gene

    NASA Astrophysics Data System (ADS)

    Dabeva, Mariana D.; Post-Beittenmiller, Martha A.; Warner, Jonathan R.

    1986-08-01

    The gene for a yeast ribosomal protein, RPL32, contains a single intron. The product of this gene appears to participate in feedback control of the splicing of the intron from the transcript. This autogenous regulation of splicing provides a striking analogy to the autogenous regulation of translation of ribosomal proteins in Escherichia coli.

  12. Characteristics of the nuclear (18S, 5.8S, 28S and 5S) and mitochondrial (12S and 16S) rRNA genes of Apis mellifera (Insecta: Hymenoptera): structure, organization, and retrotransposable elements

    PubMed Central

    Gillespie, J J; Johnston, J S; Cannone, J J; Gutell, R R

    2006-01-01

    As an accompanying manuscript to the release of the honey bee genome, we report the entire sequence of the nuclear (18S, 5.8S, 28S and 5S) and mitochondrial (12S and 16S) ribosomal RNA (rRNA)-encoding gene sequences (rDNA) and related internally and externally transcribed spacer regions of Apis mellifera (Insecta: Hymenoptera: Apocrita). Additionally, we predict secondary structures for the mature rRNA molecules based on comparative sequence analyses with other arthropod taxa and reference to recently published crystal structures of the ribosome. In general, the structures of honey bee rRNAs are in agreement with previously predicted rRNA models from other arthropods in core regions of the rRNA, with little additional expansion in non-conserved regions. Our multiple sequence alignments are made available on several public databases and provide a preliminary establishment of a global structural model of all rRNAs from the insects. Additionally, we provide conserved stretches of sequences flanking the rDNA cistrons that comprise the externally transcribed spacer regions (ETS) and part of the intergenic spacer region (IGS), including several repetitive motifs. Finally, we report the occurrence of retrotransposition in the nuclear large subunit rDNA, as R2 elements are present in the usual insertion points found in other arthropods. Interestingly, functional R1 elements usually present in the genomes of insects were not detected in the honey bee rRNA genes. The reverse transcriptase products of the R2 elements are deduced from their putative open reading frames and structurally aligned with those from another hymenopteran insect, the jewel wasp Nasonia (Pteromalidae). Stretches of conserved amino acids shared between Apis and Nasonia are illustrated and serve as potential sites for primer design, as target amplicons within these R2 elements may serve as novel phylogenetic markers for Hymenoptera. Given the impending completion of the sequencing of the Nasonia genome

  13. Dosage Sensitivity of RPL9 and Concerted Evolution of Ribosomal Protein Genes in Plants

    PubMed Central

    Devis, Deborah; Firth, Sue M.; Liang, Zhe; Byrne, Mary E.

    2015-01-01

    The ribosome in higher eukaryotes is a large macromolecular complex composed of four rRNAs and eighty different ribosomal proteins. In plants, each ribosomal protein is encoded by multiple genes. Duplicate genes within a family are often necessary to provide a threshold dose of a ribosomal protein but in some instances appear to have non-redundant functions. Here, we addressed whether divergent members of the RPL9 gene family are dosage sensitive or whether these genes have non-overlapping functions. The RPL9 family in Arabidopsis thaliana comprises two nearly identical members, RPL9B and RPL9C, and a more divergent member, RPL9D. Mutations in RPL9C and RPL9D genes lead to delayed growth early in development, and loss of both genes is embryo lethal, indicating that these are dosage-sensitive and redundant genes. Phylogenetic analysis of RPL9 as well as RPL4, RPL5, RPL27a, RPL36a, and RPS6 family genes in the Brassicaceae indicated that multicopy ribosomal protein genes have been largely retained following whole genome duplication. However, these gene families also show instances of tandem duplication, small scale deletion, and evidence of gene conversion. Furthermore, phylogenetic analysis of RPL9 genes in angiosperm species showed that genes within a species are more closely related to each other than to RPL9 genes in other species, suggesting ribosomal protein genes undergo convergent evolution. Our analysis indicates that ribosomal protein gene retention following whole genome duplication contributes to the number of genes in a family. However, small scale rearrangements influence copy number and likely drive concerted evolution of these dosage-sensitive genes. PMID:26734020

  14. Molecular phylogenetics of the spider infraorder Mygalomorphae using nuclear rRNA genes (18S and 28S): conflict and agreement with the current system of classification.

    PubMed

    Hedin, Marshal; Bond, Jason E

    2006-11-01

    Mygalomorph spiders, which include the tarantulas, trapdoor spiders, and their kin, represent one of three main spider lineages. Mygalomorphs are currently classified into 15 families, comprising roughly 2500 species and 300 genera. The few published phylogenies of mygalomorph relationships are based exclusively on morphological data and reveal areas of both conflict and congruence, suggesting the need for additional phylogenetic research utilizing new character systems. As part of a larger combined evidence study of global mygalomorph relationships, we have gathered approximately 3.7 kb of rRNA data (18S and 28S) for a sample of 80 genera, representing all 15 mygalomorph families. Taxon sampling was particularly intensive across families that are questionable in composition-Cyrtaucheniidae and Nemesiidae. The following primary results are supported by both Bayesian and parsimony analyses of combined matrices representing multiple 28S alignments: (1) the Atypoidea, a clade that includes the families Atypidae, Antrodiaetidae, and Mecicobothriidae, is recovered as a basal lineage sister to all other mygalomorphs, (2) diplurids and hexathelids form a paraphyletic grade at the base of the non-atypoid clade, but neither family is monophyletic in any of our analyses, (3) a clade consisting of all sampled nemesiids, Microstigmata and the cyrtaucheniid genera Kiama, Acontius, and Fufius is consistently recovered, (4) other sampled cyrtaucheniids are fragmented across three separate clades, including a monophyletic North American Euctenizinae and a South African clade, (5) of the Domiothelina, only idiopids are consistently recovered as monophyletic; ctenizids are polyphyletic and migids are only weakly supported. The Domiothelina is not monophyletic. The molecular results we present are consistent with more recent hypotheses of mygalomorph relationship; however, additional work remains before mygalomorph classification can be formally reassessed with confidence

  15. Ribosomes in the balance: structural equilibrium ensures translational fidelity and proper gene expression

    PubMed Central

    Musalgaonkar, Sharmishtha; Moomau, Christine A.; Dinman, Jonathan D.

    2014-01-01

    At equilibrium, empty ribosomes freely transit between the rotated and un-rotated states. In the cell, the binding of two translation elongation factors to the same general region of the ribosome stabilizes one state over the other. These stabilized states are resolved by expenditure of energy in the form of GTP hydrolysis. A prior study employing mutants of a late assembling peripheral ribosomal protein suggested that ribosome rotational status determines its affinity for elongation factors, and hence translational fidelity and gene expression. Here, mutants of the early assembling integral ribosomal protein uL2 are used to test the generality of this hypothesis. rRNA structure probing analyses reveal that mutations in the uL2 B7b bridge region shift the equilibrium toward the rotated state, propagating rRNA structural changes to all of the functional centers of ribosome. Structural disequilibrium unbalances ribosome biochemically: rotated ribosomes favor binding of the eEF2 translocase and disfavor that of the elongation ternary complex. This manifests as specific translational fidelity defects, impacting the expression of genes involved in telomere maintenance. A model is presented describing how cyclic intersubunit rotation ensures the unidirectionality of translational elongation, and how perturbation of rotational equilibrium affects specific aspects of translational fidelity and cellular gene expression. PMID:25389262

  16. DNA homologies of ribosomal RNA genes of Neurospora species

    SciTech Connect

    Mukhopadhyay, D.K.; Mimiko, R.; Dutta, S.K.

    1980-01-01

    Ribosomal RNA genes (rDNAs) of Neurospora crassa contain DNA sequences which code for 17S, 5.8S, and 26S rRNAs, in addition to internal and external spacers. As has been reported for many eukaryotes, the DNA sequences which code for 17S, 5.8S, and 26S rRNAs in Neurospora species are probably conserved while the internal and external spacer regions are probably variable sequences. Extensive electron microscopic studies of 45S precursor rRNA of several cold and warm blooded animals confirm that spacer regions vary extensively from species to species. It was desirable to know whether such differences in rDNA sequences exist between Neurospora species. Any such difference should be detectable using standard procedures for DNA homology studies rDNA sequences were isolated from N. crassa mycelial cells using the procedure described previously. The purified rDNA was /sup 3/H-labeled (by nick translation) and reassociated with total DNA isolated from the heterothallic species N. crassa and from three homothalliospecies: N. dodgei, N. lineolata, and N. africana. In addition, /sup 32/P-labeled total DNA of N. crassa was reannealed with unlabeled bulk DNA from N. crassa, N. dodgei, and N. lineolata.

  17. Expression of ribosomal genes in pea cotyledons at the initial stages of germination

    SciTech Connect

    Gumilevskaya, N.A.; Chumikhina, L.V.; Akhmatova, A.T.; Kretovich, V.L.

    1986-01-20

    The time of appearance of newly synthesized rRNAs and ribosomal proteins (r-proteins) in the ribosomes of pea cotyledons (Pisum sativum L.) during germination was investigated. The ribosomal fraction was isolated and analyzed according to the method of germination of the embryo in the presence of labeled precursors or after pulse labeling of the embryos at different stages of germination. For the identification of newly synthesized rRNAs in the ribosomes we estimated the relative stability of labeled RNAs to the action of RNase, the sedimentation rate, the ability to be methylated in vivo in the presence of (/sup 14/C)CH/sub 3/-methionine, and the localization in the subunits of dissociated ribosomes. The presence of newly synthesized r-proteins in the ribosomes was judged on the basis of the electrophoretic similarity in SDS-disc electrophoresis of labeled polypeptides of purified ribosome preparations and of genuine r-proteins, as well as according to the localization of labeled proteins in the subunits of the dissociated ribosomes. It was shown that the expression of the ribosomal genes in highly specialized cells of pea cotyledons that have completed their growth occurs at very early stages of germination.

  18. Further use of nearly complete 28S and 18S rRNA genes to classify Ecdysozoa: 37 more arthropods and a kinorhynch.

    PubMed

    Mallatt, Jon; Giribet, Gonzalo

    2006-09-01

    This work expands on a study from 2004 by Mallatt, Garey, and Shultz [Mallatt, J.M., Garey, J.R., Shultz, J.W., 2004. Ecdysozoan phylogeny and Bayesian inference: first use of nearly complete 28S and 18S rRNA gene sequences to classify the arthropods and their kin. Mol. Phylogenet. Evol. 31, 178-191] that evaluated the phylogenetic relationships in Ecdysozoa (molting animals), especially arthropods. Here, the number of rRNA gene-sequences was effectively doubled for each major group of arthropods, and sequences from the phylum Kinorhyncha (mud dragons) were also included, bringing the number of ecdysozoan taxa to over 80. The methods emphasized maximum likelihood, Bayesian inference and statistical testing with parametric bootstrapping, but also included parsimony and minimum evolution. Prominent findings from our combined analysis of both genes are as follows. The fundamental subdivisions of Hexapoda (insects and relatives) are Insecta and Entognatha, with the latter consisting of collembolans (springtails) and a clade of proturans plus diplurans. Our rRNA-gene data provide the strongest evidence to date that the sister group of Hexapoda is Branchiopoda (fairy shrimps, tadpole shrimps, etc.), not Malacostraca. The large, Pancrustacea clade (hexapods within a paraphyletic Crustacea) divided into a few basic subclades: hexapods plus branchiopods; cirripedes (barnacles) plus malacostracans (lobsters, crabs, true shrimps, isopods, etc.); and the basally located clades of (a) ostracods (seed shrimps) and (b) branchiurans (fish lice) plus the bizarre pentastomids (tongue worms). These findings about Pancrustacea agree with a recent study by Regier, Shultz, and Kambic that used entirely different genes [Regier, J.C., Shultz, J.W., Kambic, R.E., 2005a. Pancrustacean phylogeny: hexapods are terrestrial crustaceans and maxillopods are not monophyletic. Proc. R. Soc. B 272, 395-401]. In Malacostraca, the stomatopod (mantis shrimp) was not at the base of the eumalacostracans

  19. Tempo and Mode of Gene Duplication in Mammalian Ribosomal Protein Evolution

    PubMed Central

    Gajdosik, Matthew D.; Simon, Amanda; Nelson, Craig E.

    2014-01-01

    Gene duplication has been widely recognized as a major driver of evolutionary change and organismal complexity through the generation of multi-gene families. Therefore, understanding the forces that govern the evolution of gene families through the retention or loss of duplicated genes is fundamentally important in our efforts to study genome evolution. Previous work from our lab has shown that ribosomal protein (RP) genes constitute one of the largest classes of conserved duplicated genes in mammals. This result was surprising due to the fact that ribosomal protein genes evolve slowly and transcript levels are very tightly regulated. In our present study, we identified and characterized all RP duplicates in eight mammalian genomes in order to investigate the tempo and mode of ribosomal protein family evolution. We show that a sizable number of duplicates are transcriptionally active and are very highly conserved. Furthermore, we conclude that existing gene duplication models do not readily account for the preservation of a very large number of intact retroduplicated ribosomal protein (RT-RP) genes observed in mammalian genomes. We suggest that selection against dominant-negative mutations may underlie the unexpected retention and conservation of duplicated RP genes, and may shape the fate of newly duplicated genes, regardless of duplication mechanism. PMID:25369106

  20. Cloning, sequencing, gene organization, and localization of the human ribosomal protein RPL23A gene

    SciTech Connect

    Fan, Wufang; Christensen, M.; Eichler, E.

    1997-12-01

    The intron-containing gene for human ribosomal protein RPL23A has been cloned, sequenced, and localized. The gene is approximately 4.0 kb in length and contains five exons and four introns. All splice sites exactly match the AG/GT consensus rule. The transcript is about 0.6 kb and is detected in all tissues examined. In adult tissues, the RPL23A transcript is dramatically more abundant in pancreas, skeletal muscle, and heart, while much less abundant in kidney, brain, placenta, lung, and liver. A full-length cDNA clone of 576 nt was identified, and the nucleotide sequence was found to match the exon sequence precisely. The open reading frame encodes a polypeptide of 156 amino acids, which is absolutely conserved with the rat RPL23A protein. In the 5{prime} flanking region of the gene, a canonical TATA sequence and a defined CAAT box were found for the first time in a mammalian ribosomal protein gene. The intron-containing RPL23A gene was mapped to cytogenetic band 17q11 by fluorescence in situ hybridization. 33 refs., 4 figs.

  1. Nucleotide sequence of the tcml gene (ribosomal protein L3) of Saccharomyces cerevisiae.

    PubMed Central

    Schultz, L D; Friesen, J D

    1983-01-01

    The yeast tcml gene, which codes for ribosomal protein L3, has been isolated by using recombinant DNA and genetic complementation. The DNA fragment carrying this gene has been subcloned and we have determined its DNA sequence. The 20 amino acid residues at the amino terminus as inferred from the nucleotide sequence agreed exactly with the amino acid sequence data. The amino acid composition of the encoded protein agreed with that determined for purified ribosomal protein L3. Codon usage in the tcml gene was strongly biased in the direction found for several other abundant Saccharomyces cerevisiae proteins. The tcml gene has no introns, which appears to be atypical of ribosomal protein structural genes. PMID:6305925

  2. Ribosomal protein gene expression is cell type specific during development in Dictyostelium discoideum.

    PubMed

    Agarwal, A K; Parrish, S N; Blumberg, D D

    1999-10-01

    Starvation for amino acids initiates the developmental cycle in the cellular slime mold, Dictyostelium discoideum. Upon starvation one of the earliest developmental events is the selective loss of the ribosomal protein mRNAs from polysomes. This loss depends upon sequences in the 5' non-translated leader of the ribosomal protein (r-protein) mRNAs. Here evidence is presented which indicates that those cells which will become prestalk cells express the ribosomal protein genes during development under starvation conditions. Cells which enter the prespore pathway shut off r-protein synthesis. The promoter and 5' non-translated leader sequences from two ribosomal protein genes, the rp-L11 and the rp-S9 genes, are fused to the Escherichia coli beta-galactosidase reporter gene. While beta-galactosidase enzyme activity is detected in situ in most growing cells, by 15 h of development beta-galactosidase enzyme activity is largely lost from the prespore cells although strong beta-galactosidase enzyme activity is present in the prestalk cells. These observations suggest the possibility that the ribosomal protein mRNAs are excluded from polysomes in a cell-type-specific manner. PMID:10550541

  3. Gene clusters for ribosomal proteins in the mitochondrial genome of a liverwort, Marchantia polymorpha.

    PubMed Central

    Takemura, M; Oda, K; Yamato, K; Ohta, E; Nakamura, Y; Nozato, N; Akashi, K; Ohyama, K

    1992-01-01

    We detected 16 genes for ribosomal proteins in the complete sequence of the mitochondrial DNA from a liverwort, Marchantia polymorpha. The genes formed two major clusters, rps12-rps7 and rps10-rpl2-rps19-rps3-rpl16-rpl5- rps14-rps8- rpl6-rps13-rps11-rps1, very similar in organization to Escherichia coli ribosomal protein operons (str and S10-spc-alpha operons, respectively). In contrast, rps2 and rps4 genes were located separately in the liverwort mitochondrial genome (the latter was part of the alpha operon in E. coli). Furthermore, several ribosomal proteins encoded by the liverwort mitochondrial genome differed substantially in size from their counterparts in E. coli and liverwort chloroplast. PMID:1620617

  4. Molecular characterization of a human gene for S28 ribosomal binding protein

    SciTech Connect

    Wong, P.; Borst, D.E.; Chader, G.J.

    1994-09-01

    The mechanism of ribosome action and the ribosomal binding proteins which cooperatively interact in the working of this structure are not completely understood. Theoretically, mutations in genes that encode these proteins may compromise the efficiency of protein synthesis and therefore lead to a functional disorder. In the course of our search for human genes which show homology to the C. elegans CED-4 death gene, we have serendipitously identified one of the human S28 ribosomal binding protein genes as a random fragment fused to the end of one of our putative CED-4 positive homologue clones. The cloned S28 fragment consists of 381 nucleotides with a putative open reading frame of 113 amino acids. Sequence comparisons to GenBank revealed significant homologies to ribosomal binding protein genes in other species (including the rat S28 ribosomal binding protein gene) indicating that the S28 gene sequence is highly conserved. This finding is confirmed by zooblot analysis. Significant homologies also exist to two human expressed tagged sites (HUMRIBPROB; L05091 and HSAFIF072; Z21908). Analysis of the putative S28 peptide sequence allows insights into possible functional regions of the protein. The identification of 8 distinct bands upon Southern analysis of the S28 fragments suggests that there are multiple copies of the S28 gene in the human genome. Mapping of the S28 fragment on somatic cell hybrid panels identified distinct S28 gene loci on chromosomes 1, 2, 7, 10, 11, 12, 17 expression in adult tissues (pancreas, kidney, muscle, liver, lung, placenta, brain, heart, and retina) as well as in fetal tissues (kidney, liver, lung, brain, and heart).

  5. GATA1 and PU.1 Bind to Ribosomal Protein Genes in Erythroid Cells: Implications for Ribosomopathies

    PubMed Central

    Amanatiadou, Elsa P.; Papadopoulos, Giorgio L.; Strouboulis, John; Vizirianakis, Ioannis S.

    2015-01-01

    The clear connection between ribosome biogenesis dysfunction and specific hematopoiesis-related disorders prompted us to examine the role of critical lineage-specific transcription factors in the transcriptional regulation of ribosomal protein (RP) genes during terminal erythroid differentiation. By applying EMSA and ChIP methodologies in mouse erythroleukemia cells we show that GATA1 and PU.1 bind in vitro and in vivo the proximal promoter region of the RPS19 gene which is frequently mutated in Diamond-Blackfan Anemia. Moreover, ChIPseq data analysis also demonstrates that several RP genes are enriched as potential GATA1 and PU.1 gene targets in mouse and human erythroid cells, with GATA1 binding showing an association with higher ribosomal protein gene expression levels during terminal erythroid differentiation in human and mouse. Our results suggest that RP gene expression and hence balanced ribosome biosynthesis may be specifically and selectively regulated by lineage specific transcription factors during hematopoiesis, a finding which may be clinically relevant to ribosomopathies. PMID:26447946

  6. GATA1 and PU.1 Bind to Ribosomal Protein Genes in Erythroid Cells: Implications for Ribosomopathies.

    PubMed

    Amanatiadou, Elsa P; Papadopoulos, Giorgio L; Strouboulis, John; Vizirianakis, Ioannis S

    2015-01-01

    The clear connection between ribosome biogenesis dysfunction and specific hematopoiesis-related disorders prompted us to examine the role of critical lineage-specific transcription factors in the transcriptional regulation of ribosomal protein (RP) genes during terminal erythroid differentiation. By applying EMSA and ChIP methodologies in mouse erythroleukemia cells we show that GATA1 and PU.1 bind in vitro and in vivo the proximal promoter region of the RPS19 gene which is frequently mutated in Diamond-Blackfan Anemia. Moreover, ChIPseq data analysis also demonstrates that several RP genes are enriched as potential GATA1 and PU.1 gene targets in mouse and human erythroid cells, with GATA1 binding showing an association with higher ribosomal protein gene expression levels during terminal erythroid differentiation in human and mouse. Our results suggest that RP gene expression and hence balanced ribosome biosynthesis may be specifically and selectively regulated by lineage specific transcription factors during hematopoiesis, a finding which may be clinically relevant to ribosomopathies. PMID:26447946

  7. Complete sequence and gene organization of the Nosema heliothidis ribosomal RNA gene region.

    PubMed

    Dong, Shinan; Shen, Zhongyuan; Zhu, Feng; Tang, Xudong; Xu, Li

    2011-01-01

    By sequencing the entire ribosomal RNA (rRNA) gene region of Nosema heliothidis isolated from cotton bollworm (Helicoverpa armigera), we showed that its gene organization is similar to the type species, Nosema bombycis: the 5'-large subunit rRNA (2,490 bp)-internal transcribed spacer (192 bp)-small subunit rRNA (1,232 bp)-intergenic spacer (274 bp)-5S rRNA (115 bp)-3'. We constructed two phylogenetic trees, analyzed phylogenetic relationships, examined rRNA organization of microsporidia, and compared the secondary structure of small subunit rRNA with closely related microsporidia. The latter two features may provide important information for the classification and phylogenetic analysis of microsporidia. PMID:21895841

  8. Research Techniques Made Simple: Bacterial 16S Ribosomal RNA Gene Sequencing in Cutaneous Research.

    PubMed

    Jo, Jay-Hyun; Kennedy, Elizabeth A; Kong, Heidi H

    2016-03-01

    Skin serves as a protective barrier and also harbors numerous microorganisms collectively comprising the skin microbiome. As a result of recent advances in sequencing (next-generation sequencing), our understanding of microbial communities on skin has advanced substantially. In particular, the 16S ribosomal RNA gene sequencing technique has played an important role in efforts to identify the global communities of bacteria in healthy individuals and patients with various disorders in multiple topographical regions over the skin surface. Here, we describe basic principles, study design, and a workflow of 16S ribosomal RNA gene sequencing methodology, primarily for investigators who are not familiar with this approach. This article will also discuss some applications and challenges of 16S ribosomal RNA sequencing as well as directions for future development. PMID:26902128

  9. High-resolution microscopy of active ribosomal genes and key members of the rRNA processing machinery inside nucleolus-like bodies of fully-grown mouse oocytes.

    PubMed

    Shishova, Kseniya V; Khodarovich, Yuriy M; Lavrentyeva, Elena A; Zatsepina, Olga V

    2015-10-01

    Nucleolus-like bodies (NLBs) of fully-grown (germinal vesicle, GV) mammalian oocytes are traditionally considered as morphologically distinct entities, which, unlike normal nucleoli, contain transcribed ribosomal genes (rDNA) solely at their surface. In the current study, we for the first time showed that active ribosomal genes are present not only on the surface but also inside NLBs of the NSN-type oocytes. The "internal" rRNA synthesis was evidenced by cytoplasmic microinjections of BrUTP as precursor and by fluorescence in situ hybridization with a probe to the short-lived 5'ETS segment of the 47S pre-rRNA. We further showed that in the NLB mass of NSN-oocytes, distribution of active rDNA, RNA polymerase I (UBF) and rRNA processing (fibrillarin) protein factors, U3 snoRNA, pre-rRNAs and 18S/28S rRNAs is remarkably similar to that in somatic nucleoli capable to make pre-ribosomes. Overall, these observations support the occurrence of rDNA transcription, rRNA processing and pre-ribosome assembly in the NSN-type NLBs and so that their functional similarity to normal nucleoli. Unlike the NSN-type NLBs, the NLBs of more mature SN-oocytes do not contain transcribed rRNA genes, U3 snoRNA, pre-rRNAs, 18S and 28S rRNAs. These results favor the idea that in a process of transformation of NSN-oocytes to SN-oocytes, NLBs cease to produce pre-ribosomes and, moreover, lose their rRNAs. We also concluded that a denaturing fixative 70% ethanol used in the study to fix oocytes could be more appropriate for light microscopy analysis of nucleolar RNAs and proteins in mammalian fully-grown oocytes than a commonly used cross-linking aldehyde fixative, formalin. PMID:26226217

  10. Ribosome profiling reveals post-transcriptional buffering of divergent gene expression in yeast

    PubMed Central

    McManus, C. Joel; May, Gemma E.; Spealman, Pieter; Shteyman, Alan

    2014-01-01

    Understanding the patterns and causes of phenotypic divergence is a central goal in evolutionary biology. Much work has shown that mRNA abundance is highly variable between closely related species. However, the extent and mechanisms of post-transcriptional gene regulatory evolution are largely unknown. Here we used ribosome profiling to compare transcript abundance and translation efficiency in two closely related yeast species (S. cerevisiae and S. paradoxus). By comparing translation regulatory divergence to interspecies differences in mRNA sequence features, we show that differences in transcript leaders and codon bias substantially contribute to divergent translation. Globally, we find that translation regulatory divergence often buffers species differences in mRNA abundance, such that ribosome occupancy is more conserved than transcript abundance. We used allele-specific ribosome profiling in interspecies hybrids to compare the relative contributions of cis- and trans-regulatory divergence to species differences in mRNA abundance and translation efficiency. The mode of gene regulatory divergence differs for these processes, as trans-regulatory changes play a greater role in divergent mRNA abundance than in divergent translation efficiency. Strikingly, most genes with aberrant transcript abundance in F1 hybrids (either over- or underexpressed compared to both parent species) did not exhibit aberrant ribosome occupancy. Our results show that interspecies differences in translation contribute substantially to the evolution of gene expression. Compensatory differences in transcript abundance and translation efficiency may increase the robustness of gene regulation. PMID:24318730

  11. Ribosomal protein methylation in Escherichia coli: the gene prmA, encoding the ribosomal protein L11 methyltransferase, is dispensable.

    PubMed

    Vanet, A; Plumbridge, J A; Guérin, M F; Alix, J H

    1994-12-01

    The prmA gene, located at 72 min on the Escherichia coli chromosome, is the genetic determinant of ribosomal protein L11-methyltransferase activity. Mutations at this locus, prmA1 and prmA3, result in a severely undermethylated form of L11. No effect, other than the lack of methyl groups on L11, has been ascribed to these mutations. DNA sequence analysis of the mutant alleles prmA1 and prmA3 detected point mutations near the C-terminus of the protein and plasmids overproducing the wild-type and the two mutant proteins have been constructed. The wild-type PrmA protein could be crosslinked to its radiolabelled substrate, S-adenosyl-L-methionine (SAM), by u.v. irradiation indicating that it is the gene for the methyltransferase rather than a regulatory protein. One of the mutant proteins, PrmA3, was also weakly crosslinked to SAM. Both mutant enzymes when expressed from the overproducing plasmids were capable of catalysing the incorporation of 3H-labelled methyl groups from SAM to L11 in vitro. This confirmed the observation that the mutant proteins possess significant residual activity which could account for their lack of growth phenotype. However, a strain carrying an in vitro-constructed null mutation of the prmA gene, transferred to the E. coli chromosome by homologous recombination, was perfectly viable. PMID:7715456

  12. Transcription termination and RNA processing in the 3'-end spacer of mouse ribosomal RNA genes.

    PubMed Central

    Miwa, T; Kominami, R; Yoshikura, H; Sudo, K; Muramatsu, M

    1987-01-01

    The 3' termini of ribosomal RNA precursors from mouse FM3A cultured cells are mapped to eight sites within 625 bp downstream from the 3' terminus of 28 S rRNA. Three additional sites are mapped in liver RNA from C3H/He strain mice. Two of them, the sites at 570 bp and 625 bp are assumed to be termination sites in vivo, because they correspond to in vitro termination sites of RNA polymerase I, and 45 S RNAs having these 3' termini decay with kinetics distinct from others. The amount of 45 S RNA having the 3' terminus at other sites is variable among several mouse strains, despite their having the same DNA sequence in these regions. The ability to produce 3' termini in these sites seems to follow Mendel's law of inheritance. Therefore, we postulate that these nine sites are RNA processing sites which are controlled genetically. Images PMID:3031586

  13. Remodeling of ribosomal genes in somatic cells by Xenopus egg extract

    SciTech Connect

    Ostrup, Olga; Hyttel, Poul; Klaerke, Dan A.; Collas, Philippe

    2011-09-02

    Highlights: {yields} Xenopus egg extract remodels nuclei and alter cell growth characteristics. {yields} Ribosomal genes are reprogrammed within 6 h after extract exposure. {yields} rDNA reprogramming involves promoter targeting of SNF2H remodeling complex. {yields} Xenopus egg extract does not initiate stress-related response in somatic cells. {yields} Aza-cytidine elicits a stress-induced response in reprogrammed cells. -- Abstract: Extracts from Xenopus eggs can reprogram gene expression in somatic nuclei, however little is known about the earliest processes associated with the switch in the transcriptional program. We show here that an early reprogramming event is the remodeling of ribosomal chromatin and gene expression. This occurs within hours of extract treatment and is distinct from a stress response. Egg extract elicits remodeling of the nuclear envelope, chromatin and nucleolus. Nucleolar remodeling involves a rapid and stable decrease in ribosomal gene transcription, and promoter targeting of the nucleolar remodeling complex component SNF2H without affecting occupancy of the transcription factor UBF and the stress silencers SUV39H1 and SIRT1. During this process, nucleolar localization of UBF and SIRT1 is not altered. On contrary, azacytidine pre-treatment has an adverse effect on rDNA remodeling induced by extract and elicits a stress-type nuclear response. Thus, an early event of Xenopus egg extract-mediated nuclear reprogramming is the remodeling of ribosomal genes involving nucleolar remodeling complex. Condition-specific and rapid silencing of ribosomal genes may serve as a sensitive marker for evaluation of various reprogramming methods.

  14. The spc ribosomal protein operon of Escherichia coli: sequence and cotranscription of the ribosomal protein genes and a protein export gene.

    PubMed

    Cerretti, D P; Dean, D; Davis, G R; Bedwell, D M; Nomura, M

    1983-05-11

    The genes encoding the 52 ribosomal proteins (r-proteins) of Escherichia coli are organized into approximately 19 operons scattered throughout the chromosome. One of these, the spc operon, contains the genes for ten ribosomal proteins: L14, L24, L5, S14, S8, L6, L18, S5, L30 and L15 (rp1N, rp1X, rp1E, rpsN, rpsH, rp1F, rp1R, rpsE, rpmD, and rp1O). We now report the entire 5.9 kb nucleotide sequence of the spc operon. DNA sequence analysis has confirmed the genetic organization and refined the amino acid sequence of the ten r-proteins in this operon. It has also revealed the presence of two open reading frames past the last known gene (L15) of the spc operon. One of these corresponds to a gene (pr1A or secY) which recently has been shown by others to be involved in protein export. In addition, S1 mapping experiments indicate that a significant proportion of transcription initiated from the spc operon continues not only into the two putative genes, but also without termination into the downstream alpha r-protein operon. PMID:6222285

  15. Molecular characterization of Stenocarpella maydis based on nuclear ribosomal Internal Transcribed Spacer regions between the 18S and 28S nuclear rRNA gene sequences

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Diplodia ear rot of maize is caused by the fungus Stenocarpella maydis (syn. Diplodia maydis). Although considered a minor pathogen in the later 1900's, with the increased emphasis on conservation tillage, S. maydis has reestablished itself as an important ear and stalk rot pathogen. While S. maydis...

  16. Molecular Genetics of Cryptopleurine Resistance in Saccharomyces Cerevisiae: Expression of a Ribosomal Protein Gene Family

    PubMed Central

    Paulovich, A. G.; Thompson, J. R.; Larkin, J. C.; Li, Z.; Woolford-Jr., J. L.

    1993-01-01

    The Saccharomyces cerevisiae CRY1 gene encodes the 40S ribosomal subunit protein rp59 and confers sensitivity to the protein synthesis inhibitor cryptopleurine. A yeast strain containing the cry1-δ1::URA3 null allele is viable, cryptopleurine sensitive (Cry(S)), and expresses rp59 mRNA, suggesting that there is a second functional CRY gene. The CRY2 gene has been isolated from a yeast genomic library cloned in bacteriophage λ, using a CRY1 DNA probe. The DNA sequence of the CRY2 gene contains an open reading frame encoding ribosomal protein 59 that differs at five residues from rp59 encoded by the CRY1 gene. The CRY2 gene was mapped to the left arm of chromosome X, centromere-proximal to cdc6 and immediately adjacent to ribosomal protein genes RPS24A and RPL46. Ribosomal protein 59 is an essential protein; upon sporulation of a diploid doubly heterozygous for cry1-δ2::TRP1 cry2-δ1::LEU2 null alleles, no spore clones containing both null alleles were recovered. Several results indicate that CRY2 is expressed, but at lower levels than CRY1: (1) Introduction of CRY2 on high copy plasmids into Cry(R) yeast of genotype cry1 CRY2 confers a Cry(S) phenotype. Transformation of these Cry(R) yeast with CRY2 on a low copy CEN plasmid does not confer a Cry(S) phenotype. (2) Haploids containing the cry1-δ2::TRP1 null allele have a deficit of 40S ribosomal subunits, but cry2-δ1::LEU2 strains have wild-type amounts of 40S ribosomal subunits. (3) CRY2 mRNA is present at lower levels than CRY1 mRNA. (4) Higher levels of β-galactosidase are expressed from a CRY1-lacZ gene fusion than from a CRY2-lacZ gene fusion. Mutations that alter or eliminate the last amino acid of rp59 encoded by either CRY1 or CRY2 result in resistance to cryptopleurine. Because CRY2 (and cry2) is expressed at lower levels than CRY1 (and cry1), the Cry(R) phenotype of cry2 mutants is only expressed in strains containing a cry1-δ null allele. PMID:8293976

  17. Over-represented localized sequence motifs in ribosomal protein gene promoters of basal metazoans.

    PubMed

    Perina, Drago; Korolija, Marina; Roller, Maša; Harcet, Matija; Jeličić, Branka; Mikoč, Andreja; Cetković, Helena

    2011-07-01

    Equimolecular presence of ribosomal proteins (RPs) in the cell is needed for ribosome assembly and is achieved by synchronized expression of ribosomal protein genes (RPGs) with promoters of similar strengths. Over-represented motifs of RPG promoter regions are identified as targets for specific transcription factors. Unlike RPs, those motifs are not conserved between mammals, drosophila, and yeast. We analyzed RPGs proximal promoter regions of three basal metazoans with sequenced genomes: sponge, cnidarian, and placozoan and found common features, such as 5'-terminal oligopyrimidine tracts and TATA-boxes. Furthermore, we identified over-represented motifs, some of which displayed the highest similarity to motifs abundant in human RPG promoters and not present in Drosophila or yeast. Our results indicate that humans over-represented motifs, as well as corresponding domains of transcription factors, were established very early in metazoan evolution. The fast evolving nature of RPGs regulatory network leads to formation of other, lineage specific, over-represented motifs. PMID:21457775

  18. Mutational robustness of 16S ribosomal RNA, shown by experimental horizontal gene transfer in Escherichia coli

    PubMed Central

    Kitahara, Kei; Yasutake, Yoshiaki; Miyazaki, Kentaro

    2012-01-01

    The bacterial ribosome consists of three rRNA molecules and 57 proteins and plays a crucial role in translating mRNA-encoded information into proteins. Because of the ribosome’s structural and mechanistic complexity, it is believed that each ribosomal component coevolves to maintain its function. Unlike 5S rRNA, 16S and 23S rRNAs appear to lack mutational robustness, because they form the structural core of the ribosome. However, using Escherichia coli Δ7 (null mutant of operons) as a host, we have recently shown that an active hybrid ribosome whose 16S rRNA has been specifically substituted with that from non–E. coli bacteria can be reconstituted in vivo. To investigate the mutational robustness of 16S rRNA and the structural basis for its functionality, we used a metagenomic approach to screen for 16S rRNA genes that complement the growth of E. coli Δ7. Various functional genes were obtained from the Gammaproteobacteria and Betaproteobacteria lineages. Despite the large sequence diversity (80.9–99.0% identity with E. coli 16S rRNA) of the functional 16S rRNA molecules, the doubling times (DTs) of each mutant increased only modestly with decreasing sequence identity (average increase in DT, 4.6 s per mutation). The three-dimensional structure of the 30S ribosome showed that at least 40.7% (628/1,542) of the nucleotides were variable, even at ribosomal protein-binding sites, provided that the secondary structures were properly conserved. Our results clearly demonstrate that 16S rRNA functionality largely depends on the secondary structure but not on the sequence itself. PMID:23112186

  19. In vitro expression of Escherichia coli ribosomal protein genes: autogenous inhibition of translation.

    PubMed Central

    Yates, J L; Arfsten, A E; Nomura, M

    1980-01-01

    Escherichia coli ribosomal protein L1 (0.5 micro M) was found to inhibit the synthesis of both proteins of the L11 operon, L11 and L1, but not the synthesis of other proteins directed by lambda rifd 18 DNA. Similarly, S4 (1 micro M) selectively inhibited the synthesis of three proteins of the alpha operon, S13, S11, and S4, directed by lambda spcI DNA or a restriction enzyme fragment obtained from this DNA. S8 (3.6 micro M) also showed preferential inhibitory effects on the synthesis of some proteins encoded in the spc operon, L24 and L5 (and probably S14 and S8), directed by lambda spcl DNA or a restriction enzyme fragment carrying the genes for these proteins. The inhibitory effect of L1 was observed only with L1 and not with other proteins examined, including S4 and S8. Similarly, the effect of S4 was not observed with L1 or S8, and that of S8 was not seen with L1 or S4. Inhibition was shown to take place at the level of translation rather than transcription. Thus, at least some ribosomal proteins (L1 S4, and S8) have the ability to cause selective translational inhibition of the synthesis of certain ribosomal proteins whose genes are in the same operon as their own. These results support the hypothesis that certain free ribosomal proteins not assembled into ribosomes act as "autogenous" feedback inhibitors to regulate the synthesis of ribosomal proteins. Images PMID:6445562

  20. Preservation of Gene Duplication Increases the Regulatory Spectrum of Ribosomal Protein Genes and Enhances Growth under Stress.

    PubMed

    Parenteau, Julie; Lavoie, Mathieu; Catala, Mathieu; Malik-Ghulam, Mustafa; Gagnon, Jules; Abou Elela, Sherif

    2015-12-22

    In baker's yeast, the majority of ribosomal protein genes (RPGs) are duplicated, and it was recently proposed that such duplications are preserved via the functional specialization of the duplicated genes. However, the origin and nature of duplicated RPGs' (dRPGs) functional specificity remain unclear. In this study, we show that differences in dRPG functions are generated by variations in the modality of gene expression and, to a lesser extent, by protein sequence. Analysis of the sequence and expression patterns of non-intron-containing RPGs indicates that each dRPG is controlled by specific regulatory sequences modulating its expression levels in response to changing growth conditions. Homogenization of dRPG sequences reduces cell tolerance to growth under stress without changing the number of expressed genes. Together, the data reveal a model where duplicated genes provide a means for modulating the expression of ribosomal proteins in response to stress. PMID:26686636

  1. High-resolution view of bacteriophage lambda gene expression by ribosome profiling

    PubMed Central

    Liu, Xiaoqiu; Jiang, Huifeng; Gu, Zhenglong; Roberts, Jeffrey W.

    2013-01-01

    Bacteriophage lambda is one of the most extensively studied organisms and has been a primary model for understanding basic modes of genetic regulation. Here, we examine the progress of lambda gene expression during phage development by ribosome profiling and, thereby, provide a very-high-resolution view of lambda gene expression. The known genes are expressed in a predictable fashion, authenticating the analysis. However, many previously unappreciated potential open reading frames become apparent in the expression analysis, revealing an unexpected complexity in the pattern of lambda gene function. PMID:23812753

  2. Ribosomal protein genes are highly enriched among genes with allele-specific expression in the interspecific F1 hybrid catfish.

    PubMed

    Chen, Ailu; Wang, Ruijia; Liu, Shikai; Peatman, Eric; Sun, Luyang; Bao, Lisui; Jiang, Chen; Li, Chao; Li, Yun; Zeng, Qifan; Liu, Zhanjiang

    2016-06-01

    Interspecific hybrids provide a rich source for the analysis of allele-specific expression (ASE). In this work, we analyzed ASE in F1 hybrid catfish using RNA-Seq datasets. While the vast majority of genes were expressed with both alleles, 7-8 % SNPs exhibited significant differences in allele ratios of expression. Of the 66,251 and 177,841 SNPs identified from the datasets of the liver and gill, 5420 (8.2 %) and 13,390 (7.5 %) SNPs were identified as significant ASE-SNPs, respectively. With these SNPs, a total of 1519 and 3075 ASE-genes were identified. Gene Ontology analysis revealed that genes encoding cytoplasmic ribosomal proteins (RP) were highly enriched among ASE genes. Parent-of-origin was determined for 27 and 30 ASE RP genes in the liver and gill, respectively. The results indicated that genes from both channel catfish and blue catfish were involved in ASE. However, each RP gene appeared to be almost exclusively expressed from only one parent, indicating that ribosomes in the hybrid catfish were in the "hybrid" form. Overall representation of RP transcripts among the transcriptome appeared lower in the F1 hybrid catfish than in channel catfish or blue catfish, suggesting that the "hybrid" ribosomes may work more efficiently for translation in the F1 hybrid catfish. PMID:26747053

  3. Structural alterations of the ribosomal RNA genes in leukemic cells.

    PubMed

    Smirnova, I A

    1992-01-01

    Cloned 6.7 kb EcoR1 fragment of mice rDNA was used as a hybridization probe for rDNA structure analysis in mice, rat and calf haemopoietic tumor and normal cells. EcoR1, BglII and Pst1 restriction fragment length polymorphism (RFLP) was found in neoplastic rDNA and was not revealed in normal ones. The rRNA gene rearrangements were observed not only in spacer region but in coding sequences of the genes. Leukemic cells reveal also rDNA amplification. A role of genetic rearrangements of rDNA for mechanisms of carcinogenesis is suggested. PMID:1342066

  4. The Arabidopsis HUELLENLOS Gene, Which Is Essential for Normal Ovule Development, Encodes a Mitochondrial Ribosomal Protein

    PubMed Central

    Skinner, Debra J.; Baker, Shawn C.; Meister, Robert J.; Broadhvest, Jean; Schneitz, Kay; Gasser, Charles S.

    2001-01-01

    The HUELLENLOS (HLL) gene participates in patterning and growth of the Arabidopsis ovule. We have isolated the HLL gene and shown that it encodes a protein homologous to the L14 proteins of eubacterial ribosomes. The Arabidopsis genome also includes a highly similar gene, HUELLENLOS PARALOG (HLP), and genes for both cytosolic (L23) and chloroplast ribosome L14 proteins. Phylogenetic analysis shows that HLL and HLP differ significantly from these other two classes of such proteins. HLL and HLP fusions to green fluorescent protein were localized to mitochondria. Ectopic expression of HLP complemented the hll mutant, indicating that HLP and HLL share redundant functions. We conclude that HLL and HLP encode L14 subunits of mitochondrial ribosomes. HLL mRNA was at significantly higher levels than HLP mRNA in pistils, with the opposite pattern in leaves. This differential expression can explain the confinement of effects of hll mutations to gynoecia and ovules. Our elucidation of the nature of HLL shows that metabolic defects can have specific effects on developmental patterning. PMID:11752383

  5. Microbial Community Analysis with Ribosomal Gene Fragments from Shotgun Metagenomes

    PubMed Central

    Guo, Jiarong; Cole, James R.; Zhang, Qingpeng; Brown, C. Titus

    2015-01-01

    Shotgun metagenomic sequencing does not depend on gene-targeted primers or PCR amplification; thus, it is not affected by primer bias or chimeras. However, searching rRNA genes from large shotgun Illumina data sets is computationally expensive, and no approach exists for unsupervised community analysis of small-subunit (SSU) rRNA gene fragments retrieved from shotgun data. We present a pipeline, SSUsearch, to achieve the faster identification of short-subunit rRNA gene fragments and enabled unsupervised community analysis with shotgun data. It also includes classification and copy number correction, and the output can be used by traditional amplicon analysis platforms. Shotgun metagenome data using this pipeline yielded higher diversity estimates than amplicon data but retained the grouping of samples in ordination analyses. We applied this pipeline to soil samples with paired shotgun and amplicon data and confirmed bias against Verrucomicrobia in a commonly used V6-V8 primer set, as well as discovering likely bias against Actinobacteria and for Verrucomicrobia in a commonly used V4 primer set. This pipeline can utilize all variable regions in SSU rRNA and also can be applied to large-subunit (LSU) rRNA genes for confirmation of community structure. The pipeline can scale to handle large amounts of soil metagenomic data (5 Gb memory and 5 central processing unit hours to process 38 Gb [1 lane] of trimmed Illumina HiSeq2500 data) and is freely available at https://github.com/dib-lab/SSUsearch under a BSD license. PMID:26475107

  6. Rice Ribosomal Protein Large Subunit Genes and Their Spatio-temporal and Stress Regulation

    PubMed Central

    Moin, Mazahar; Bakshi, Achala; Saha, Anusree; Dutta, Mouboni; Madhav, Sheshu M.; Kirti, P. B.

    2016-01-01

    Ribosomal proteins (RPs) are well-known for their role in mediating protein synthesis and maintaining the stability of the ribosomal complex, which includes small and large subunits. In the present investigation, in a genome-wide survey, we predicted that the large subunit of rice ribosomes is encoded by at least 123 genes including individual gene copies, distributed throughout the 12 chromosomes. We selected 34 candidate genes, each having 2–3 identical copies, for a detailed characterization of their gene structures, protein properties, cis-regulatory elements and comprehensive expression analysis. RPL proteins appear to be involved in interactions with other RP and non-RP proteins and their encoded RNAs have a higher content of alpha-helices in their predicted secondary structures. The majority of RPs have binding sites for metal and non-metal ligands. Native expression profiling of 34 ribosomal protein large (RPL) subunit genes in tissues covering the major stages of rice growth shows that they are predominantly expressed in vegetative tissues and seedlings followed by meiotically active tissues like flowers. The putative promoter regions of these genes also carry cis-elements that respond specifically to stress and signaling molecules. All the 34 genes responded differentially to the abiotic stress treatments. Phytohormone and cold treatments induced significant up-regulation of several RPL genes, while heat and H2O2 treatments down-regulated a majority of them. Furthermore, infection with a bacterial pathogen, Xanthomonas oryzae, which causes leaf blight also induced the expression of 80% of the RPL genes in leaves. Although the expression of RPL genes was detected in all the tissues studied, they are highly responsive to stress and signaling molecules indicating that their encoded proteins appear to have roles in stress amelioration besides house-keeping. This shows that the RPL gene family is a valuable resource for manipulation of stress tolerance in

  7. Rice Ribosomal Protein Large Subunit Genes and Their Spatio-temporal and Stress Regulation.

    PubMed

    Moin, Mazahar; Bakshi, Achala; Saha, Anusree; Dutta, Mouboni; Madhav, Sheshu M; Kirti, P B

    2016-01-01

    Ribosomal proteins (RPs) are well-known for their role in mediating protein synthesis and maintaining the stability of the ribosomal complex, which includes small and large subunits. In the present investigation, in a genome-wide survey, we predicted that the large subunit of rice ribosomes is encoded by at least 123 genes including individual gene copies, distributed throughout the 12 chromosomes. We selected 34 candidate genes, each having 2-3 identical copies, for a detailed characterization of their gene structures, protein properties, cis-regulatory elements and comprehensive expression analysis. RPL proteins appear to be involved in interactions with other RP and non-RP proteins and their encoded RNAs have a higher content of alpha-helices in their predicted secondary structures. The majority of RPs have binding sites for metal and non-metal ligands. Native expression profiling of 34 ribosomal protein large (RPL) subunit genes in tissues covering the major stages of rice growth shows that they are predominantly expressed in vegetative tissues and seedlings followed by meiotically active tissues like flowers. The putative promoter regions of these genes also carry cis-elements that respond specifically to stress and signaling molecules. All the 34 genes responded differentially to the abiotic stress treatments. Phytohormone and cold treatments induced significant up-regulation of several RPL genes, while heat and H2O2 treatments down-regulated a majority of them. Furthermore, infection with a bacterial pathogen, Xanthomonas oryzae, which causes leaf blight also induced the expression of 80% of the RPL genes in leaves. Although the expression of RPL genes was detected in all the tissues studied, they are highly responsive to stress and signaling molecules indicating that their encoded proteins appear to have roles in stress amelioration besides house-keeping. This shows that the RPL gene family is a valuable resource for manipulation of stress tolerance in rice

  8. Revising the Taxonomic Distribution, Origin and Evolution of Ribosome Inactivating Protein Genes

    PubMed Central

    Lapadula, Walter J.; Sánchez Puerta, María Virginia; Juri Ayub, Maximiliano

    2013-01-01

    Ribosome inactivating proteins are enzymes that depurinate a specific adenine residue in the alpha-sarcin-ricin loop of the large ribosomal RNA, being ricin and Shiga toxins the most renowned examples. They are widely distributed in plants and their presence has also been confirmed in a few bacterial species. According to this taxonomic distribution, the current model about the origin and evolution of RIP genes postulates that an ancestral RIP domain was originated in flowering plants, and later acquired by some bacteria via horizontal gene transfer. Here, we unequivocally detected the presence of RIP genes in fungi and metazoa. These findings, along with sequence and phylogenetic analyses, led us to propose an alternative, more parsimonious, hypothesis about the origin and evolutionary history of the RIP domain, where several paralogous RIP genes were already present before the three domains of life evolved. This model is in agreement with the current idea of the Last Universal Common Ancestor (LUCA) as a complex, genetically redundant organism. Differential loss of paralogous genes in descendants of LUCA, rather than multiple horizontal gene transfer events, could account for the complex pattern of RIP genes across extant species, as it has been observed for other genes. PMID:24039805

  9. A 5.8S nuclear ribosomal RNA gene sequence database: applications to ecology and evolution

    NASA Technical Reports Server (NTRS)

    Cullings, K. W.; Vogler, D. R.

    1998-01-01

    We complied a 5.8S nuclear ribosomal gene sequence database for animals, plants, and fungi using both newly generated and GenBank sequences. We demonstrate the utility of this database as an internal check to determine whether the target organism and not a contaminant has been sequenced, as a diagnostic tool for ecologists and evolutionary biologists to determine the placement of asexual fungi within larger taxonomic groups, and as a tool to help identify fungi that form ectomycorrhizae.

  10. Molecular characterisation of three regions of the nuclear ribosomal DNA unit and the mitochondrial cox1 gene of Sarcocystis fusiformis from water buffaloes (Bubalus bubalis) in Egypt.

    PubMed

    Gjerde, Bjørn; Hilali, Mosaad; Mawgood, Sahar Abdel

    2015-09-01

    A total of 33 macroscopically visible (3-11 × 1-5 mm) sarcocysts of Sarcocystis fusiformis were excised from the oesophagus of 12 freshly slaughtered water buffalos in Giza, Egypt. Genomic DNA was extracted from the sarcocysts, and all isolates were characterised at the mitochondrial cytochrome c oxidase subunit I (cox1) gene through PCR amplification and direct sequencing, whereas a few selected isolates were characterised at the 18S and 28S ribosomal (r) RNA genes and the internal transcribed spacer 1 (ITS1) region of the nuclear rDNA unit following cloning. Among the 33 cox1 sequences (1,038-bp long), there was a total of 13 haplotypes, differing from each other by one to seven substitutions and sharing an identity of 99.3-99.9 %. In comparison, the sequence identity was 98.8-99.0 % among eight complete 18S rRNA gene sequences (1,873-1,879-bp long), 98.1-100 % among 28 complete ITS1 sequences (853-864-bp long) and 97.4-99.6 % among five partial 28S rRNA gene sequences (1,607-1,622 bp). At the three nuclear loci, the intraspecific (and intra-isolate) sequence variation was due to both substitutions and indels, which necessitated cloning of the PCR products before sequencing. Some additional clones of the 18S and 28S rRNA genes were highly divergent from the more typical clones, but the true nature of these aberrant clones could not be determined. Sequence comparisons and phylogenetic analyses based on either 18S rRNA gene or cox1 nucleotide sequences, placed S. fusiformis closest to Sarcocystis cafferi from the African buffalo, but only the analyses based on cox1 data separated the two taxa clearly from each other and showed that they were separate species (monophyletic clusters and 93 % sequence identity at cox1 versus interleaved sequences and 98.7-99.1 % sequence identity at the 18S rRNA gene). Two cats experimentally infected with sarcocysts of S. fusiformis started shedding small numbers of sporocysts 8-10 days post-infection (dpi) and were euthanized 15

  11. Genomic location of the major ribosomal protein gene locus determines Vibrio cholerae global growth and infectivity.

    PubMed

    Soler-Bistué, Alfonso; Mondotte, Juan A; Bland, Michael Jason; Val, Marie-Eve; Saleh, María-Carla; Mazel, Didier

    2015-04-01

    The effects on cell physiology of gene order within the bacterial chromosome are poorly understood. In silico approaches have shown that genes involved in transcription and translation processes, in particular ribosomal protein (RP) genes, localize near the replication origin (oriC) in fast-growing bacteria suggesting that such a positional bias is an evolutionarily conserved growth-optimization strategy. Such genomic localization could either provide a higher dosage of these genes during fast growth or facilitate the assembly of ribosomes and transcription foci by keeping physically close the many components of these macromolecular machines. To explore this, we used novel recombineering tools to create a set of Vibrio cholerae strains in which S10-spec-α (S10), a locus bearing half of the ribosomal protein genes, was systematically relocated to alternative genomic positions. We show that the relative distance of S10 to the origin of replication tightly correlated with a reduction of S10 dosage, mRNA abundance and growth rate within these otherwise isogenic strains. Furthermore, this was accompanied by a significant reduction in the host-invasion capacity in Drosophila melanogaster. Both phenotypes were rescued in strains bearing two S10 copies highly distal to oriC, demonstrating that replication-dependent gene dosage reduction is the main mechanism behind these alterations. Hence, S10 positioning connects genome structure to cell physiology in Vibrio cholerae. Our results show experimentally for the first time that genomic positioning of genes involved in the flux of genetic information conditions global growth control and hence bacterial physiology and potentially its evolution. PMID:25875621

  12. Internal ribosome entry site-based vectors for combined gene therapy

    PubMed Central

    Renaud-Gabardos, Edith; Hantelys, Fransky; Morfoisse, Florent; Chaufour, Xavier; Garmy-Susini, Barbara; Prats, Anne-Catherine

    2015-01-01

    Gene therapy appears as a promising strategy to treat incurable diseases. In particular, combined gene therapy has shown improved therapeutic efficiency. Internal ribosome entry sites (IRESs), RNA elements naturally present in the 5’ untranslated regions of a few mRNAs, constitute a powerful tool to co-express several genes of interest. IRESs are translational enhancers allowing the translational machinery to start protein synthesis by internal initiation. This feature allowed the design of multi-cistronic vectors expressing several genes from a single mRNA. IRESs exhibit tissue specificity, and drive translation in stress conditions when the global cell translation is blocked, which renders them useful for gene transfer in hypoxic conditions occurring in ischemic diseases and cancer. IRES-based viral and non viral vectors have been used successfully in preclinical and clinical assays of combined gene therapy and resulted in therapeutic benefits for various pathologies including cancers, cardiovascular diseases and degenerative diseases. PMID:25699230

  13. The human ubiquitin-52 amino acid fusion protein gene shares several structural features with mammalian ribosomal protein genes.

    PubMed Central

    Baker, R T; Board, P G

    1991-01-01

    Complementary DNA clones encoding ubiquitin fused to a 52 amino acid tail protein were isolated from human placental and adrenal gland cDNA libraries. The deduced human 52 amino acid tail protein is very similar to the homologous protein from other species, including the conservation of the putative metal-binding, nucleic acid-binding domain observed in these proteins. Northern blot analysis with a tail-specific probe indicated that the previously identified UbA mRNA species most likely represents comigrating transcripts of the 52 amino acid tail (UbA52) and 80 amino acid tail (UbA80) ubiquitin fusion genes. The UbA52 gene was isolated from a human genomic library and consists of five exons distributed over 3400 base pairs. One intron is in the 5' non-coding region, two interrupt the single ubiquitin coding unit, and the fourth intron is within the tail coding region. Several members of the Alu family of repetitive DNA are associated with the gene. The UbA52 promoter has several features in common with mammalian ribosomal protein genes, including its location in a CpG-rich island, initiation of transcription within a polypyrimidine tract, the lack of a consensus TATA motif, and the presence of Sp1 binding sites, observations that are consistent with the recent identification of the ubiquitin-free tail proteins as ribosomal proteins. Thus, in spite of its unusual feature of being translationally fused to ubiquitin, the 52 amino acid tail ribosomal protein is expressed from a structurally typical ribosomal protein gene. Images PMID:1850507

  14. Ribosomal DNA and Stellate gene copy number variation on the Y chromosome of Drosophila melanogaster.

    PubMed Central

    Lyckegaard, E M; Clark, A G

    1989-01-01

    Multigene families on the Y chromosome face an unusual array of evolutionary forces. Both ribosomal DNA and Stellate, the two families examined here, have multiple copies of similar sequences on the X and Y chromosomes. Although the rate of sequence divergence on the Y chromosome depends on rates of mutation, gene conversion and exchange with the X chromosome, as well as purifying selection, the regulation of gene copy number may also depend on other pleiotropic functions, such as maintenance of chromosome pairing. Gene copy numbers were estimated for a series of 34 Y chromosome replacement lines using densitometric measurements of slot blots of genomic DNA from adult Drosophila melanogaster. Scans of autoradiographs of the same blots probed with the cloned alcohol dehydrogenase gene, a single copy gene, served as internal standards. Copy numbers span a 6-fold range for ribosomal DNA and a 3-fold range for Stellate DNA. Despite this magnitude of variation, there was no association between copy number and segregation variation of the sex chromosomes. Images PMID:2494656

  15. Phylogenetic reconstruction of the wolf spiders (Araneae: Lycosidae) using sequences from the 12S rRNA, 28S rRNA, and NADH1 genes: implications for classification, biogeography, and the evolution of web building behavior.

    PubMed

    Murphy, Nicholas P; Framenau, Volker W; Donnellan, Stephen C; Harvey, Mark S; Park, Yung-Chul; Austin, Andrew D

    2006-03-01

    Current knowledge of the evolutionary relationships amongst the wolf spiders (Araneae: Lycosidae) is based on assessment of morphological similarity or phylogenetic analysis of a small number of taxa. In order to enhance the current understanding of lycosid relationships, phylogenies of 70 lycosid species were reconstructed by parsimony and Bayesian methods using three molecular markers; the mitochondrial genes 12S rRNA, NADH1, and the nuclear gene 28S rRNA. The resultant trees from the mitochondrial markers were used to assess the current taxonomic status of the Lycosidae and to assess the evolutionary history of sheet-web construction in the group. The results suggest that a number of genera are not monophyletic, including Lycosa, Arctosa, Alopecosa, and Artoria. At the subfamilial level, the status of Pardosinae needs to be re-assessed, and the position of a number of genera within their respective subfamilies is in doubt (e.g., Hippasa and Arctosa in Lycosinae and Xerolycosa, Aulonia and Hygrolycosa in Venoniinae). In addition, a major clade of strictly Australasian taxa may require the creation of a new subfamily. The analysis of sheet-web building in Lycosidae revealed that the interpretation of this trait as an ancestral state relies on two factors: (1) an asymmetrical model favoring the loss of sheet-webs and (2) that the suspended silken tube of Pirata is directly descended from sheet-web building. Paralogous copies of the nuclear 28S rRNA gene were sequenced, confounding the interpretation of the phylogenetic analysis and suggesting that a cautionary approach should be taken to the further use of this gene for lycosid phylogenetic analysis. PMID:16503280

  16. Characteristic features of the nucleotide sequences of yeast mitochondrial ribosomal protein genes as analyzed by computer program GeneMark.

    PubMed

    Isono, K; McIninch, J D; Borodovsky, M

    1994-01-01

    The nucleotide sequence data for yeast mitochondrial ribosomal protein (MRP) genes were analyzed by the computer program GeneMark which predicts the presence of likely genes in sequence data by calculating statistical biases in the appearance of consecutive nucleotides. The program uses a set of standard sequence data for this calculation. We used this program for the analysis of yeast nucleotide sequence data containing MRP genes, hoping to obtain information as to whether they share features in common that are different from other yeast genes. Sequence data sets for ordinary yeast genes and for 27 known MRP genes were used. The MRP genes were nicely predicted as likely genes regardless of the data sets used, whereas other yeast genes were predicted to be likely genes only when the data set for ordinary yeast genes was used. The assembled sequence data for chromosomes II, III, VIII and XI as well as the segmented data for chromosome V were analyzed in a similar manner. In addition to the known MRP genes, eleven ORF's were predicted to be likely MRP genes. Thus, the method seems very powerful in analyzing genes of heterologous origins. PMID:7719921

  17. Haploinsufficiency screen highlights two distinct groups of ribosomal protein genes essential for embryonic stem cell fate

    PubMed Central

    Fortier, Simon; MacRae, Tara; Bilodeau, Mélanie; Sargeant, Tobias; Sauvageau, Guy

    2015-01-01

    In a functional genomics screen of mouse embryonic stem cells (ESCs) with nested hemizygous chromosomal deletions, we reveal that ribosomal protein (RP) genes are the most significant haploinsufficient determinants for embryoid body (EB) formation. Hemizygocity for three RP genes (Rps5, Rps14, or Rps28), distinguished by the proximity of their corresponding protein to the ribosome's mRNA exit site, is associated with the most profound phenotype. This EB phenotype was fully rescued by BAC or cDNA complementation but not by the reduction of p53 levels, although such reduction was effective with most other RP-deleted clones corresponding to non-mRNA exit-site proteins. RNA-sequencing studies further revealed that undifferentiated ESCs hemizygous for Rps5 showed reduced expression levels of several mesoderm-specific genes as compared with wild-type counterparts. Together, these results reveal that RP gene dosage limits the differentiation, not the self-renewal, of mouse ESCs. They also highlight two separate mechanisms underlying this process, one of which is p53 independent. PMID:25646475

  18. A Neurospora crassa ribosomal protein gene, homologous to yeast CRY1, contains sequences potentially coordinating its transcription with rRNA genes.

    PubMed Central

    Tyler, B M; Harrison, K

    1990-01-01

    We have isolated and sequenced a Neurospora crassa ribosomal protein gene (designated crp-2) strongly homologous to the rp59 gene (CRY1) of yeast and the S14 ribosomal protein gene of mammals. The inferred sequence of the crp-2 protein is more homologous (83%) to the mammalian S14 sequence than to the yeast rp59 sequence (69%). The gene has three intervening sequences (IVSs) two of which are offset 7 bp from the position of IVSs in the mammalian genes. None correspond to the position of the IVS in the yeast gene. Crp-2 was mapped by RFLP analysis to the right arm of linkage group III. The 5' region of the gene contains three copies of a sequence, the Ribo box, previously shown to be required for transcription of both 5S and 40S rRNA genes. We speculate that the Ribo box may coordinate ribosomal protein and rRNA gene transcription. Images PMID:1977135

  19. Plastid-Nuclear Interaction and Accelerated Coevolution in Plastid Ribosomal Genes in Geraniaceae.

    PubMed

    Weng, Mao-Lun; Ruhlman, Tracey A; Jansen, Robert K

    2016-01-01

    Plastids and mitochondria have many protein complexes that include subunits encoded by organelle and nuclear genomes. In animal cells, compensatory evolution between mitochondrial and nuclear-encoded subunits was identified and the high mitochondrial mutation rates were hypothesized to drive compensatory evolution in nuclear genomes. In plant cells, compensatory evolution between plastid and nucleus has rarely been investigated in a phylogenetic framework. To investigate plastid-nuclear coevolution, we focused on plastid ribosomal protein genes that are encoded by plastid and nuclear genomes from 27 Geraniales species. Substitution rates were compared for five sets of genes representing plastid- and nuclear-encoded ribosomal subunit proteins targeted to the cytosol or the plastid as well as nonribosomal protein controls. We found that nonsynonymous substitution rates (dN) and the ratios of nonsynonymous to synonymous substitution rates (ω) were accelerated in both plastid- (CpRP) and nuclear-encoded subunits (NuCpRP) of the plastid ribosome relative to control sequences. Our analyses revealed strong signals of cytonuclear coevolution between plastid- and nuclear-encoded subunits, in which nonsynonymous substitutions in CpRP and NuCpRP tend to occur along the same branches in the Geraniaceae phylogeny. This coevolution pattern cannot be explained by physical interaction between amino acid residues. The forces driving accelerated coevolution varied with cellular compartment of the sequence. Increased ω in CpRP was mainly due to intensified positive selection whereas increased ω in NuCpRP was caused by relaxed purifying selection. In addition, the many indels identified in plastid rRNA genes in Geraniaceae may have contributed to changes in plastid subunits. PMID:27190001

  20. Plastid–Nuclear Interaction and Accelerated Coevolution in Plastid Ribosomal Genes in Geraniaceae

    PubMed Central

    Weng, Mao-Lun; Ruhlman, Tracey A.; Jansen, Robert K.

    2016-01-01

    Plastids and mitochondria have many protein complexes that include subunits encoded by organelle and nuclear genomes. In animal cells, compensatory evolution between mitochondrial and nuclear-encoded subunits was identified and the high mitochondrial mutation rates were hypothesized to drive compensatory evolution in nuclear genomes. In plant cells, compensatory evolution between plastid and nucleus has rarely been investigated in a phylogenetic framework. To investigate plastid–nuclear coevolution, we focused on plastid ribosomal protein genes that are encoded by plastid and nuclear genomes from 27 Geraniales species. Substitution rates were compared for five sets of genes representing plastid- and nuclear-encoded ribosomal subunit proteins targeted to the cytosol or the plastid as well as nonribosomal protein controls. We found that nonsynonymous substitution rates (dN) and the ratios of nonsynonymous to synonymous substitution rates (ω) were accelerated in both plastid- (CpRP) and nuclear-encoded subunits (NuCpRP) of the plastid ribosome relative to control sequences. Our analyses revealed strong signals of cytonuclear coevolution between plastid- and nuclear-encoded subunits, in which nonsynonymous substitutions in CpRP and NuCpRP tend to occur along the same branches in the Geraniaceae phylogeny. This coevolution pattern cannot be explained by physical interaction between amino acid residues. The forces driving accelerated coevolution varied with cellular compartment of the sequence. Increased ω in CpRP was mainly due to intensified positive selection whereas increased ω in NuCpRP was caused by relaxed purifying selection. In addition, the many indels identified in plastid rRNA genes in Geraniaceae may have contributed to changes in plastid subunits. PMID:27190001

  1. Interaction of nucleolin with ribosomal RNA genes and its role in RNA polymerase I transcription

    PubMed Central

    Cong, Rong; Das, Sadhan; Ugrinova, Iva; Kumar, Sanjeev; Mongelard, Fabien; Wong, Jiemin; Bouvet, Philippe

    2012-01-01

    Nucleolin is a multi-functional nucleolar protein that is required for ribosomal RNA gene (rRNA) transcription in vivo, but the mechanism by which nucleolin modulates RNA polymerase I (RNAPI) transcription is not well understood. Nucleolin depletion results in an increase in the heterochromatin mark H3K9me2 and a decrease in H4K12Ac and H3K4me3 euchromatin histone marks in rRNA genes. ChIP-seq experiments identified an enrichment of nucleolin in the ribosomal DNA (rDNA) coding and promoter region. Nucleolin is preferentially associated with unmethylated rRNA genes and its depletion leads to the accumulation of RNAPI at the beginning of the transcription unit and a decrease in UBF along the coding and promoter regions. Nucleolin is able to affect the binding of transcription termination factor-1 on the promoter-proximal terminator T0, thus inhibiting the recruitment of TIP5 and HDAC1 and the establishment of a repressive heterochromatin state. These results reveal the importance of nucleolin for the maintenance of the euchromatin state and transcription elongation of rDNA. PMID:22859736

  2. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools

    PubMed Central

    Quast, Christian; Pruesse, Elmar; Yilmaz, Pelin; Gerken, Jan; Schweer, Timmy; Yarza, Pablo; Peplies, Jörg; Glöckner, Frank Oliver

    2013-01-01

    SILVA (from Latin silva, forest, http://www.arb-silva.de) is a comprehensive web resource for up to date, quality-controlled databases of aligned ribosomal RNA (rRNA) gene sequences from the Bacteria, Archaea and Eukaryota domains and supplementary online services. The referred database release 111 (July 2012) contains 3 194 778 small subunit and 288 717 large subunit rRNA gene sequences. Since the initial description of the project, substantial new features have been introduced, including advanced quality control procedures, an improved rRNA gene aligner, online tools for probe and primer evaluation and optimized browsing, searching and downloading on the website. Furthermore, the extensively curated SILVA taxonomy and the new non-redundant SILVA datasets provide an ideal reference for high-throughput classification of data from next-generation sequencing approaches. PMID:23193283

  3. High temperature, differentiation, and endoplasmic reticulum stress decrease but epigenetic and antioxidative agents increase Aspergillus ribosomal protein gene expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genome-wide gene expression assays using next-generation sequencing techniques have allowed the identification of transcriptomes in many species. Transcript abundance of ribosomal protein (RP) genes can serve as a proxy for the capacity of general transcription and synthesis of cellular proteins tha...

  4. Two C or not two C: recurrent disruption of Zn-ribbons, gene duplication, lineage-specific gene loss, and horizontal gene transfer in evolution of bacterial ribosomal proteins

    PubMed Central

    Makarova, Kira S; Ponomarev, Vladimir A; Koonin, Eugene V

    2001-01-01

    Background Ribosomal proteins are encoded in all genomes of cellular life forms and are, generally, well conserved during evolution. In prokaryotes, the genes for most ribosomal proteins are clustered in several highly conserved operons, which ensures efficient co-regulation of their expression. Duplications of ribosomal-protein genes are infrequent, and given their coordinated expression and functioning, it is generally assumed that ribosomal-protein genes are unlikely to undergo horizontal transfer. However, with the accumulation of numerous complete genome sequences of prokaryotes, several paralogous pairs of ribosomal protein genes have been identified. Here we analyze all such cases and attempt to reconstruct the evolutionary history of these ribosomal proteins. Results Complete bacterial genomes were searched for duplications of ribosomal proteins. Ribosomal proteins L36, L33, L31, S14 are each duplicated in several bacterial genomes and ribosomal proteins L11, L28, L7/L12, S1, S15, S18 are so far duplicated in only one genome each. Sequence analysis of the four ribosomal proteins, for which paralogs were detected in several genomes, two of the ribosomal proteins duplicated in one genome (L28 and S18), and the ribosomal protein L32 showed that each of them comes in two distinct versions. One form contains a predicted metal-binding Zn-ribbon that consists of four conserved cysteines (in some cases replaced by histidines), whereas, in the second form, these metal-chelating residues are completely or partially replaced. Typically, genomes containing paralogous genes for these ribosomal proteins encode both versions, designated C+ and C-, respectively. Analysis of phylogenetic trees for these seven ribosomal proteins, combined with comparison of genomic contexts for the respective genes, indicates that in most, if not all cases, their evolution involved a duplication of the ancestral C+ form early in bacterial evolution, with subsequent alternative loss of the C

  5. Molecular Identification of Ptychodera flava (Hemichordata: Enteropneusta): Reconsideration in Light of Nucleotide Polymorphism in the 18S Ribosomal RNA Gene.

    PubMed

    Urata, Makoto

    2015-06-01

    Seven nuclear and mitochondrial DNA markers were examined in 12 specimens of Ptychodera flava, a model acorn worm used in molecular biology, collected in Japan from three local populations with different modes of living. A comparison of intraspecific results did not show genetically isolated populations despite the species' enclave habitats and asexual reproduction. Moreover, both the nuclear 18S ribosomal RNA gene and mitochondrial 16S ribosomal RNA gene sequences were identical to those from Moorea in French Polynesia, nearly 10,000 kilometers away from Japan. I also provide the first definitive information regarding polymorphisms in 18S ribosomal RNA gene, the external transcribed spacer (ETS), internal transcribed spacers (ITS), and mitochondrial cytochrome c oxidase subunit 1 (mtCO1) sequence in hemichordates using newly designed primer sets, and I show both high larval vagility and certain criteria for the molecular identification of this species. PMID:26003987

  6. Identification of ribosomal RNA methyltransferase gene ermF in Riemerella anatipestifer.

    PubMed

    Luo, Hongyan; Liu, Mafeng; Wang, Lanying; Zhou, Wangshu; Wang, Mingshu; Cheng, Anchun; Jia, Renyong; Chen, Shun; Sun, Kunfeng; Yang, Qiao; Chen, Xiaoyue; Zhu, Dekang

    2015-01-01

    Riemerella anatipestifer is a major bacterial pathogen of waterfowl, globally responsible for avian septicaemic disease. As chemotherapy is the predominant method for the prevention and treatment of R. anatipestifer infection in poultry, the widespread use of antibiotics has favoured the emergence of antibiotic-resistant strains. However, little is known about R. anatipestifer susceptibility to macrolide antibiotics and its resistance mechanism. We report for the first time the identification of a macrolide resistance mechanism in R. anatipestifer that is mediated by the ribosomal RNA methyltransferase ermF. We identified the presence of the ermF gene in 64/206 (31%) R. anatipestifer isolates from different regions in China. An ermF deletion strain was constructed to investigate the function of the ermF gene on the resistance to high levels of macrolides. The ermF mutant strain showed significantly decreased resistance to macrolide and lincosamide, exhibiting 1024-, 1024-, 4- and >2048-fold reduction in the minimum inhibitory concentrations for erythromycin, azithromycin, tylosin and lincomycin, respectively. Furthermore, functional analysis of ermF expression in E. coli XL1-blue showed that the R. anatipestifer ermF gene was functional in E. coli XL1-blue and conferred resistance to high levels of erythromycin (100 µg/ml), supporting the hypothesis that the ermF gene is associated with high-level macrolide resistance. Our work suggests that ribosomal RNA modification mediated by the ermF methyltransferase is the predominant mechanism of resistance to erythromycin in R. anatipestifer isolates. PMID:25690020

  7. Chromosome mapping of ribosomal genes and histone H4 in the genus Radacridium (Romaleidae)

    PubMed Central

    Anjos, Allison; Loreto, Vilma; de Souza, Maria José

    2013-01-01

    In this study, two species of Romaleidae grasshoppers, Radacridium mariajoseae and R.nordestinum, were analyzed after CMA3/DA/DAPI sequential staining and fluorescence in situ hybridization (FISH) to determine the location of the 18S and 5S rDNA and histone H4 genes. Both species presented karyotypes composed of 2n = 23, X0 with exclusively acrocentric chromosomes. CMA3+ blocks were detected after CMA3/DA/DAPI staining in only one medium size autosome bivalent and in the X chromosome in R. mariajoseae. On the other hand, all chromosomes, except the L1 bivalent, of R. nordestinum presented CMA3+ blocks. FISH analysis showed that the 18S genes are restricted to the X chromosome in R. mariajoseae, whereas these genes were located in the L2, S9 and S10 autosomes in R. nordestinum. In R. mariajoseae, the 5S rDNA sites were localized in the in L1 and L2 bivalents and in the X chromosome. In R. nordestinum, the 5S genes were located in the L2, L3, M4 and M5 pairs. In both species the histone H4 genes were present in a medium size bivalent. Together, these data evidence a great variability of chromosome markers and show that the 18S and 5S ribosomal genes are dispersed in the Radacridium genome without a significant correlation. PMID:24130439

  8. RINT-1 interacts with MSP58 within nucleoli and plays a role in ribosomal gene transcription.

    PubMed

    Yang, Chuan-Pin; Kuo, Yu-Liang; Lee, Yi-Chao; Lee, Kuen-Haur; Chiang, Chi-Wu; Wang, Ju-Ming; Hsu, Che-Chia; Chang, Wen-Chang; Lin, Ding-Yen

    2016-09-16

    The nucleolus is the cellular site of ribosomal (r)DNA transcription and ribosome biogenesis. The 58-kDa microspherule protein (MSP58) is a nucleolar protein involved in rDNA transcription and cell proliferation. However, regulation of MSP58-mediated rDNA transcription remains unknown. Using a yeast two-hybrid system with MSP58 as bait, we isolated complementary (c)DNA encoding Rad50-interacting protein 1 (RINT-1), as a MSP58-binding protein. RINT-1 was implicated in the cell cycle checkpoint, membrane trafficking, Golgi apparatus and centrosome dynamic integrity, and telomere length control. Both in vitro and in vivo interaction assays showed that MSP58 directly interacts with RINT-1. Interestingly, microscopic studies revealed the co-localization of MSP58, RINT-1, and the upstream binding factor (UBF), a rRNA transcription factor, in the nucleolus. We showed that ectopic expression of MSP58 or RINT-1 resulted in decreased rRNA expression and rDNA promoter activity, whereas knockdown of MSP58 or RINT-1 by siRNA exerted the opposite effect. Coexpression of MSP58 and RINT-1 robustly decreased rRNA synthesis compared to overexpression of either protein alone, whereas depletion of RINT-1 from MSP58-transfected cells enhanced rRNA synthesis. We also found that MSP58, RINT-1, and the UBF were associated with the rDNA promoter using a chromatin immunoprecipitation assay. Because aberrant ribosome biogenesis contributes to neoplastic transformation, our results revealed a novel protein complex involved in the regulation of rRNA gene expression, suggesting a role for MSP58 and RINT-1 in cancer development. PMID:27530925

  9. Structural and Functional Characterization of Ribosomal Protein Gene Introns in Sponges

    PubMed Central

    Perina, Drago; Korolija, Marina; Mikoč, Andreja; Roller, Maša; Pleše, Bruna; Imešek, Mirna; Morrow, Christine; Batel, Renato; Ćetković, Helena

    2012-01-01

    Ribosomal protein genes (RPGs) are a powerful tool for studying intron evolution. They exist in all three domains of life and are much conserved. Accumulating genomic data suggest that RPG introns in many organisms abound with non-protein-coding-RNAs (ncRNAs). These ancient ncRNAs are small nucleolar RNAs (snoRNAs) essential for ribosome assembly. They are also mobile genetic elements and therefore probably important in diversification and enrichment of transcriptomes through various mechanisms such as intron/exon gain/loss. snoRNAs in basal metazoans are poorly characterized. We examined 449 RPG introns, in total, from four demosponges: Amphimedon queenslandica, Suberites domuncula, Suberites ficus and Suberites pagurorum and showed that RPG introns from A. queenslandica share position conservancy and some structural similarity with “higher” metazoans. Moreover, our study indicates that mobile element insertions play an important role in the evolution of their size. In four sponges 51 snoRNAs were identified. The analysis showed discrepancies between the snoRNA pools of orthologous RPG introns between S. domuncula and A. queenslandica. Furthermore, these two sponges show as much conservancy of RPG intron positions between each other as between themselves and human. Sponges from the Suberites genus show consistency in RPG intron position conservation. However, significant differences in some of the orthologous RPG introns of closely related sponges were observed. This indicates that RPG introns are dynamic even on these shorter evolutionary time scales. PMID:22880015

  10. Two distinct promoter architectures centered on dynamic nucleosomes control ribosomal protein gene transcription.

    PubMed

    Knight, Britta; Kubik, Slawomir; Ghosh, Bhaswar; Bruzzone, Maria Jessica; Geertz, Marcel; Martin, Victoria; Dénervaud, Nicolas; Jacquet, Philippe; Ozkan, Burak; Rougemont, Jacques; Maerkl, Sebastian J; Naef, Félix; Shore, David

    2014-08-01

    In yeast, ribosome production is controlled transcriptionally by tight coregulation of the 138 ribosomal protein genes (RPGs). RPG promoters display limited sequence homology, and the molecular basis for their coregulation remains largely unknown. Here we identify two prevalent RPG promoter types, both characterized by upstream binding of the general transcription factor (TF) Rap1 followed by the RPG-specific Fhl1/Ifh1 pair, with one type also binding the HMG-B protein Hmo1. We show that the regulatory properties of the two promoter types are remarkably similar, suggesting that they are determined to a large extent by Rap1 and the Fhl1/Ifh1 pair. Rapid depletion experiments allowed us to define a hierarchy of TF binding in which Rap1 acts as a pioneer factor required for binding of all other TFs. We also uncovered unexpected features underlying recruitment of Fhl1, whose forkhead DNA-binding domain is not required for binding at most promoters, and Hmo1, whose binding is supported by repeated motifs. Finally, we describe unusually micrococcal nuclease (MNase)-sensitive nucleosomes at all RPG promoters, located between the canonical +1 and -1 nucleosomes, which coincide with sites of Fhl1/Ifh1 and Hmo1 binding. We speculate that these "fragile" nucleosomes play an important role in regulating RPG transcriptional output. PMID:25085421

  11. Structural and functional characterization of ribosomal protein gene introns in sponges.

    PubMed

    Perina, Drago; Korolija, Marina; Mikoč, Andreja; Roller, Maša; Pleše, Bruna; Imešek, Mirna; Morrow, Christine; Batel, Renato; Ćetković, Helena

    2012-01-01

    Ribosomal protein genes (RPGs) are a powerful tool for studying intron evolution. They exist in all three domains of life and are much conserved. Accumulating genomic data suggest that RPG introns in many organisms abound with non-protein-coding-RNAs (ncRNAs). These ancient ncRNAs are small nucleolar RNAs (snoRNAs) essential for ribosome assembly. They are also mobile genetic elements and therefore probably important in diversification and enrichment of transcriptomes through various mechanisms such as intron/exon gain/loss. snoRNAs in basal metazoans are poorly characterized. We examined 449 RPG introns, in total, from four demosponges: Amphimedon queenslandica, Suberites domuncula, Suberites ficus and Suberites pagurorum and showed that RPG introns from A. queenslandica share position conservancy and some structural similarity with "higher" metazoans. Moreover, our study indicates that mobile element insertions play an important role in the evolution of their size. In four sponges 51 snoRNAs were identified. The analysis showed discrepancies between the snoRNA pools of orthologous RPG introns between S. domuncula and A. queenslandica. Furthermore, these two sponges show as much conservancy of RPG intron positions between each other as between themselves and human. Sponges from the Suberites genus show consistency in RPG intron position conservation. However, significant differences in some of the orthologous RPG introns of closely related sponges were observed. This indicates that RPG introns are dynamic even on these shorter evolutionary time scales. PMID:22880015

  12. [Characterization of Black and Dichothrix Cyanobacteria Based on the 16S Ribosomal RNA Gene Sequence

    NASA Technical Reports Server (NTRS)

    Ortega, Maya

    2010-01-01

    My project focuses on characterizing different cyanobacteria in thrombolitic mats found on the island of Highborn Cay, Bahamas. Thrombolites are interesting ecosystems because of the ability of bacteria in these mats to remove carbon dioxide from the atmosphere and mineralize it as calcium carbonate. In the future they may be used as models to develop carbon sequestration technologies, which could be used as part of regenerative life systems in space. These thrombolitic communities are also significant because of their similarities to early communities of life on Earth. I targeted two cyanobacteria in my research, Dichothrix spp. and whatever black is, since they are believed to be important to carbon sequestration in these thrombolitic mats. The goal of my summer research project was to molecularly identify these two cyanobacteria. DNA was isolated from each organism through mat dissections and DNA extractions. I ran Polymerase Chain Reactions (PCR) to amplify the 16S ribosomal RNA (rRNA) gene in each cyanobacteria. This specific gene is found in almost all bacteria and is highly conserved, meaning any changes in the sequence are most likely due to evolution. As a result, the 16S rRNA gene can be used for bacterial identification of different species based on the sequence of their 16S rRNA gene. Since the exact sequence of the Dichothrix gene was unknown, I designed different primers that flanked the gene based on the known sequences from other taxonomically similar cyanobacteria. Once the 16S rRNA gene was amplified, I cloned the gene into specialized Escherichia coli cells and sent the gene products for sequencing. Once the sequence is obtained, it will be added to a genetic database for future reference to and classification of other Dichothrix sp.

  13. Transformation of Chloroplast Ribosomal RNA Genes in Chlamydomonas: Molecular and Genetic Characterization of Integration Events

    PubMed Central

    Newman, S. M.; Boynton, J. E.; Gillham, N. W.; Randolph-Anderson, B. L.; Johnson, A. M.; Harris, E. H.

    1990-01-01

    Transformation of chloroplast ribosomal RNA (rRNA) genes in Chlamydomonas has been achieved by the biolistic process using cloned chloroplast DNA fragments carrying mutations that confer antibiotic resistance. The sites of exchange employed during the integration of the donor DNA into the recipient genome have been localized using a combination of antibiotic resistance mutations in the 16S and 23S rRNA genes and restriction fragment length polymorphisms that flank these genes. Complete or nearly complete replacement of a region of the chloroplast genome in the recipient cell by the corresponding sequence from the donor plasmid was the most common integration event. Exchange events between the homologous donor and recipient sequences occurred preferentially near the vector:insert junctions. Insertion of the donor rRNA genes and flanking sequences into one inverted repeat of the recipient genome was followed by intramolecular copy correction so that both copies of the inverted repeat acquired identical sequences. Increased frequencies of rRNA gene transformants were achieved by reducing the copy number of the chloroplast genome in the recipient cells and by decreasing the heterology between donor and recipient DNA sequences flanking the selectable markers. In addition to producing bona fide chloroplast rRNA transformants, the biolistic process induced mutants resistant to low levels of streptomycin, typical of nuclear mutations in Chlamydomonas. PMID:1981764

  14. Circular code motifs in transfer and 16S ribosomal RNAs: a possible translation code in genes.

    PubMed

    Michel, Christian J

    2012-04-01

    In 1996, a common trinucleotide circular code, called X, is identified in genes of eukaryotes and prokaryotes (Arquès and Michel, 1996). This circular code X is a set of 20 trinucleotides allowing the reading frames in genes to be retrieved locally, i.e. anywhere in genes and in particular without start codons. This reading frame retrieval needs a window length l of 12 nucleotides (l ≥ 12). With a window length strictly less than 12 nucleotides (l < 12), some words of X, called ambiguous words, are found in the shifted frames (the reading frame shifted by one or two nucleotides) preventing the reading frame in genes to be retrieved. Since 1996, these ambiguous words of X were never studied. In the first part of this paper, we identify all the ambiguous words of the common trinucleotide circular code X. With a length l varying from 1 to 11 nucleotides, the type and the occurrence number (multiplicity) of ambiguous words of X are given in each shifted frame. Maximal ambiguous words of X, words which are not factors of another ambiguous words, are also determined. Two probability definitions based on these results show that the common trinucleotide circular code X retrieves the reading frame in genes with a probability of about 90% with a window length of 6 nucleotides, and a probability of 99.9% with a window length of 9 nucleotides (100% with a window length of 12 nucleotides, by definition of a circular code). In the second part of this paper, we identify X circular code motifs (shortly X motifs) in transfer RNA and 16S ribosomal RNA: a tRNA X motif of 26 nucleotides including the anticodon stem-loop and seven 16S rRNA X motifs of length greater or equal to 15 nucleotides. Window lengths of reading frame retrieval with each trinucleotide of these X motifs are also determined. Thanks to the crystal structure 3I8G (Jenner et al., 2010), a 3D visualization of X motifs in the ribosome shows several spatial configurations involving mRNA X motifs, A-tRNA and E-tRNA X

  15. Molecular phylogeny of labyrinthulids and thraustochytrids based on the sequencing of 18S ribosomal RNA gene.

    PubMed

    Honda, D; Yokochi, T; Nakahara, T; Raghukumar, S; Nakagiri, A; Schaumann, K; Higashihara, T

    1999-01-01

    Labyrinthulids and thraustochytrids are unicellular heterotrophs, formerly considered as fungi, but presently are recognized as members in the stramenopiles of the kingdom Protista sensu lato. We determined the 18S ribosomal RNA gene sequences of 14 strains from different species of the six genera and analyzed the molecular phylogenetic relationships. The results conflict with the current classification based on morphology, at the genus and species levels. These organisms are separated, based on signature sequences and unique inserted sequences, into two major groups, which were named the labyrinthulid phylogenetic group and the thraustochytrid phylogenetic group. Although these groupings are in disagreement with many conventional taxonomic characters, they correlated better with the sugar composition of the cell wall. Thus, the currently used taxonomic criteria need serious reconsideration. PMID:10568038

  16. Small-subunit ribosomal RNA gene sequences of Phaeodarea challenge the monophyly of Haeckel's Radiolaria.

    PubMed

    Polet, Stephane; Berney, Cédric; Fahrni, José; Pawlowski, Jan

    2004-03-01

    In his grand monograph of Radiolaria, Ernst Haeckel originally included Phaeodarea together with Acantharea and Polycystinea, all three taxa characterized by the presence of a central capsule and the possession of axopodia. Cytological and ultrastructural studies, however, questioned the monophyly of Radiolaria, suggesting an independent evolutionary origin of the three taxa, and the first molecular data on Acantharea and Polycystinea brought controversial results. To test further the monophyly of Radiolaria, we sequenced the complete small subunit ribosomal RNA gene of three phaeodarians and three polycystines. Our analyses reveal that phaeodarians clearly branch among the recently described phylum Cercozoa, separately from Acantharea and Polycystinea. This result enhances the morphological variability within the phylum Cercozoa, which already contains very heterogeneous groups of protists. Our study also confirms the common origin of Acantharea and Polycystinea, which form a sister-group to the Cercozoa, and allows a phylogenetic reinterpretation of the morphological features of the three radiolarian groups. PMID:15144058

  17. Sequence and organization of 5S ribosomal RNA-encoding genes of Arabidopsis thaliana.

    PubMed

    Campell, B R; Song, Y; Posch, T E; Cullis, C A; Town, C D

    1992-03-15

    We have isolated a genomic clone containing Arabidopsis thaliana 5S ribosomal RNA (rRNA)-encoding genes (rDNA) by screening an A. thaliana library with a 5S rDNA probe from flax. The clone isolated contains seven repeat units of 497 bp, plus 11 kb of flanking genomic sequence at one border. Sequencing of individual subcloned repeat units shows that the sequence of the 5S rRNA coding region is very similar to that reported for other flowering plants. Four A. thaliana ecotypes were found to contain approx. 1000 copies of 5S rDNA per haploid genome. Southern-blot analysis of genomic DNA indicates that 5S rDNA occurs in long tandem arrays, and shows the presence of numerous restriction-site polymorphisms among the six ecotypes studied. PMID:1348233

  18. A phylogeny of cockroaches and related insects based on DNA sequence of mitochondrial ribosomal RNA genes.

    PubMed Central

    Kambhampati, S

    1995-01-01

    Cockroaches are among the most ancient winged insects, the earliest fossils dating back to about 400 million years. Several conflicting phylogenies for cockroach families, subfamilies, and genera have been proposed in the past. In addition, the relationship of Cryptocercidae to other cockroach families and the relationship between the cockroach, Cryptocercus punctulatus, and the termite, Mastotermes darwiniensis, have generated debate. In this paper, a phylogeny for cockroaches, mantids, and termites based on DNA sequence of the mitochondrial ribosomal RNA genes is presented. The results indicated that cockroaches are a monophyletic group, whose sister group is Mantoidea. The inferred relationship among cockroach families was in agreement with the presently accepted phylogeny. However, there was only partial congruence at the subfamily and the generic levels. The phylogeny inferred here does not support a close relationship between C. punctulatus and M. darwiniensis. The apparent synapomorphies of these two species are likely a manifestation of convergent evolution because there are similarities in biology and habitat. PMID:7534409

  19. Biodiversity characterization of cellulolytic bacteria present on native Chaco soil by comparison of ribosomal RNA genes.

    PubMed

    Talia, Paola; Sede, Silvana M; Campos, Eleonora; Rorig, Marcela; Principi, Dario; Tosto, Daniela; Hopp, H Esteban; Grasso, Daniel; Cataldi, Angel

    2012-04-01

    Sequence analysis of the 16S ribosomal RNA gene was used to study bacterial diversity of a pristine forest soil and of two cultures of the same soil enriched with cellulolytic bacteria. Our analysis revealed high bacterial diversity in the native soil sample, evidencing at least 10 phyla, in which Actinobacteria, Proteobacteria and Acidobacteria accounted for more than 76% of all sequences. In both enriched samples, members of Proteobacteria were the most frequently represented. The majority of bacterial genera in both enriched samples were identified as Brevundimonas and Caulobacter, but members of Devosia, Sphingomonas, Variovorax, Acidovorax, Pseudomonas, Xanthomonas, Stenotrophomonas, Achromobacter and Delftia were also found. In addition, it was possible to identify cellulolytic taxa such as Acidothermus, Micromonospora, Streptomyces, Paenibacillus and Pseudomonas, which indicates that this ecosystem could be an attractive source for study of novel enzymes for cellulose degradation. PMID:22202170

  20. A variant of Plasmodium ovale; analysis of its 18S ribosomal RNA gene sequence.

    PubMed

    Miyake, H; Suwa, S; Kimura, M; Wataya, Y

    1997-01-01

    We report here a new variant of human malaria parasite found by comparison of diagnostic results obtained from a new DNA diagnostic method named microtiter plate-hybridization (MPH) and traditional microscopic method. Total five cases of malaria were diagnosed as microscopy-positive but MPH-negative; one case was found in epidemiological research in Vietnam and four cases were obtained from imported malaria in Japan. Although they were quite similar to typical P. ovale morphologically in microscopy, sequence analysis of PCR-amplified DNA fragment revealed that their 18S ribosomal RNA gene sequence was different from published sequence of P. ovale. Combination of MPH and microscopic examination provides us a new method for detection of a new type of malaria parasite which is difficult to distinguish morphologically. PMID:9586115

  1. The novel organization and complete sequence of the ribosomal RNA gene of Nosema bombycis.

    PubMed

    Huang, Wei-Fone; Tsai, Shu-Jen; Lo, Chu-Fang; Soichi, Yamane; Wang, Chung-Hsiung

    2004-05-01

    We present here for the first time the complete DNA sequence data (4301bp) of the ribosomal RNA (rRNA) gene of the microsporidian type species, Nosema bombycis. Sequences for the large subunit gene (LSUrRNA: 2497bp, GenBank Accession No. ), the internal transcribed spacer (ITS: 179bp, GenBank Accession No. ), the small subunit gene (SSUrRNA: 1232bp), intergenic spacer (IGS: 279bp), and 5S region (114bp) are also given, and the secondary structure of the large subunit is discussed. The organization of the N. bombycis rRNA gene is LSUrRNA-ITS-SSUrRNA-IGS-5S. This novel arrangement, in which the LSU is 5' of the SSU, is the reverse of the organizational sequence (i.e., SSU-ITS-LSU) found in all previously reported microsporidian rRNAs, including Nosema apis. This unique character in the type species may have taxonomic implications for the members of the genus Nosema. PMID:15050536

  2. Cloning and characterization of a gene from Rhizobium melilotii 2011 coding for ribosomal protein S1.

    PubMed Central

    Schnier, J; Thamm, S; Lurz, R; Hussain, A; Faist, G; Dobrinski, B

    1988-01-01

    A 7 kb chromosomal DNA fragment from R. melilotii was cloned, which complemented temperature-sensitivity of an E. coli amber mutant in rpsA, the gene for ribosomal protein S1 (ES1). From complementation and maxicell analysis a 58 kd protein was identified as the homolog of protein S1 (RS1). DNA sequence analysis of the R. melilotii rpsA gene identified a protein of 568 amino acids, which showed 47% identical amino acid homology to protein S1 from E. coli. The RS1 protein lacked the two Cys residues which had been reported to play an important role for the function of ES1. Two repeats containing Shine-Dalgarno sequences were identified upstream of the structural gene. Binding studies with RNA polymerase from E. coli and Pseudomonas putida located one RNA-polymerase binding site close to the RS1 gene and another one several hundred basepairs upstream. One possible promoter was also identified by DNA sequence comparison with the corresponding E. coli promoter. Images PMID:3368316

  3. Integrative analyses shed new light on human ribosomal protein gene regulation.

    PubMed

    Li, Xin; Zheng, Yiyu; Hu, Haiyan; Li, Xiaoman

    2016-01-01

    Ribosomal protein genes (RPGs) are important house-keeping genes that are well-known for their coordinated expression. Previous studies on RPGs are largely limited to their promoter regions. Recent high-throughput studies provide an unprecedented opportunity to study how human RPGs are transcriptionally modulated and how such transcriptional regulation may contribute to the coordinate gene expression in various tissues and cell types. By analyzing the DNase I hypersensitive sites under 349 experimental conditions, we predicted 217 RPG regulatory regions in the human genome. More than 86.6% of these computationally predicted regulatory regions were partially corroborated by independent experimental measurements. Motif analyses on these predicted regulatory regions identified 31 DNA motifs, including 57.1% of experimentally validated motifs in literature that regulate RPGs. Interestingly, we observed that the majority of the predicted motifs were shared by the predicted distal and proximal regulatory regions of the same RPGs, a likely general mechanism for enhancer-promoter interactions. We also found that RPGs may be differently regulated in different cells, indicating that condition-specific RPG regulatory regions still need to be discovered and investigated. Our study advances the understanding of how RPGs are coordinately modulated, which sheds light to the general principles of gene transcriptional regulation in mammals. PMID:27346035

  4. Sumoylation of Rap1 mediates the recruitment of TFIID to promote transcription of ribosomal protein genes.

    PubMed

    Chymkowitch, Pierre; Nguéa, Aurélie P; Aanes, Håvard; Koehler, Christian J; Thiede, Bernd; Lorenz, Susanne; Meza-Zepeda, Leonardo A; Klungland, Arne; Enserink, Jorrit M

    2015-06-01

    Transcription factors are abundant Sumo targets, yet the global distribution of Sumo along the chromatin and its physiological relevance in transcription are poorly understood. Using Saccharomyces cerevisiae, we determined the genome-wide localization of Sumo along the chromatin. We discovered that Sumo-enriched genes are almost exclusively involved in translation, such as tRNA genes and ribosomal protein genes (RPGs). Genome-wide expression analysis showed that Sumo positively regulates their transcription. We also discovered that the Sumo consensus motif at RPG promoters is identical to the DNA binding motif of the transcription factor Rap1. We demonstrate that Rap1 is a molecular target of Sumo and that sumoylation of Rap1 is important for cell viability. Furthermore, Rap1 sumoylation promotes recruitment of the basal transcription machinery, and sumoylation of Rap1 cooperates with the target of rapamycin kinase complex 1 (TORC1) pathway to promote RPG transcription. Strikingly, our data reveal that sumoylation of Rap1 functions in a homeostatic feedback loop that sustains RPG transcription during translational stress. Taken together, Sumo regulates the cellular translational capacity by promoting transcription of tRNA genes and RPGs. PMID:25800674

  5. Integrative analyses shed new light on human ribosomal protein gene regulation

    PubMed Central

    Li, Xin; Zheng, Yiyu; Hu, Haiyan; Li, Xiaoman

    2016-01-01

    Ribosomal protein genes (RPGs) are important house-keeping genes that are well-known for their coordinated expression. Previous studies on RPGs are largely limited to their promoter regions. Recent high-throughput studies provide an unprecedented opportunity to study how human RPGs are transcriptionally modulated and how such transcriptional regulation may contribute to the coordinate gene expression in various tissues and cell types. By analyzing the DNase I hypersensitive sites under 349 experimental conditions, we predicted 217 RPG regulatory regions in the human genome. More than 86.6% of these computationally predicted regulatory regions were partially corroborated by independent experimental measurements. Motif analyses on these predicted regulatory regions identified 31 DNA motifs, including 57.1% of experimentally validated motifs in literature that regulate RPGs. Interestingly, we observed that the majority of the predicted motifs were shared by the predicted distal and proximal regulatory regions of the same RPGs, a likely general mechanism for enhancer-promoter interactions. We also found that RPGs may be differently regulated in different cells, indicating that condition-specific RPG regulatory regions still need to be discovered and investigated. Our study advances the understanding of how RPGs are coordinately modulated, which sheds light to the general principles of gene transcriptional regulation in mammals. PMID:27346035

  6. A ribosomal protein gene cluster is encoded in the mitochondrial DNA of Dictyostelium discoideum: UGA termination codons and similarity of gene order to Acanthamoeba castellanii.

    PubMed

    Iwamoto, M; Pi, M; Kurihara, M; Morio, T; Tanaka, Y

    1998-04-01

    We sequenced a region of about 14.5 kb downstream from the ribosomal protein L11 gene (rpl11) in the mitochondrial DNA (54+/-2 kb) of the cellular slime mold Dictyostelium discoideum. Sequence analysis revealed that eleven ribosomal protein genes and six open reading frames (ORFs) formed a cluster arranged in the order: rpl11-orf189-rps12-rps7-rpl2-rps19-+ ++orf425-orf1740-rpl16-rpl14-orf188- rps14-rps8-rpl6-rps13-orf127-orf796. This order was very similar to that of homologous genes in Acanthamoeba castellanii mitochondrial DNA. The N-terminal region of ORF425 and the C-terminal region of ORF1740 had partial similarities to the S3 ribosomal protein of other organisms. The termination codons of rpl16 and orf188 were UGA, which has not hitherto been found in genes encoded in D. discoideum mitochondrial DNA. PMID:9560439

  7. SINA: Accurate high-throughput multiple sequence alignment of ribosomal RNA genes

    PubMed Central

    Pruesse, Elmar; Peplies, Jörg; Glöckner, Frank Oliver

    2012-01-01

    Motivation: In the analysis of homologous sequences, computation of multiple sequence alignments (MSAs) has become a bottleneck. This is especially troublesome for marker genes like the ribosomal RNA (rRNA) where already millions of sequences are publicly available and individual studies can easily produce hundreds of thousands of new sequences. Methods have been developed to cope with such numbers, but further improvements are needed to meet accuracy requirements. Results: In this study, we present the SILVA Incremental Aligner (SINA) used to align the rRNA gene databases provided by the SILVA ribosomal RNA project. SINA uses a combination of k-mer searching and partial order alignment (POA) to maintain very high alignment accuracy while satisfying high throughput performance demands. SINA was evaluated in comparison with the commonly used high throughput MSA programs PyNAST and mothur. The three BRAliBase III benchmark MSAs could be reproduced with 99.3, 97.6 and 96.1 accuracy. A larger benchmark MSA comprising 38 772 sequences could be reproduced with 98.9 and 99.3% accuracy using reference MSAs comprising 1000 and 5000 sequences. SINA was able to achieve higher accuracy than PyNAST and mothur in all performed benchmarks. Availability: Alignment of up to 500 sequences using the latest SILVA SSU/LSU Ref datasets as reference MSA is offered at http://www.arb-silva.de/aligner. This page also links to Linux binaries, user manual and tutorial. SINA is made available under a personal use license. Contact: epruesse@mpi-bremen.de Supplementary information: Supplementary data are available at Bioinformatics online. PMID:22556368

  8. Characterization and Physical Mapping of Ribosomal RNA Gene Families in Plantago

    PubMed Central

    DHAR, MANOJ K.; FRIEBE, BERND; KAUL, SANJANA; GILL, BIKRAM S.

    2006-01-01

    • Background and Aims The organization of rRNA genes in cultivated Plantago ovata Forsk. and several of its wild allies was analysed to gain insight into the phylogenetic relationships of these species in the genus which includes some 200 species. • Methods Specific primers were designed to amplify the internal transcribed spacer (ITS1 and ITS2) regions from seven Plantago species and the resulting fragments were cloned and sequenced. Similarly, using specific primers, the 5S rRNA genes from these species were amplified and subsequently cloned. Fluorescence in-situ hybridization (FISH) was used for physical mapping of 5S and 45S ribosomal RNA genes. • Results The ITS1 region is 19–29 bp longer than the ITS2 in different Plantago species. The 5S rRNA gene-repeating unit varies in length from 289 to 581 bp. Coding regions are highly conserved across species, but the non-transcribed spacers (NTS) do not match any database sequences. The clone from the cultivated species P. ovata was used for physical mapping of these genes by FISH. Four species have one FISH site while three have two FISH sites. In P. lanceolata and P. rhodosperma, the 5S and 45S (18S-5·8S-25S) sites are coupled. • Conclusions Characterization of 5S and 45S rRNA genes has indicated a possible origin of P. ovata, the only cultivated species of the genus and also the only species with x = 4, from a species belonging to subgenus Psyllium. Based on the studies reported here, P. ovata is closest to P. arenaria, although on the basis of other data the two species have been placed in different subgenera. FISH mapping can be used as an efficient tool to help determine phylogenetic relationships in the genus Plantago and show the interrelationship between P. lanceolata and P. lagopus. PMID:16481363

  9. Compensation for differences in gene copy number among yeast ribosomal proteins is encoded within their promoters

    PubMed Central

    Zeevi, Danny; Sharon, Eilon; Lotan-Pompan, Maya; Lubling, Yaniv; Shipony, Zohar; Raveh-Sadka, Tali; Keren, Leeat; Levo, Michal; Weinberger, Adina; Segal, Eran

    2011-01-01

    Coordinate regulation of ribosomal protein (RP) genes is key for controlling cell growth. In yeast, it is unclear how this regulation achieves the required equimolar amounts of the different RP components, given that some RP genes exist in duplicate copies, while others have only one copy. Here, we tested whether the solution to this challenge is partly encoded within the DNA sequence of the RP promoters, by fusing 110 different RP promoters to a fluorescent gene reporter, allowing us to robustly detect differences in their promoter activities that are as small as ∼10%. We found that single-copy RP promoters have significantly higher activities, suggesting that proper RP stoichiometry is indeed partly encoded within the RP promoters. Notably, we also partially uncovered how this regulation is encoded by finding that RP promoters with higher activity have more nucleosome-disfavoring sequences and characteristic spatial organizations of these sequences and of binding sites for key RP regulators. Mutations in these elements result in a significant decrease of RP promoter activity. Thus, our results suggest that intrinsic (DNA-dependent) nucleosome organization may be a key mechanism by which genomes encode biologically meaningful promoter activities. Our approach can readily be applied to uncover how transcriptional programs of other promoters are encoded. PMID:22009988

  10. Dictyostelium ribosomal protein genes and the elongation factor 1B gene show coordinate developmental regulation which is under post-transcriptional control.

    PubMed

    Agarwal, A K; Blumberg, D D

    1999-06-01

    Starvation for amino acids initiates the developmental program in the cellular slime mold, Dictyostelium discoideum [19, 20]. One of the earliest developmental events is the decline in ribosomal protein synthesis [2, 17, 29, 30]. The ribosomal protein mRNAs are excluded from polysomes with 20 min to 1 h following the removal of nutrients, and their mRNA levels decline sharply at about 9 h into the 24-h developmental cycle [28, 31, 35, 36]. It has been generally assumed that the decline in r-protein mRNA levels during late development reflected a decline in the transcription rate [12, 32, 43]. Here we demonstrate that this is not the case. The transcription rates of three ribosomal protein genes, rpL11, rpL23 and rpS9 as well as an elongation factor 1B gene have been determined during growth and development in Dictyostelium. Throughout growth and development the transcription rate of the ribosomal protein genes remains relatively constant at 0.2%-0.5% of the rate of rRNA transcription while the elongation factor 1B gene is transcribed at 0.4%-0.6% of the rRNA rate. This low but constant transcription rate is in contrast to a spore coat protein gene Psp D, which is transcribed at 6% of the rRNA rate in late developing cells. The elongation factor 1B gene appears to be co-regulated with the ribosomal protein genes both in terms of its transcription rate and mRNA accumulation. Dictyostelium has been a popular model for understanding signal transduction and the growth to differentiation transition, thus it is of significance that the regulation of ribosome biosynthesis in Dictyostelium resembles that of higher eukaryotes in being regulated largely at the post-transcriptional level in response to starvation as opposed to yeasts where the regulation is largely transcriptional [27]. PMID:10374261

  11. Comparisons of Ribosomal Protein Gene Promoters Indicate Superiority of Heterologous Regulatory Sequences for Expressing Transgenes in Phytophthora infestans

    PubMed Central

    Khachatoorian, Careen; Judelson, Howard S.

    2015-01-01

    Molecular genetics approaches in Phytophthora research can be hampered by the limited number of known constitutive promoters for expressing transgenes and the instability of transgene activity. We have therefore characterized genes encoding the cytoplasmic ribosomal proteins of Phytophthora and studied their suitability for expressing transgenes in P. infestans. Phytophthora spp. encode a standard complement of 79 cytoplasmic ribosomal proteins. Several genes are duplicated, and two appear to be pseudogenes. Half of the genes are expressed at similar levels during all stages of asexual development, and we discovered that the majority share a novel promoter motif named the PhRiboBox. This sequence is enriched in genes associated with transcription, translation, and DNA replication, including tRNA and rRNA biogenesis. Promoters from the three P. infestans genes encoding ribosomal proteins S9, L10, and L23 and their orthologs from P. capsici were tested for their ability to drive transgenes in stable transformants of P. infestans. Five of the six promoters yielded strong expression of a GUS reporter, but the stability of expression was higher using the P. capsici promoters. With the RPS9 and RPL10 promoters of P. infestans, about half of transformants stopped making GUS over two years of culture, while their P. capsici orthologs conferred stable expression. Since cross-talk between native and transgene loci may trigger gene silencing, we encourage the use of heterologous promoters in transformation studies. PMID:26716454

  12. Ribosome profiling reveals an important role for translational control in circadian gene expression

    PubMed Central

    Jang, Christopher; Lahens, Nicholas F.; Hogenesch, John B.; Sehgal, Amita

    2015-01-01

    Physiological and behavioral circadian rhythms are driven by a conserved transcriptional/translational negative feedback loop in mammals. Although most core clock factors are transcription factors, post-transcriptional control introduces delays that are critical for circadian oscillations. Little work has been done on circadian regulation of translation, so to address this deficit we conducted ribosome profiling experiments in a human cell model for an autonomous clock. We found that most rhythmic gene expression occurs with little delay between transcription and translation, suggesting that the lag in the accumulation of some clock proteins relative to their mRNAs does not arise from regulated translation. Nevertheless, we found that translation occurs in a circadian fashion for many genes, sometimes imposing an additional level of control on rhythmically expressed mRNAs and, in other cases, conferring rhythms on noncycling mRNAs. Most cyclically transcribed RNAs are translated at one of two major times in a 24-h day, while rhythmic translation of most noncyclic RNAs is phased to a single time of day. Unexpectedly, we found that the clock also regulates the formation of cytoplasmic processing (P) bodies, which control the fate of mRNAs, suggesting circadian coordination of mRNA metabolism and translation. PMID:26338483

  13. Ribosomal RNA genes of Trypanosoma brucei. Cloning of a rRNA gene containing a mobile element.

    PubMed Central

    Hasan, G; Turner, M J; Cordingley, J S

    1982-01-01

    An ordered restriction map of the ribosomal RNA genes of Trypanosoma brucei brucei is presented. Bgl II fragments of T.b.brucei genomic DNA were cloned into pAT 153, and the clones containing rDNA identified. Restriction maps were established and the sense strands identified. One clone was shown by heteroduplex mapping to contain a 1.1 kb inserted sequence which was demonstrated to be widely distributed throughout the genomes of members of the subgenus Trypanozoon. However, in two other subgenera of Trypanosoma, Nannomonas and Schizotrypanum, the sequence is far less abundant. Analysis of the genomic DNA from two serodemes of T.b.brucei showed that the sequence was present in the rRNA of only one of them, implying that the sequence is a mobile element and that its appearance in rDNA is a comparitively recent occurrence. Images PMID:6294613

  14. Mutation of the rice ASL2 gene encoding plastid ribosomal protein L21 causes chloroplast developmental defects and seedling death.

    PubMed

    Lin, D; Jiang, Q; Zheng, K; Chen, S; Zhou, H; Gong, X; Xu, J; Teng, S; Dong, Y

    2015-05-01

    The plastid ribosome proteins (PRPs) play important roles in plastid protein biosynthesis, chloroplast differentiation and early chloroplast development. However, the specialised functions of individual protein components of the chloroplast ribosome in rice (Oryza sativa) remain unresolved. In this paper, we identified a novel rice PRP mutant named asl2 (Albino seedling lethality 2) exhibiting an albino, seedling death phenotype. In asl2 mutants, the alteration of leaf colour was associated with chlorophyll (Chl) content and abnormal chloroplast development. Through map-based cloning and complementation, the mutated ASL2 gene was isolated and found to encode the chloroplast 50S ribosome protein L21 (RPL21c), a component of the chloroplast ribosome large subunit, which was localised in chloroplasts. ASL2 was expressed at a higher level in the plumule and leaves, implying its tissue-specific expression. Additionally, the expression of ASL2 was regulated by light. The transcript levels of the majority of genes for Chl biosynthesis, photosynthesis and chloroplast development were strongly affected in asl2 mutants. Collectively, the absence of functional ASL2 caused chloroplast developmental defects and seedling death. This report establishes the important role of RPL21c in chloroplast development in rice. PMID:25280352

  15. Nucleotide sequence of the L1 ribosomal protein gene of Xenopus laevis: remarkable sequence homology among introns.

    PubMed Central

    Loreni, F; Ruberti, I; Bozzoni, I; Pierandrei-Amaldi, P; Amaldi, F

    1985-01-01

    Ribosomal protein L1 is encoded by two genes in Xenopus laevis. The comparison of two cDNA sequences shows that the two L1 gene copies (L1a and L1b) have diverged in many silent sites and very few substitution sites; moreover a small duplication occurred at the very end of the coding region of the L1b gene which thus codes for a product five amino acids longer than that coded by L1a. Quantitatively the divergence between the two L1 genes confirms that a whole genome duplication took place in Xenopus laevis approximately 30 million years ago. A genomic fragment containing one of the two L1 gene copies (L1a), with its nine introns and flanking regions, has been completely sequenced. The 5' end of this gene has been mapped within a 20-pyridimine stretch as already found for other vertebrate ribosomal protein genes. Four of the nine introns have a 60-nucleotide sequence with 80% homology; within this region some boxes, one of which is 16 nucleotides long, are 100% homologous among the four introns. This feature of L1a gene introns is interesting since we have previously shown that the activity of this gene is regulated at a post-transcriptional level and it involves the block of the normal splicing of some intron sequences. Images Fig. 3. Fig. 5. PMID:3841512

  16. Diversity and Recombination of Dispersed Ribosomal DNA and Protein Coding Genes in Microsporidia

    PubMed Central

    Ironside, Joseph Edward

    2013-01-01

    Microsporidian strains are usually classified on the basis of their ribosomal DNA (rDNA) sequences. Although rDNA occurs as multiple copies, in most non-microsporidian species copies within a genome occur as tandem arrays and are homogenised by concerted evolution. In contrast, microsporidian rDNA units are dispersed throughout the genome in some species, and on this basis are predicted to undergo reduced concerted evolution. Furthermore many microsporidian species appear to be asexual and should therefore exhibit reduced genetic diversity due to a lack of recombination. Here, DNA sequences are compared between microsporidia with different life cycles in order to determine the effects of concerted evolution and sexual reproduction upon the diversity of rDNA and protein coding genes. Comparisons of cloned rDNA sequences between microsporidia of the genus Nosema with different life cycles provide evidence of intragenomic variability coupled with strong purifying selection. This suggests a birth and death process of evolution. However, some concerted evolution is suggested by clustering of rDNA sequences within species. Variability of protein-coding sequences indicates that considerable intergenomic variation also occurs between microsporidian cells within a single host. Patterns of variation in microsporidian DNA sequences indicate that additional diversity is generated by intragenomic and/or intergenomic recombination between sequence variants. The discovery of intragenomic variability coupled with strong purifying selection in microsporidian rRNA sequences supports the hypothesis that concerted evolution is reduced when copies of a gene are dispersed rather than repeated tandemly. The presence of intragenomic variability also renders the use of rDNA sequences for barcoding microsporidia questionable. Evidence of recombination in the single-copy genes of putatively asexual microsporidia suggests that these species may undergo cryptic sexual reproduction, a

  17. Invasion of protein coding genes by green algal ribosomal group I introns.

    PubMed

    McManus, Hilary A; Lewis, Louise A; Fučíková, Karolina; Haugen, Peik

    2012-01-01

    The spread of group I introns depends on their association with intron-encoded homing endonucleases. Introns that encode functional homing endonuclease genes (HEGs) are highly invasive, whereas introns that only encode the group I ribozyme responsible for self-splicing are generally stably inherited (i.e., vertical inheritance). A number of recent case studies have provided new knowledge on the evolution of group I introns, however, there are still large gaps in understanding of their distribution on the tree of life, and how they have spread into new hosts and genic sites. During a larger phylogenetic survey of chlorophyceaen green algae, we found that 23 isolates contain at least one group I intron in the rbcL chloroplast gene. Structural analyses show that the introns belong to one of two intron lineages, group IA2 intron-HEG (GIY-YIG family) elements inserted after position 462 in the rbcL gene, and group IA1 introns inserted after position 699. The latter intron type sometimes encodes HNH homing endonucleases. The distribution of introns was analyzed on an exon phylogeny and patterns were recovered that are consistent with vertical inheritance and possible horizontal transfer. The rbcL 462 introns are thus far reported only within the Volvocales, Hydrodictyaceae and Bracteacoccus, and closely related isolates of algae differ in the presence of rbcL introns. Phylogenetic analysis of the intron conserved regions indicates that the rbcL699 and rbcL462 introns have distinct evolutionary origins. The rbcL699 introns were likely derived from ribosomal RNA L2449 introns, whereas the rbcL462 introns form a close relationship with psbA introns. PMID:22056605

  18. Conservation and divergence of transcriptional coregulations between box C/D snoRNA and ribosomal protein genes in Ascomycota

    PubMed Central

    Diao, Li-Ting; Xiao, Zhen-Dong; Leng, Xiao-Min; Li, Bin; Li, Jun-Hao; Luo, Yu-Ping; Li, Si-Guang; Yu, Chuan-He; Zhou, Hui

    2014-01-01

    Coordinated assembly of the ribosome is essential for proper translational activity in eukaryotic cells. It is therefore critical to coordinate the expression of components of ribosomal programs with the cell's nutritional status. However, coordinating expression of these components is poorly understood. Here, by combining experimental and computational approaches, we systematically identified box C/D snoRNAs in four fission yeasts and found that the expression of box C/D snoRNA and ribosomal protein (RP) genes were orchestrated by a common Homol-D box, thereby ensuring a constant balance of these two genetic components. Interestingly, such transcriptional coregulations could be observed in most Ascomycota species and were mediated by different cis-regulatory elements. Via the reservation of cis elements, changes in spatial configuration, the substitution of cis elements, and gain or loss of cis elements, the regulatory networks of box C/D snoRNAs evolved to correspond with those of the RP genes, maintaining transcriptional coregulation between box C/D snoRNAs and RP genes. Our results indicate that coregulation via common cis elements is an important mechanism to coordinate expression of the RP and snoRNA genes, which ensures a constant balance of these two components. PMID:25002674

  19. Ribosome-inactivating proteins

    PubMed Central

    Walsh, Matthew J; Dodd, Jennifer E; Hautbergue, Guillaume M

    2013-01-01

    Ribosome-inactivating proteins (RIPs) were first isolated over a century ago and have been shown to be catalytic toxins that irreversibly inactivate protein synthesis. Elucidation of atomic structures and molecular mechanism has revealed these proteins to be a diverse group subdivided into two classes. RIPs have been shown to exhibit RNA N-glycosidase activity and depurinate the 28S rRNA of the eukaryotic 60S ribosomal subunit. In this review, we compare archetypal RIP family members with other potent toxins that abolish protein synthesis: the fungal ribotoxins which directly cleave the 28S rRNA and the newly discovered Burkholderia lethal factor 1 (BLF1). BLF1 presents additional challenges to the current classification system since, like the ribotoxins, it does not possess RNA N-glycosidase activity but does irreversibly inactivate ribosomes. We further discuss whether the RIP classification should be broadened to include toxins achieving irreversible ribosome inactivation with similar turnovers to RIPs, but through different enzymatic mechanisms. PMID:24071927

  20. Ribosome traffic on mRNAs maps to gene ontology: genome-wide quantification of translation initiation rates and polysome size regulation.

    PubMed

    Ciandrini, Luca; Stansfield, Ian; Romano, M Carmen

    2013-01-01

    To understand the complex relationship governing transcript abundance and the level of the encoded protein, we integrate genome-wide experimental data of ribosomal density on mRNAs with a novel stochastic model describing ribosome traffic dynamics during translation elongation. This analysis reveals that codon arrangement, rather than simply codon bias, has a key role in determining translational efficiency. It also reveals that translation output is governed both by initiation efficiency and elongation dynamics. By integrating genome-wide experimental data sets with simulation of ribosome traffic on all Saccharomyces cerevisiae ORFs, mRNA-specific translation initiation rates are for the first time estimated across the entire transcriptome. Our analysis identifies different classes of mRNAs characterised by their initiation rates, their ribosome traffic dynamics, and by their response to ribosome availability. Strikingly, this classification based on translational dynamics maps onto key gene ontological classifications, revealing evolutionary optimisation of translation responses to be strongly influenced by gene function. PMID:23382661

  1. Ribosome Traffic on mRNAs Maps to Gene Ontology: Genome-wide Quantification of Translation Initiation Rates and Polysome Size Regulation

    PubMed Central

    Ciandrini, Luca

    2013-01-01

    To understand the complex relationship governing transcript abundance and the level of the encoded protein, we integrate genome-wide experimental data of ribosomal density on mRNAs with a novel stochastic model describing ribosome traffic dynamics during translation elongation. This analysis reveals that codon arrangement, rather than simply codon bias, has a key role in determining translational efficiency. It also reveals that translation output is governed both by initiation efficiency and elongation dynamics. By integrating genome-wide experimental data sets with simulation of ribosome traffic on all Saccharomyces cerevisiae ORFs, mRNA-specific translation initiation rates are for the first time estimated across the entire transcriptome. Our analysis identifies different classes of mRNAs characterised by their initiation rates, their ribosome traffic dynamics, and by their response to ribosome availability. Strikingly, this classification based on translational dynamics maps onto key gene ontological classifications, revealing evolutionary optimisation of translation responses to be strongly influenced by gene function. PMID:23382661

  2. End-targeting proteomics of isolated chromatin segments of a mammalian ribosomal RNA gene promoter

    PubMed Central

    Ide, Satoru; Dejardin, Jerome

    2015-01-01

    The unbiased identification of proteins associated with specific loci is crucial for understanding chromatin-based processes. The proteomics of isolated chromatin fragment (PICh) method has previously been developed to purify telomeres and identify associated proteins. This approach is based on the affinity capture of endogenous chromatin segments by hybridization with oligonucleotide containing locked nucleic acids. However, PICh is only efficient with highly abundant genomic targets, limiting its applicability. Here we develop an approach for identifying factors bound to the promoter region of the ribosomal RNA genes that we call end-targeting PICh (ePICh). Using ePICh, we could specifically enrich the RNA polymerase I pre-initiation complex, including the selectivity factor 1. The high purity of the ePICh material allowed the identification of ZFP106, a novel factor regulating transcription initiation by targeting RNA polymerase I to the promoter. Our results demonstrate that ePICh can uncover novel proteins controlling endogenous regulatory elements in mammals. PMID:25812914

  3. End-targeting proteomics of isolated chromatin segments of a mammalian ribosomal RNA gene promoter.

    PubMed

    Ide, Satoru; Dejardin, Jerome

    2015-01-01

    The unbiased identification of proteins associated with specific loci is crucial for understanding chromatin-based processes. The proteomics of isolated chromatin fragment (PICh) method has previously been developed to purify telomeres and identify associated proteins. This approach is based on the affinity capture of endogenous chromatin segments by hybridization with oligonucleotide containing locked nucleic acids. However, PICh is only efficient with highly abundant genomic targets, limiting its applicability. Here we develop an approach for identifying factors bound to the promoter region of the ribosomal RNA genes that we call end-targeting PICh (ePICh). Using ePICh, we could specifically enrich the RNA polymerase I pre-initiation complex, including the selectivity factor 1. The high purity of the ePICh material allowed the identification of ZFP106, a novel factor regulating transcription initiation by targeting RNA polymerase I to the promoter. Our results demonstrate that ePICh can uncover novel proteins controlling endogenous regulatory elements in mammals. PMID:25812914

  4. Nucleotide excision repair of the 5 S ribosomal RNA gene assembled into a nucleosome.

    PubMed

    Liu, X; Smerdon, M J

    2000-08-01

    A-175-base pair fragment containing the Xenopus borealis somatic 5 S ribosomal RNA gene was used as a model system to determine the effect of nucleosome assembly on nucleotide excision repair (NER) of the major UV photoproduct (cyclobutane pyrimidine dimer (CPD)) in DNA. Xenopus oocyte nuclear extracts were used to carry out repair in vitro on reconstituted, positioned 5 S rDNA nucleosomes. Nucleosome structure strongly inhibits NER at many CPD sites in the 5 S rDNA fragment while having little effect at a few sites. The time course of CPD removal at 35 different sites indicates that >85% of the CPDs in the naked DNA fragment have t(12) values <2 h, whereas <26% of the t(12) values in nucleosomes are <2 h, and 15% are >8 h. Moreover, removal of histone tails from these mononucleosomes has little effect on the repair rates. Finally, nucleosome inhibition of repair shows no correlation with the rotational setting of a 14-nucleotide-long pyrimidine tract located 30 base pairs from the nucleosome dyad. These results suggest that inhibition of NER by mononucleosomes is not significantly influenced by the rotational orientation of CPDs on the histone surface, and histone tails play little (or no) role in this inhibition. PMID:10821833

  5. Direct TFIIA-TFIID protein contacts drive budding yeast ribosomal protein gene transcription.

    PubMed

    Layer, Justin H; Weil, P Anthony

    2013-08-01

    We have previously shown that yeast TFIID provides coactivator function on the promoters of ribosomal protein-encoding genes (RPGs) by making direct contact with the transactivator repressor activator protein 1 (Rap1). Further, our structural studies of assemblies generated with purified Rap1, TFIID, and TFIIA on RPG enhancer-promoter DNA indicate that Rap1-TFIID interaction induces dramatic conformational rearrangements of enhancer-promoter DNA and TFIID-bound TFIIA. These data indicate a previously unknown yet critical role for yeast TFIIA in the integration of activator-TFIID contacts with promoter conformation and downstream preinitiation complex formation and/or function. Here we describe the use of systematic mutagenesis to define how specific TFIIA contacts contribute to these processes. We have verified that TFIIA is required for RPG transcription in vivo and in vitro, consistent with the existence of a critical Rap1-TFIIA-TFIID interaction network. We also identified essential points of contact for TFIIA and Rap1 within the Rap1 binding domain of the Taf4 subunit of TFIID. These data suggest a mechanism for how interactions between TFIID, TFIIA, and Rap1 contribute to the high rate of transcription initiation seen on RPGs in vivo. PMID:23814059

  6. Phylogeny of chloromonas (chlorophyceae): A study of 18S ribosomal RNA gene sequences

    SciTech Connect

    Buchheim, M.A.; Buchheim, J.A.; Chapman, R.L.

    1997-04-01

    The unicellular, biflagellate genus Chloromonas differs from its ally, Chlamydomonas, primarily by the absence of pyrenoids in the vegetative stage of the former. As with most green flagellate genera, little is known about phylogenetic affinities within and among Chloromonas species. Phylogenetic analyses of nuclear-encoded small-subunit ribosomal RNA gene sequences demonstrate that a sampling of five Chloromonas taxa, obtained from major culture collections, do not form a monophyletic group. However, only three of these isolates, Chloromonas clathrata, Chloromonas serbinowi, and Chloromonas rosae, are diagnosable morphologically as Chloromonas species by the absence of a pyrenoid in the vegetative stage. The three diagnosable Chloromonas taxa form an alliance with two pyrenoid-bearing chlamydomonads, Chlamydomonas augustae and Chlamydomonas macrostellata. With the exception of Chloromonas serbinowi, which represents the basal lineage within the clade, each of the diagnosable Chloromonas taxa and their pyrenoid-bearing Chlamydomonas allies were isolated originally from mountain soils, snow, or cold peat. These observations suggest that hibitat, independent of pyrenoid status, may be most closely linked to the natural history of this clade of chlamydomonad flagellates. 51 refs., 3 figs., 3 tabs.

  7. An update to polyketide synthase and non-ribosomal synthetase genes and nomenclature in Fusarium.

    PubMed

    Hansen, Frederik T; Gardiner, Donald M; Lysøe, Erik; Fuertes, Patricia Romans; Tudzynski, Bettina; Wiemann, Philipp; Sondergaard, Teis Esben; Giese, Henriette; Brodersen, Ditlev E; Sørensen, Jens Laurids

    2015-02-01

    Members of the genus Fusarium produce a plethora of bioactive secondary metabolites, which can be harmful to humans and animals or have potential in drug development. In this study we have performed comparative analyses of polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) from ten different Fusarium species including F. graminearum (two strains), F. verticillioides, F. solani, F. culmorum, F. pseudograminearum, F. fujikuroi, F. acuminatum, F. avenaceum, F. equiseti, and F. oxysporum (12 strains). This led to identification of 52 NRPS and 52 PKSs orthology groups, respectively, and although not all PKSs and NRPSs are assumed to be intact or functional, the analyses illustrate the huge secondary metabolite potential in Fusarium. In our analyses we identified a core collection of eight NRPSs (NRPS2-4, 6, 10-13) and two PKSs (PKS3 and PKS7) that are conserved in all strains analyzed in this study. The identified PKSs and NRPSs were named based on a previously developed classification system (www.FusariumNRPSPKS.dk). We suggest this system be used when PKSs and NRPSs have to be classified in future sequenced Fusarium strains. This system will facilitate identification of orthologous and non-orthologous NRPSs and PKSs from newly sequenced Fusarium genomes and will aid the scientific community by providing a common nomenclature for these two groups of genes/enzymes. PMID:25543026

  8. Comparative cytogenetics in four species of Palinuridae: B chromosomes, ribosomal genes and telomeric sequences.

    PubMed

    Salvadori, Susanna; Coluccia, Elisabetta; Deidda, Federica; Cau, Angelo; Cannas, Rita; Deiana, Anna Maria

    2012-12-01

    The evolutionary pathway of Palinuridae (Crustacea, Decapoda) is still controversial, uncertain and unexplored, expecially from a karyological point of view. Here we describe the South African spiny lobster Jasus lalandii karyotype: n and 2n values, heterochromatin distribution, nucleolar organizer region (NOR) location and telomeric repeat structure and location. To compare the genomic and chromosomal organization in Palinuridae we located NORs in Panulirus regius, Palinurus gilchristi and Palinurus mauritanicus: all species showed multiple NORs. In J. lalandii NORs were located on three chromosome pairs, with interindividual polymorphism. In P. regius and in the two Palinurus species NORs were located on two chromosome pairs. In the two last species 45S ribosomal gene loci were also found on B chromosomes. In addition, the nature and location of telomeric repeats were investigated by FISH in J. lalandii, P. gilchristi, P. mauritanicus Palinurus elephas, and P. regius (Palinuridae, Achelata), and in Scyllarus arctus (Scyllaridae, Achelata): all these Achelata species showed the (TTAGG)n pentameric repeats. Furthermore, in J. lalandii these repeats occurred in all the telomeres and in some interstitial chromosomal sites, associated with NORs. PMID:23224906

  9. Primary structure of dihydrofolate reductase and mitochondrial ribosomal protein L36 genes from the basidiomycete Coprinus cinereus.

    PubMed

    Aimi, Tadanori; Fukuhara, Shoji; Ishiguro, Maki; Kitamoto, Yutaka; Morinaga, Tsutomu

    2004-08-01

    We amplified and sequenced the dihydrofolate reductase (DHFR) gene of the basidiomycete Coprinus cinereus. Downstream of the DHFR coding region, a mitochondrial (mt) ribosomal protein L36 (RPL36) gene was discovered in the opposite orientation to DHFR gene. Putative polyadenylation signals of the two genes overlapped, both containing the 8-bp palindrome 5'-aatatatt-3'. The finding that C. cinereus DHFR gene is closely clustered with a mt protein gene strongly suggests that C. cinereus DHFR is closely related to mt function and evolution. The amino acid sequence of C. cinereus DHFR is most homologous to eukaryotic proteins such as Cryptococcus neoformans and Pneumocystis carinii DHFRs. However, the sequence of C. cinereus mt RPL36 closely resembles RPL36 of bacteria and cyanobacteria such as Synechocystis sp. and Escherichia coli. This result strongly supports the serial endosymbiotic theory of the development of ancestral eukaryotes, and suggests that C. cinereus mt RPL36 gene originated from the ancestral eubacterial genome. PMID:15620217

  10. The RICE MINUTE-LIKE1 (RML1) gene, encoding a ribosomal large subunit protein L3B, regulates leaf morphology and plant architecture in rice

    PubMed Central

    Zheng, Ming; Wang, Yihua; Liu, Xi; Sun, Juan; Wang, Yunlong; Xu, Yang; Lv, Jia; Long, Wuhua; Zhu, Xiaopin; Guo, Xiuping; Jiang, Ling; Wang, Chunming; Wan, Jianmin

    2016-01-01

    Mutations of ribosomal proteins (RPs) are known to cause developmental abnormalities in yeast, mammals, and dicotyledonous plants; however, their effects have not been studied in rice. Here, we identifiy a ribosomal biogenesis mutant, rice minute-like1 (rml1) that displays a minute phenotype as evidenced by retarded growth and defects in the vascular system. We determine that RML1 encodes a ribosome large subunit protein 3B (RPL3B) in rice by means of map-based cloning and genetic complementation. RPL3B is abundantly expressed in all the tissues, whereas RPL3A, another RPL3 gene family member, is expressed at low levels. Notably, the expression level of RPL3A in the rml1 mutant is similar to that in the wild-type, suggesting that RPL3A provides no functional compensation for RPL3B in rml1 plants. Ribosomal profiles show that mutation of RPL3B leads to a significant reduction in free 60S ribosomal subunits and polysomes, indicating a ribosomal insufficiency in the rml1 mutant. Our results demonstrate that the ribosomal protein gene RPL3B is required for maintaining normal leaf morphology and plant architecture in rice through its regulation of ribosome biogenesis. PMID:27241493

  11. The RICE MINUTE-LIKE1 (RML1) gene, encoding a ribosomal large subunit protein L3B, regulates leaf morphology and plant architecture in rice.

    PubMed

    Zheng, Ming; Wang, Yihua; Liu, Xi; Sun, Juan; Wang, Yunlong; Xu, Yang; Lv, Jia; Long, Wuhua; Zhu, Xiaopin; Guo, Xiuping; Jiang, Ling; Wang, Chunming; Wan, Jianmin

    2016-05-01

    Mutations of ribosomal proteins (RPs) are known to cause developmental abnormalities in yeast, mammals, and dicotyledonous plants; however, their effects have not been studied in rice. Here, we identifiy a ribosomal biogenesis mutant, rice minute-like1 (rml1) that displays a minute phenotype as evidenced by retarded growth and defects in the vascular system. We determine that RML1 encodes a ribosome large subunit protein 3B (RPL3B) in rice by means of map-based cloning and genetic complementation. RPL3B is abundantly expressed in all the tissues, whereas RPL3A, another RPL3 gene family member, is expressed at low levels. Notably, the expression level of RPL3A in the rml1 mutant is similar to that in the wild-type, suggesting that RPL3A provides no functional compensation for RPL3B in rml1 plants. Ribosomal profiles show that mutation of RPL3B leads to a significant reduction in free 60S ribosomal subunits and polysomes, indicating a ribosomal insufficiency in the rml1 mutant. Our results demonstrate that the ribosomal protein gene RPL3B is required for maintaining normal leaf morphology and plant architecture in rice through its regulation of ribosome biogenesis. PMID:27241493

  12. Dysfunction in Ribosomal Gene Expression in the Hypothalamus and Hippocampus following Chronic Social Defeat Stress in Male Mice as Revealed by RNA-Seq

    PubMed Central

    Smagin, Dmitry A.; Kovalenko, Irina L.; Galyamina, Anna G.; Bragin, Anatoly O.; Orlov, Yuriy L.; Kudryavtseva, Natalia N.

    2016-01-01

    Chronic social defeat stress leads to the development of anxiety- and depression-like states in male mice and is accompanied by numerous molecular changes in brain. The influence of 21-day period of social stress on ribosomal gene expression in five brain regions was studied using the RNA-Seq database. Most Rps, Rpl, Mprs, and Mprl genes were upregulated in the hypothalamus and downregulated in the hippocampus, which may indicate ribosomal dysfunction following chronic social defeat stress. There were no differentially expressed ribosomal genes in the ventral tegmental area, midbrain raphe nuclei, or striatum. This approach may be used to identify a pharmacological treatment of ribosome biogenesis abnormalities in the brain of patients with “ribosomopathies.” PMID:26839715

  13. Dysfunction in Ribosomal Gene Expression in the Hypothalamus and Hippocampus following Chronic Social Defeat Stress in Male Mice as Revealed by RNA-Seq.

    PubMed

    Smagin, Dmitry A; Kovalenko, Irina L; Galyamina, Anna G; Bragin, Anatoly O; Orlov, Yuriy L; Kudryavtseva, Natalia N

    2016-01-01

    Chronic social defeat stress leads to the development of anxiety- and depression-like states in male mice and is accompanied by numerous molecular changes in brain. The influence of 21-day period of social stress on ribosomal gene expression in five brain regions was studied using the RNA-Seq database. Most Rps, Rpl, Mprs, and Mprl genes were upregulated in the hypothalamus and downregulated in the hippocampus, which may indicate ribosomal dysfunction following chronic social defeat stress. There were no differentially expressed ribosomal genes in the ventral tegmental area, midbrain raphe nuclei, or striatum. This approach may be used to identify a pharmacological treatment of ribosome biogenesis abnormalities in the brain of patients with "ribosomopathies." PMID:26839715

  14. Human C4orf14 interacts with the mitochondrial nucleoid and is involved in the biogenesis of the small mitochondrial ribosomal subunit

    PubMed Central

    He, J.; Cooper, H. M.; Reyes, A.; Di Re, M.; Kazak, L.; Wood, S. R.; Mao, C. C.; Fearnley, I. M.; Walker, J. E.; Holt, I. J.

    2012-01-01

    The bacterial homologue of C4orf14, YqeH, has been linked to assembly of the small ribosomal subunit. Here, recombinant C4orf14 isolated from human cells, co-purified with the small, 28S subunit of the mitochondrial ribosome and the endogenous protein co-fractionated with the 28S subunit in sucrose gradients. Gene silencing of C4orf14 specifically affected components of the small subunit, leading to decreased protein synthesis in the organelle. The GTPase of C4orf14 was critical to its interaction with the 28S subunit, as was GTP. Therefore, we propose that C4orf14, with bound GTP, binds to components of the 28S subunit facilitating its assembly, and GTP hydrolysis acts as the release mechanism. C4orf14 was also found to be associated with human mitochondrial nucleoids, and C4orf14 gene silencing caused mitochondrial DNA depletion. In vitro C4orf14 is capable of binding to DNA. The association of C4orf14 with mitochondrial translation factors and the mitochondrial nucleoid suggests that the 28S subunit is assembled at the mitochondrial nucleoid, enabling the direct transfer of messenger RNA from the nucleoid to the ribosome in the organelle. PMID:22447445

  15. Cinnamomin: a multifunctional type II ribosome-inactivating protein.

    PubMed

    He, Wen-Jun; Liu, Wang-Yi

    2003-07-01

    Plant ribosome-inactivating proteins (RIPs) are a group of toxic proteins that can irreversibly inactivate ribosomes by specifically removing the conserved adenine base from the "Sarcin/Ricin domain" of the 28S RNA in ribosome. Cinnamomin is a novel type II RIP isolated in our laboratory from the mature seeds of camphor tree. Besides site-specific deadenylation of the A4324 in the Sarcin/Ricin domain of rat ribosome, this protein could also release the adenine base from DNA molecules at multiple sites and from AMP, ADP, dAMP and adenosine. Furthermore, cinnamomin displays cytotoxicity to carcinoma cells and insect larvae by modifying their ribosomal RNA. These functions possessed by cinnamomin shed a new light on the possible application of cinnamomin in the field of immunotoxin design and transgenic reagents. In this review, we introduce the major recent results on cinnamomin obtained in our laboratory, including purification of this protein, characterization of its enzymatic mechanism, structure and function, gene pattern, physiological role and its biological implications in cytotoxicity. PMID:12672471

  16. High-Resolution Analysis of Coronavirus Gene Expression by RNA Sequencing and Ribosome Profiling

    PubMed Central

    Jones, Joshua D.; Chung, Betty Y.-W.; Siddell, Stuart G.; Brierley, Ian

    2016-01-01

    Members of the family Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV), are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global “snap-shot” of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59), a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the ribosomal

  17. High-Resolution Analysis of Coronavirus Gene Expression by RNA Sequencing and Ribosome Profiling.

    PubMed

    Irigoyen, Nerea; Firth, Andrew E; Jones, Joshua D; Chung, Betty Y-W; Siddell, Stuart G; Brierley, Ian

    2016-02-01

    Members of the family Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV), are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global "snap-shot" of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59), a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the ribosomal

  18. Characterization of the ribosomal RNA gene of Kudoa neothunni (Myxosporea: Multivalvulida) in tunas (Thunnus spp.) and Kudoa scomberi n. sp. in a chub mackerel (Scomber japonicus).

    PubMed

    Li, Ying-Chun; Sato, Hiroshi; Tanaka, Shuhei; Ohnishi, Takahiro; Kamata, Yoichi; Sugita-Konishi, Yoshiko

    2013-05-01

    Kudoa neothunni is the first described Kudoa species having six shell valves and polar capsules, previously assigned to the genus Hexacapsula Arai and Matsumoto, 1953. Since its genetic analyses remain to be conducted, the present study characterizes the ribosomal RNA gene (rDNA) using two isolates from a yellowfin tuna (Thunnus albacares) with post-harvest myoliquefaction and a northern bluefin tuna (Thunnus thynnus) without tissue degradation. Spores of the two isolates localized in the myofiber of trunk muscles, forming pseudocysts, and showed typical morphology of K. neothunni with six equal-sized shell valves radially arranged in apical view: spores (n = 15) measuring 9.5-11.4 μm in width, 7.3-8.6 μm in suture width, 8.9-10.9 μm in thickness, and 7.3-7.7 μm in length; and polar capsules measuring 3.6-4.1 μm by 1.8-2.3 μm. In lateral view, the spores were pyramidal in shape without apical protrusions. Their 18S and 5.8S rDNA sequences were essentially identical, but variations in the ITS1 (62.4 % similarity across 757-bp length), ITS2 (66.9 % similarity across 599-bp length), and 28S (99.0 % similarity across 2,245-bp length) rDNA regions existed between the two isolates. On phylogenetic trees based on the 18S or 28S rDNA sequence, K. neothunni formed a clade with Kudoa spp. with more than four shell valves and polar capsules, particularly K. grammatorcyni and K. scomberomori. Semiquadrate spores of a kudoid species with four shell valves and polar capsules were detected from minute cysts (0.30-0.75 mm by 0.20-0.40 mm) embedded in the trunk muscle of a chub mackerel (Scomber japonicus) fished in the Sea of Japan. Morphologically, it resembled K. caudata described from a chub mackerel fished in the southeastern Pacific Ocean off Peru; however, it lacked filamentous projections on the shell valves of spores. Additionally, it morphologically resembled K. thunni described from a yellowfin tuna also fished in the Pacific Ocean; spores (n

  19. Evidence for ribosomal frameshifting and a novel overlapping gene in the genomes of insect-specific flaviviruses

    SciTech Connect

    Firth, Andrew E.; Blitvich, Bradley J.; Wills, Norma M.; Miller, Cathy L.; Atkins, John F.

    2010-03-30

    Flaviviruses have a positive-sense, single-stranded RNA genome of approx11 kb, encoding a large polyprotein that is cleaved to produce approx10 mature proteins. Cell fusing agent virus, Kamiti River virus, Culex flavivirus and several recently discovered flaviviruses have no known vertebrate host and apparently infect only insects. We present compelling bioinformatic evidence for a 253-295 codon overlapping gene (designated fifo) conserved throughout these insect-specific flaviviruses and immunofluorescent detection of its product. Fifo overlaps the NS2A/NS2B coding sequence in the - 1/+ 2 reading frame and is most likely expressed as a trans-frame fusion protein via ribosomal frameshifting at a conserved GGAUUUY slippery heptanucleotide with 3'-adjacent RNA secondary structure (which stimulates efficient frameshifting in vitro). The discovery bears striking parallels to the recently discovered ribosomal frameshifting site in the NS2A coding sequence of the Japanese encephalitis serogroup of flaviviruses and suggests that programmed ribosomal frameshifting may be more widespread in flaviviruses than currently realized.

  20. Helianthus annuus ssp. texanus has chloroplast DNA and nuclear ribosomal RNA genes of Helianthus debilis ssp. cucumerifolius.

    PubMed Central

    Rieseberg, L H; Beckstrom-Sternberg, S; Doan, K

    1990-01-01

    Heiser [Heiser, C. B. (1951) Evolution 5, 42-51] hypothesized that Helianthus annuus ssp. texanus was derived by the introduction of H. annuus into Texas and subsequent introgression of genes from Helianthus debilis ssp. cucumerifolius into H. annuus. Although often considered to be one of the best cases of introgression in plants, alternative hypotheses to introgression, such as convergence or the joint retention of the ancestral condition, could not be ruled out in the original study. To test for the occurrence of introgression we examined 14 populations of H. annuus ssp. texanus, 14 allopatric populations of H. annuus, and three populations of H. debilis ssp. cucumerifolius with reference to diagnostic chloroplast DNA and nuclear ribosomal DNA markers. Thirteen of the 14 populations of H. annuus ssp. texanus had chloroplast DNA and/or ribosomal DNA markers of H. debilis ssp. cucumerifolius. In contrast, no chloroplast DNA or ribosomal DNA markers of H. debilis ssp. cucumerifolius were found in the 14 allopatric populations of H. annuus. Our findings provide strong support, therefore, for the hypothesized introgressive origin of H. annuus ssp. texanus. PMID:11607056

  1. Structure and expression of the nuclear gene coding for the chloroplast ribosomal protein L21: developmental regulation of a housekeeping gene by alternative promoters.

    PubMed Central

    Lagrange, T; Franzetti, B; Axelos, M; Mache, R; Lerbs-Mache, S

    1993-01-01

    We have cloned and sequenced the nuclear gene of the chloroplast ribosomal protein L21 (rpl21) of Spinacia oleracea. The gene consists of five exons and four introns. All introns are located in the sequence which corresponds to the Escherichia coli-like central core of the protein. L21 mRNA is present in photosynthetic (leaves) and nonphotosynthetic (roots and seeds) plant organs, although large quantitative differences exist. Primer extension and S1 nuclease mapping experiments revealed the existence of two types of transcripts in leaves. The two corresponding start sites were defined as P1 and P2. In roots and seeds, we found only the shorter of the two transcripts (initiated at P2). The nucleotide sequence surrounding P2 resembles promoters for housekeeping and vertebrate r-protein genes. Analysis of several promoter constructions by transient expression confirmed that both transcripts originate from transcription initiation. Results are interpreted to mean that the expression of the rpl21 gene is regulated by alternative promoters. One of the promoters (P2) is constitutive, and the other one (P1) is specifically induced in leaves, i.e., its activation should be related to the transformation of amyloplasts or proplastids to chloroplasts. The gene thus represents the first example of a housekeeping gene which is regulated by the organ-specific usage of alternative promoters. Primer extension analysis and S1 nuclease mapping of another nucleus-encoded chloroplast ribosomal protein gene (rps1) give evidence that the same type of regulation by two-promoter usage might be a more general phenomenon of plant chloroplast-related ribosomal protein genes. Preliminary results indicate that presence of conserved sequences within the rpl21 and rps1 promoter regions which compete for the same DNA binding activities. Images PMID:8455634

  2. Ribotoxic stress response: activation of the stress-activated protein kinase JNK1 by inhibitors of the peptidyl transferase reaction and by sequence-specific RNA damage to the alpha-sarcin/ricin loop in the 28S rRNA.

    PubMed Central

    Iordanov, M S; Pribnow, D; Magun, J L; Dinh, T H; Pearson, J A; Chen, S L; Magun, B E

    1997-01-01

    Inhibition of protein synthesis per se does not potentiate the stress-activated protein kinases (SAPKs; also known as cJun NH2-terminal kinases [JNKs]). The protein synthesis inhibitor anisomycin, however, is a potent activator of SAPKs/JNKs. The mechanism of this activation is unknown. We provide evidence that in order to activate SAPK/JNK1, anisomycin requires ribosomes that are translationally active at the time of contact with the drug, suggesting a ribosomal origin of the anisomycin-induced signaling to SAPK/JNK1. In support of this notion, we have found that aminohexose pyrimidine nucleoside antibiotics, which bind to the same region in the 28S rRNA that is the target site for anisomycin, are also potent activators of SAPK/JNK1. Binding of an antibiotic to the 28S rRNA interferes with the functioning of the molecule by altering the structural interactions of critical regions. We hypothesized, therefore, that such alterations in the 28S rRNA may act as recognition signals to activate SAPK/JNK1. To test this hypothesis, we made use of two ribotoxic enzymes, ricin A chain and alpha-sarcin, both of which catalyze sequence-specific RNA damage in the 28S rRNA. Consistent with our hypothesis, ricin A chain and alpha-sarcin were strong agonists of SAPK/JNK1 and of its activator SEK1/MKK4 and induced the expression of the immediate-early genes c-fos and c-jun. As in the case of anisomycin, ribosomes that were active at the time of exposure to ricin A chain or alpha-sarcin were able to initiate signal transduction from the damaged 28S rRNA to SAPK/JNK1 while inactive ribosomes were not. PMID:9154836

  3. Identification of group-I introns in the 28s rDNA of the entomopathogenic fungus Beauveria brongniartii.

    PubMed

    Neuvéglise, C; Brygoo, Y

    1994-12-01

    The length of the 28s ribosomal DNA differs significantly between two strains (Bt102 and Bt114) of the entomopathogenic fungus Beauveria brongniartii. RFLP analysis on PCR products revealed the presence of three insertional elements of 350-450 bp in strain Bt114. One of the insertions has been cloned and sequenced and shown to possess all the characteristic sequences and secondary structures of a group-IC intron. Its length is 428 bp and it is devoid of any long open reading frame. The distribution of this intron elsewhere in the genome of Bt114, as well as in the chromosomal ribosomal DNA, was studied. It seems to be present as seven copies in different genes not corresponding to the mitochondrial DNA. The presence of the intron in other strains of B. brongniartii was examined by the hybridization method. Some of them seemed to possess introns with a similar core although others presented no homology with the cloned fragment. PMID:7750145

  4. A pathogenic non-coding RNA induces changes in dynamic DNA methylation of ribosomal RNA genes in host plants.

    PubMed

    Martinez, German; Castellano, Mayte; Tortosa, Maria; Pallas, Vicente; Gomez, Gustavo

    2014-02-01

    Viroids are plant-pathogenic non-coding RNAs able to interfere with as yet poorly known host-regulatory pathways and to cause alterations recognized as diseases. The way in which these RNAs coerce the host to express symptoms remains to be totally deciphered. In recent years, diverse studies have proposed a close interplay between viroid-induced pathogenesis and RNA silencing, supporting the belief that viroid-derived small RNAs mediate the post-transcriptional cleavage of endogenous mRNAs by acting as elicitors of symptoms expression. Although the evidence supporting the role of viroid-derived small RNAs in pathogenesis is robust, the possibility that this phenomenon can be a more complex process, also involving viroid-induced alterations in plant gene expression at transcriptional levels, has been considered. Here we show that plants infected with the 'Hop stunt viroid' accumulate high levels of sRNAs derived from ribosomal transcripts. This effect was correlated with an increase in the transcription of ribosomal RNA (rRNA) precursors during infection. We observed that the transcriptional reactivation of rRNA genes correlates with a modification of DNA methylation in their promoter region and revealed that some rRNA genes are demethylated and transcriptionally reactivated during infection. This study reports a previously unknown mechanism associated with viroid (or any other pathogenic RNA) infection in plants providing new insights into aspects of host alterations induced by the viroid infectious cycle. PMID:24178032

  5. Adjacent Gene Pairing Plays a Role in the Coordinated Expression of Ribosome Biogenesis Genes MPP10 and YJR003C in Saccharomyces cerevisiae ▿

    PubMed Central

    Arnone, James T.; McAlear, Michael A.

    2011-01-01

    The rRNA and ribosome biogenesis (RRB) regulon from Saccharomyces cerevisiae contains some 200 genes, the expression of which is tightly regulated under changing cellular conditions. RRB gene promoters are enriched for the RRPE and PAC consensus motifs, and a significant fraction of RRB genes are found as adjacent gene pairs. A genetic analysis of the MPP10 promoter revealed that both the RRPE and PAC motifs are important for coordinated expression of MPP10 following heat shock, osmotic stress, and glucose replenishment. The association of the RRPE binding factor Stb3 with the MPP10 promoter was found to increase after glucose replenishment and to decrease following heat shock. Similarly, bulk histone H3 clearing and histone H4K12 acetylation levels at the MPP10 promoter were found to increase or decrease following glucose replenishment or heat shock, respectively. Interestingly, substitutions in the PAC and RRPE sequences at the MPP10 promoter were also found to impact the regulated expression of the adjacent RRB gene YJR003, whose promoter lies in the opposite orientation and some 3.8 kb away. Furthermore, the regulated expression of YJR003C could be disrupted by inserting a reporter cassette that increased its distance from MPP10. Given that a high incidence of gene pairing was also found within the ribosomal protein (RP) and RRB regulons across different yeast species, our results indicate that immediately adjacent positioning of genes can be functionally significant for their coregulated expression. PMID:21115740

  6. An internal ribosome entry site (IRES) mutant library for tuning expression level of multiple genes in mammalian cells.

    PubMed

    Koh, Esther Y C; Ho, Steven C L; Mariati; Song, Zhiwei; Bi, Xuezhi; Bardor, Muriel; Yang, Yuansheng

    2013-01-01

    A set of mutated Encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) elements with varying strengths is generated by mutating the translation initiation codons of 10(th), 11(th), and 12(th) AUG to non-AUG triplets. They are able to control the relative expression of multiple genes over a wide range in mammalian cells in both transient and stable transfections. The relative strength of each IRES mutant remains similar in different mammalian cell lines and is not gene specific. The expressed proteins have correct molecular weights. Optimization of light chain over heavy chain expression by these IRES mutants enhances monoclonal antibody expression level and quality in stable transfections. Uses of this set of IRES mutants can be extended to other applications such as synthetic biology, investigating interactions between proteins and its complexes, cell engineering, multi-subunit protein production, gene therapy, and reprogramming of somatic cells into stem cells. PMID:24349195

  7. An Internal Ribosome Entry Site (IRES) Mutant Library for Tuning Expression Level of Multiple Genes in Mammalian Cells

    PubMed Central

    Koh, Esther Y. C.; Ho, Steven C. L.; Mariati; Song, Zhiwei; Bi, Xuezhi; Bardor, Muriel; Yang, Yuansheng

    2013-01-01

    A set of mutated Encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) elements with varying strengths is generated by mutating the translation initiation codons of 10th, 11th, and 12th AUG to non-AUG triplets. They are able to control the relative expression of multiple genes over a wide range in mammalian cells in both transient and stable transfections. The relative strength of each IRES mutant remains similar in different mammalian cell lines and is not gene specific. The expressed proteins have correct molecular weights. Optimization of light chain over heavy chain expression by these IRES mutants enhances monoclonal antibody expression level and quality in stable transfections. Uses of this set of IRES mutants can be extended to other applications such as synthetic biology, investigating interactions between proteins and its complexes, cell engineering, multi-subunit protein production, gene therapy, and reprogramming of somatic cells into stem cells. PMID:24349195

  8. The complete nucleotide sequence of a 16S ribosomal RNA gene from a blue-green alga, Anacystis nidulans.

    PubMed

    Tomioka, N; Sugiura, M

    1983-01-01

    The complete nucleotide sequence of a 16S ribosomal RNA gene from a blue-green alga, Anacystis nidulans, has been determined. Its coding region is estimated to be 1,487 base pairs long, which is nearly identical to those reported for chloroplast 16S rRNA genes and is about 4% shorter than that of the Escherichia coli gene. The 16S rRNA sequence of A. nidulans has 83% homology with that of tobacco chloroplast and 74% homology with that of E. coli. Possible stem and loop structures of A. nidulans 16S rRNA sequences resemble more closely those of chloroplast 16S rRNAs than those of E. coli 16S rRNA. These observations support the endosymbiotic theory of chloroplast origin. PMID:6412038

  9. siRNA Knockdown of Ribosomal Protein Gene RPL19 Abrogates the Aggressive Phenotype of Human Prostate Cancer

    PubMed Central

    Bee, Alix; Brewer, Daniel; Beesley, Carol; Dodson, Andrew; Forootan, Shiva; Dickinson, Timothy; Gerard, Patricia; Lane, Brian; Yao, Sheng; Cooper, Colin S.; Djamgoz, Mustafa B. A.; Gosden, Christine M.; Ke, Youqiang; Foster, Christopher S.

    2011-01-01

    We provide novel functional data that posttranscriptional silencing of gene RPL19 using RNAi not only abrogates the malignant phenotype of PC-3M prostate cancer cells but is selective with respect to transcription and translation of other genes. Reducing RPL19 transcription modulates a subset of genes, evidenced by gene expression array analysis and Western blotting, but does not compromise cell proliferation or apoptosis in-vitro. However, growth of xenografted tumors containing the knocked-down RPL19 in-vivo is significantly reduced. Analysis of the modulated genes reveals induction of the non-malignant phenotype principally to involve perturbation of networks of transcription factors and cellular adhesion genes. The data provide evidence that extra-ribosomal regulatory functions of RPL19, beyond protein synthesis, are critical regulators of cellular phenotype. Targeting key members of affected networks identified by gene expression analysis raises the possibility of therapeutically stabilizing a benign phenotype generated by modulating the expression of an individual gene and thereafter constraining a malignant phenotype while leaving non-malignant tissues unaffected. PMID:21799931

  10. The C-terminal silencing domain of Rap1p is essential for the repression of ribosomal protein genes in response to a defect in the secretory pathway.

    PubMed Central

    Mizuta, K; Tsujii, R; Warner, J R; Nishiyama, M

    1998-01-01

    We have previously shown that a functional secretory pathway is essential for continued ribosome synthesis in Saccharomyces cerevisiae. When a temperature-sensitive mutant defective in the secretory pathway is transferred to the non-permissive temperature, transcription of both rRNA genes and ribosomal protein genes is nearly abolished. In order to define the cis -acting element(s) of ribosomal protein genes sensitive to a defect in the secretory pathway, we have constructed a series of fusion genes containing the CYH2 promoter region, with various deletions, fused to lacZ. Each fusion gene for which transcription is detected is subject to the repression. Rap1p is the transcriptional activator for most ribosomal protein genes, as well as having an important role in silencing in the vicinity of telomeres and at the silent mating-type loci. To assess its role in the repression of transcription by the defect in the secretory pathway, we have introduced rap1 mutations. The replacement of wild-type Rap1p by Rap1p truncated at the C-terminal region caused substantial attenuation of the repression. Furthermore, we have demonstrated that the Rap1p-truncation affects the repression of TCM1 , encoding ribosomal protein L3, which has no Rap1p-binding site in its upstream regulatory region. These results suggest that the repression of transcription of ribosomal protein genes by a secretory defect is mediated through Rap1p, but does not require a Rap1p-binding site within the UAS. PMID:9461469

  11. A Minimal Model of Ribosome Allocation Dynamics Captures Trade-offs in Expression between Endogenous and Synthetic Genes.

    PubMed

    Gorochowski, Thomas E; Avcilar-Kucukgoze, Irem; Bovenberg, Roel A L; Roubos, Johannes A; Ignatova, Zoya

    2016-07-15

    Cells contain a finite set of resources that must be distributed across many processes to ensure survival. Among them, the largest proportion of cellular resources is dedicated to protein translation. Synthetic biology often exploits these resources in executing orthogonal genetic circuits, yet the burden this places on the cell is rarely considered. Here, we develop a minimal model of ribosome allocation dynamics capturing the demands on translation when expressing a synthetic construct together with endogenous genes required for the maintenance of cell physiology. Critically, it contains three key variables related to design parameters of the synthetic construct covering transcript abundance, translation initiation rate, and elongation time. We show that model-predicted changes in ribosome allocation closely match experimental shifts in synthetic protein expression rate and cellular growth. Intriguingly, the model is also able to accurately infer transcript levels and translation times after further exposure to additional ambient stress. Our results demonstrate that a simple model of resource allocation faithfully captures the redistribution of protein synthesis resources when faced with the burden of synthetic gene expression and environmental stress. The tractable nature of the model makes it a versatile tool for exploring the guiding principles of efficient heterologous expression and the indirect interactions that can arise between synthetic circuits and their host chassis because of competition for shared translational resources. PMID:27112032

  12. Molecular ecology of tetracycline resistance: development and validation of primers for detection of tetracycline resistance genes encoding ribosomal protection proteins.

    PubMed

    Aminov, R I; Garrigues-Jeanjean, N; Mackie, R I

    2001-01-01

    Phylogenetic analysis of tetracycline resistance genes encoding the ribosomal protection proteins (RPPs) revealed the monophyletic origin of these genes. The most deeply branching class, exemplified by tet and otrA, consisted of genes from the antibiotic-producing organisms Streptomyces rimosus and Streptomyces lividans. With a high degree of confidence, the corresponding genes of the other seven classes (Tet M, Tet S, Tet O, Tet W, Tet Q, Tet T, and TetB P) formed phylogenetically distinct separate clusters. Based on this phylogenetic analysis, a set of PCR primers for detection, retrieval, and sequence analysis of the corresponding gene fragments from a variety of bacterial and environmental sources was developed and characterized. A pair of degenerate primers targeted all tetracycline resistance genes encoding RPPs except otrA and tet, and seven other primer pairs were designed to target the specific classes. The primers were used to detect the circulation of these genes in the rumina of cows, in swine feed and feces, and in swine fecal streptococci. Classes Tet O and Tet W were found in the intestinal contents of both animals, while Tet M was confined to pigs and Tet Q was confined to the rumen. The tet(O) and tet(W) genes circulating in the microbiota of the rumen and the gastrointestinal tract of pigs were identical despite the differences in animal hosts and antibiotic use regimens. Swine fecal streptococci uniformly possessed the tet(O) gene, and 22% of them also carried tet(M). This population could be considered one of the main reservoirs of these two resistance genes in the pig gastrointestinal tract. All classes of RPPs except Tet T and TetB P were found in the commercial components of swine feed. This is the first demonstration of the applicability of molecular ecology techniques to estimation of the gene pool and the flux of antibiotic resistance genes in production animals. PMID:11133424

  13. Fine mapping of 28S rRNA sites specifically cleaved in cells undergoing apoptosis.

    PubMed Central

    Houge, G; Robaye, B; Eikhom, T S; Golstein, J; Mellgren, G; Gjertsen, B T; Lanotte, M; Døskeland, S O

    1995-01-01

    Bona fide apoptosis in rat and human leukemia cells, rat thymocytes, and bovine endothelial cells was accompanied by limited and specific cleavage of polysome-associated and monosome-associated 28S rRNA, with 18S rRNA being spared. Specific 28S rRNA cleavage was observed in all instances of apoptotic death accompanied by internucleosomal DNA fragmentation, with cleavage of 28S rRNA and of DNA being linked temporally. This indicates that 28S rRNA fragmentation may be as general a feature of apoptosis as internucleosomal DNA fragmentation and that concerted specific cleavage of intra- and extranuclear polynucleotides occurs in apoptosis. Apoptosis-associated cleavage sites were mapped to the 28S rRNA divergent domains D2, D6 (endothelial cells), and D8. The D2 cuts occurred in hairpin loop junctions considered to be buried in the intact ribosome, suggesting that this rRNA region becomes a target for RNase attack in apoptotic cells. D8 was cleaved in two exposed UU(U) sequences in bulge loops. Treatment with agents causing necrotic cell death or aging of cell lysates failed to produce any detectable limited D2 cleavage but did produce a more generalized cleavage in the D8 region. Of potential functional interest was the finding that the primary cuts in D2 exactly flanked a 0.3-kb hypervariable subdomain (D2c), allowing excision of the latter. The implication of hypervariable rRNA domains in apoptosis represents the first association of any functional process with these enigmatic parts of the ribosomes. PMID:7891700

  14. Translation of branched-chain aminotransferase-1 transcripts is impaired in cells haploinsufficient for ribosomal protein genes.

    PubMed

    Pereboom, Tamara C; Bondt, Albert; Pallaki, Paschalina; Klasson, Tim D; Goos, Yvonne J; Essers, Paul B; Groot Koerkamp, Marian J A; Gazda, Hanna T; Holstege, Frank C P; Costa, Lydie Da; MacInnes, Alyson W

    2014-05-01

    Diamond-Blackfan anemia (DBA) is a bone marrow failure syndrome linked to mutations in ribosomal protein (RP) genes that result in the impaired proliferation of hematopoietic progenitor cells. The etiology of DBA is not completely understood; however, the ribosomal nature of the genes involved has led to speculation that these mutations may alter the landscape of messenger RNA (mRNA) translation. Here, we performed comparative microarray analysis of polysomal mRNA transcripts isolated from lymphoblastoid cell lines derived from DBA patients carrying various haploinsufficient mutations in either RPS19 or RPL11. Different spectrums of changes were observed depending on the mutant gene, with large differences found in RPS19 cells and very few in RPL11 cells. However, we find that the small number of altered transcripts in RPL11 overlap for the most part with those altered in RPS19 cells. We show specifically that levels of branched-chain aminotransferase-1 (BCAT1) transcripts are significantly decreased on the polysomes of both RPS19 and RPL11 cells and that translation of BCAT1 protein is especially impaired in cells with small RP gene mutations, and we provide evidence that this effect may be due in part to the unusually long 5'UTR of the BCAT1 transcript. The BCAT1 enzyme carries out the final step in the biosynthesis and the first step of degradation of the branched-chain amino acids leucine, isoleucine, and valine. Interestingly, several animal models of DBA have reported that leucine ameliorates the anemia phenotypes generated by RPS19 loss. Our study suggests that RP mutations affect the synthesis of specific proteins involved in regulating amino acid levels that are important for maintaining the normal proliferative capacity of hematopoietic cells. PMID:24463277

  15. The Evolutionary Dynamics of Ribosomal Genes, Histone H3, and Transposable Rex Elements in the Genome of Atlantic Snappers.

    PubMed

    Costa, Gideão Wagner Werneck Félix da; Cioffi, Marcelo de Bello; Bertollo, Luiz Antonio Carlos; Molina, Wagner Franco

    2016-03-01

    Lutjanidae is a family of primarily marine and carnivorous fishes distributed in the Atlantic, Indian, and Pacific oceans, with enormous economic and ecological importance. In order to better clarify the conservative chromosomal evolution of Lutjanidae, we analyzed the evolutionary dynamics of 5 repetitive DNA classes in 5 Lutjanus and in 1 Ocyurus species from the Western Atlantic. The ribosomal 18S sites were generally located in a single chromosome pair, except for L. jocu and L. alexandrei where they are found in 2 pairs. In turn, the 5S rDNA sites are unique, terminal and nonsyntenic with the 18S rDNA sites. In 3 species analyzed, H3 hisDNA genes were found in 1 chromosomal pair. However, while L. jocu presented 2 H3 sites, O. chrysurus showed a noteworthy dispersion of this gene in almost all chromosomes of the karyotype. Retrotransposons Rex1 and Rex3 do not exhibit any association with the explosive distribution of H3 sequences in O. chrysurus. The low compartmentalization of Rex elements, in addition to the general nondynamic distribution of ribosomal and H3 genes, corroborate the karyotype conservatism in Lutjanidae species, also at the microstructural level. However, some "disturbing evolutionary waves" can break down this conservative scenario, as evidenced by the massive random dispersion of H3 hisDNA in the genome of O. chrysurus. The implication of the genomic expansion of H3 histone genes and their functionality remain unknown, although suggesting that they have higher evolutionary dynamics than previously thought. PMID:26792596

  16. Improved methods of AAV-mediated gene targeting for human cell lines using ribosome-skipping 2A peptide

    PubMed Central

    Karnan, Sivasundaram; Ota, Akinobu; Konishi, Yuko; Wahiduzzaman, Md; Hosokawa, Yoshitaka; Konishi, Hiroyuki

    2016-01-01

    The adeno-associated virus (AAV)-based targeting vector has been one of the tools commonly used for genome modification in human cell lines. It allows for relatively efficient gene targeting associated with 1–4-log higher ratios of homologous-to-random integration of targeting vectors (H/R ratios) than plasmid-based targeting vectors, without actively introducing DNA double-strand breaks. In this study, we sought to improve the efficiency of AAV-mediated gene targeting by introducing a 2A-based promoter-trap system into targeting constructs. We generated three distinct AAV-based targeting vectors carrying 2A for promoter trapping, each targeting a GFP-based reporter module incorporated into the genome, PIGA exon 6 or PIGA intron 5. The absolute gene targeting efficiencies and H/R ratios attained using these vectors were assessed in multiple human cell lines and compared with those attained using targeting vectors carrying internal ribosome entry site (IRES) for promoter trapping. We found that the use of 2A for promoter trapping increased absolute gene targeting efficiencies by 3.4–28-fold and H/R ratios by 2–5-fold compared to values obtained with IRES. In CRISPR-Cas9-assisted gene targeting using plasmid-based targeting vectors, the use of 2A did not enhance the H/R ratios but did upregulate the absolute gene targeting efficiencies compared to the use of IRES. PMID:26657635

  17. Yeast ribosomal proteins: XIII. Saccharomyces cerevisiae YL8A gene, interrupted with two introns, encodes a homolog of mammalian L7.

    PubMed Central

    Mizuta, K; Hashimoto, T; Otaka, E

    1992-01-01

    We isolated and sequenced a gene, YL8A, encoding ribosomal protein YL8 of Saccharomyces cerevisiae. It is one of the two duplicated genes encoding YL8 and is located on chromosome VII while the other is on chromosome XVI. The haploid strains carrying disrupted YL8A grew more slowly than the parent strain. The open reading frame is interrupted with two introns. The predicted amino acid sequence reveals that yeast YL8 is a homolog of mammalian ribosomal protein L7, E.coli L30 and others. Images PMID:1549461

  18. Organization and nucleotide sequences of ten ribosomal protein genes from the region equivalent to the S10 operon in the archaebacterium, Halobacterium halobium.

    PubMed

    Miyokawa, T; Urayama, T; Shimooka, K; Itoh, T

    1996-08-01

    A determination was made of the nucleotide sequence of the 7340-bp region of a ribosomal protein gene cluster of Halobacterium halobium, which is equivalent to the S10 operon of Escherichia coli. The sequence was analyzed with the codonpreference program deduced from the halobacterial codon usage table that showed a very high GC content of the third codon position. The sequence was comprised of a string of 13 tightly linked ORFs. Most of the ORFs were homologous with ribosomal protein genes (ORF1-ORF2-rpl3-rpl4-rpl23--rpl2- rps19-rpl22-rps3-rpl29-ORF11-rps17-r pl14). The 13-gene string was preceded by three putative AT-rich promoter sequences. The order of the genes in H. halobium essentially agreed with that of the corresponding genes of E. coli (S10-operon), except for certain deletions or insertions of additional protein genes. PMID:8876975

  19. Phylogenetic Information Content of Copepoda Ribosomal DNA Repeat Units: ITS1 and ITS2 Impact

    PubMed Central

    Zagoskin, Maxim V.; Lazareva, Valentina I.; Grishanin, Andrey K.; Mukha, Dmitry V.

    2014-01-01

    The utility of various regions of the ribosomal repeat unit for phylogenetic analysis was examined in 16 species representing four families, nine genera, and two orders of the subclass Copepoda (Crustacea). Fragments approximately 2000 bp in length containing the ribosomal DNA (rDNA) 18S and 28S gene fragments, the 5.8S gene, and the internal transcribed spacer regions I and II (ITS1 and ITS2) were amplified and analyzed. The DAMBE (Data Analysis in Molecular Biology and Evolution) software was used to analyze the saturation of nucleotide substitutions; this test revealed the suitability of both the 28S gene fragment and the ITS1/ITS2 rDNA regions for the reconstruction of phylogenetic trees. Distance (minimum evolution) and probabilistic (maximum likelihood, Bayesian) analyses of the data revealed that the 28S rDNA and the ITS1 and ITS2 regions are informative markers for inferring phylogenetic relationships among families of copepods and within the Cyclopidae family and associated genera. Split-graph analysis of concatenated ITS1/ITS2 rDNA regions of cyclopoid copepods suggested that the Mesocyclops, Thermocyclops, and Macrocyclops genera share complex evolutionary relationships. This study revealed that the ITS1 and ITS2 regions potentially represent different phylogenetic signals. PMID:25215300

  20. The AAA-ATPase molecular chaperone Cdc48/p97 disassembles sumoylated centromeres, decondenses heterochromatin, and activates ribosomal RNA genes

    PubMed Central

    Mérai, Zsuzsanna; Chumak, Nina; García-Aguilar, Marcelina; Hsieh, Tzung-Fu; Nishimura, Toshiro; Schoft, Vera K.; Bindics, János; Ślusarz, Lucyna; Arnoux, Stéphanie; Opravil, Susanne; Mechtler, Karl; Zilberman, Daniel; Fischer, Robert L.; Tamaru, Hisashi

    2014-01-01

    Centromeres mediate chromosome segregation and are defined by the centromere-specific histone H3 variant (CenH3)/centromere protein A (CENP-A). Removal of CenH3 from centromeres is a general property of terminally differentiated cells, and the persistence of CenH3 increases the risk of diseases such as cancer. However, active mechanisms of centromere disassembly are unknown. Nondividing Arabidopsis pollen vegetative cells, which transport engulfed sperm by extended tip growth, undergo loss of CenH3; centromeric heterochromatin decondensation; and bulk activation of silent rRNA genes, accompanied by their translocation into the nucleolus. Here, we show that these processes are blocked by mutations in the evolutionarily conserved AAA-ATPase molecular chaperone, CDC48A, homologous to yeast Cdc48 and human p97 proteins, both of which are implicated in ubiquitin/small ubiquitin-like modifier (SUMO)-targeted protein degradation. We demonstrate that CDC48A physically associates with its heterodimeric cofactor UFD1-NPL4, known to bind ubiquitin and SUMO, as well as with SUMO1-modified CenH3 and mutations in NPL4 phenocopy cdc48a mutations. In WT vegetative cell nuclei, genetically unlinked ribosomal DNA (rDNA) loci are uniquely clustered together within the nucleolus and all major rRNA gene variants, including those rDNA variants silenced in leaves, are transcribed. In cdc48a mutant vegetative cell nuclei, however, these rDNA loci frequently colocalized with condensed centromeric heterochromatin at the external periphery of the nucleolus. Our results indicate that the CDC48ANPL4 complex actively removes sumoylated CenH3 from centromeres and disrupts centromeric heterochromatin to release bulk rRNA genes into the nucleolus for ribosome production, which fuels single nucleus-driven pollen tube growth and is essential for plant reproduction. PMID:25344531

  1. The AAA-ATPase molecular chaperone Cdc48/p97 disassembles sumoylated centromeres, decondenses heterochromatin, and activates ribosomal RNA genes.

    PubMed

    Mérai, Zsuzsanna; Chumak, Nina; García-Aguilar, Marcelina; Hsieh, Tzung-Fu; Nishimura, Toshiro; Schoft, Vera K; Bindics, János; Slusarz, Lucyna; Arnoux, Stéphanie; Opravil, Susanne; Mechtler, Karl; Zilberman, Daniel; Fischer, Robert L; Tamaru, Hisashi

    2014-11-11

    Centromeres mediate chromosome segregation and are defined by the centromere-specific histone H3 variant (CenH3)/centromere protein A (CENP-A). Removal of CenH3 from centromeres is a general property of terminally differentiated cells, and the persistence of CenH3 increases the risk of diseases such as cancer. However, active mechanisms of centromere disassembly are unknown. Nondividing Arabidopsis pollen vegetative cells, which transport engulfed sperm by extended tip growth, undergo loss of CenH3; centromeric heterochromatin decondensation; and bulk activation of silent rRNA genes, accompanied by their translocation into the nucleolus. Here, we show that these processes are blocked by mutations in the evolutionarily conserved AAA-ATPase molecular chaperone, CDC48A, homologous to yeast Cdc48 and human p97 proteins, both of which are implicated in ubiquitin/small ubiquitin-like modifier (SUMO)-targeted protein degradation. We demonstrate that CDC48A physically associates with its heterodimeric cofactor UFD1-NPL4, known to bind ubiquitin and SUMO, as well as with SUMO1-modified CenH3 and mutations in NPL4 phenocopy cdc48a mutations. In WT vegetative cell nuclei, genetically unlinked ribosomal DNA (rDNA) loci are uniquely clustered together within the nucleolus and all major rRNA gene variants, including those rDNA variants silenced in leaves, are transcribed. In cdc48a mutant vegetative cell nuclei, however, these rDNA loci frequently colocalized with condensed centromeric heterochromatin at the external periphery of the nucleolus. Our results indicate that the CDC48A(NPL4) complex actively removes sumoylated CenH3 from centromeres and disrupts centromeric heterochromatin to release bulk rRNA genes into the nucleolus for ribosome production, which fuels single nucleus-driven pollen tube growth and is essential for plant reproduction. PMID:25344531

  2. Cloning, sequencing, and characterization of ribosomal protein and RNA polymerase genes from the region analogous to the alpha-operon of escherichia coli in halophilic archaea, halobacterium halobium.

    PubMed

    Sano, K; Taguchi, A; Furumoto, H; Uda, T; Itoh, T

    1999-10-14

    A determination was made of the nucleotide sequence of the 3215-bp region of a ribosomal protein gene cluster (HS13, HS4, HS11, and HeL18), RNA polymerase (RNA poly D), and tRNA genes (tRNAser and tRNAarg) of halophilic Archaea Halobacterium halobium, which is analogous to the alpha-operon of Escherichia coli (tRNAser-HS13-HS4-HS11-RNA poly D-tRNAarg-HeL18). The seven-gene string was preceded by a pseudoknot-like structure similar to the proposed S4 ribosomal protein binding site of the alpha-operon mRNA leader in E. coli. Using an inducible expression system H. halobium HS4 was produced in large amounts in E. coli, and immunoblot analysis showed the S4 to constitute a 21-kDa polypeptide component of the ribosome. Analysis of the deduced amino acids sequence revealed that the HS13, HS4, and HS11 sequences including the RNA polymerase subunit are more similar to their eukaryotic than to their bacterial counterparts. HeL18, located downstream of the gene cluster analogous to the E. coli alpha-operon (S13-S11-S4-RNA poly D-L17), was similar to both the eukaryotic (eL18) and eubacterial ribosomal protein L15 located in the spc-operon, but not to L17 positioned as the terminal gene of the bacterial alpha-operon. PMID:10527834

  3. Direct evidence for Mendelian inheritance of the variations in the ribosomal protein gene introns in yellowfin tuna (Thunnus albacares).

    PubMed

    Chow, S; Scholey, V P; Nakazawa, A; Margulies, D; Wexler, J B; Olson, R J; Hazama, K

    2001-01-01

    Restriction fragment length polymorphism found in the S7 ribosomal protein gene introns of yellowfin tuna (Thunnus albacares) was compared between a single pair of parents and their offspring. The sizes of the first intron ( RP1) and second intron ( RP2) amplified by polymerase chain reaction were 810 bp and 1400 bp, respectively. The dam and sire had different restriction types from one another in HhaI and RsaI digestions for RP1 and in DdeI, HhaI, and ScrFI digestions for RP2. Putative genotypes in both introns of 64 larvae were found to be segregated in Mendelian proportions. Genotype distributions in a wild yellowfin tuna sample ( n = 34) were in Hardy-Weinberg proportions, and observed heterozygosity ranged from 0.149 to 0.388. This study presents novel Mendelian markers, which are feasible for tuna population genetic study and pedigree analysis. PMID:14961386

  4. Cloning of the 16S ribosomal RNA gene of a psychrophilic bacterium from the Alaskan Fox Permafrost Tunnel

    NASA Astrophysics Data System (ADS)

    Marsic, Damien; Pikuta, Elena V.; Hoover, Richard B.; Ng, Joseph D.

    2002-02-01

    Extreme cold environments on Earth, such as polar regions or deep ocean harbor a variety of life forms that have developed unique molecular mechanisms that allow them not only to survive, but also to proliferate under hostile conditions. Such organisms are specially relevant to astrobiology studies because they help determine the environmental limits within which life can exist. They can also have a huge potential for biotechnological applications, because of the unique properties of their macromolecules. In this study we focused on a newly isolated bacterium from the Fox Permafrost Tunnel, FTR1, that grows anaerobically at +2 degree(s)C. We describe the molecular phylogenetic analysis of this microorganism, through the cloning, sequencing and analysis of its 16S ribosomal RNA gene. Our results suggests that FTR1 is a novel species belonging to the Carnobacterium genus.

  5. Influence of intron length on interaction characters between post-spliced intron and its CDS in ribosomal protein genes

    NASA Astrophysics Data System (ADS)

    Zhao, Xiaoqing; Li, Hong; Bao, Tonglaga; Ying, Zhiqiang

    2012-09-01

    Many experiment evidences showed that sequence structures of introns and intron loss/gain can influence gene expression, but current mechanisms did not refer to the functions of post-spliced introns directly. We propose that postspliced introns play their functions in gene expression by interacting with their mRNA sequences and the interaction is characterized by the matched segments between introns and their CDS. In this study, we investigated the interaction characters with length series by improved Smith-Waterman local alignment software for the ribosomal protein genes in C. elegans and D. melanogaster. Our results showed that RF values of five intron groups are significantly high in the central non-conserved region and very low in 5'-end and 3'-end splicing region. It is interesting that the number of the optimal matched regions gradually increases with intron length. Distributions of the optimal matched regions are different for five intron groups. Our study revealed that there are more interaction regions between longer introns and their CDS than shorter, and it provides a positive pattern for regulating the gene expression.

  6. Complete Sequence Construction of the Highly Repetitive Ribosomal RNA Gene Repeats in Eukaryotes Using Whole Genome Sequence Data.

    PubMed

    Agrawal, Saumya; Ganley, Austen R D

    2016-01-01

    The ribosomal RNA genes (rDNA) encode the major rRNA species of the ribosome, and thus are essential across life. These genes are highly repetitive in most eukaryotes, forming blocks of tandem repeats that form the core of nucleoli. The primary role of the rDNA in encoding rRNA has been long understood, but more recently the rDNA has been implicated in a number of other important biological phenomena, including genome stability, cell cycle, and epigenetic silencing. Noncoding elements, primarily located in the intergenic spacer region, appear to mediate many of these phenomena. Although sequence information is available for the genomes of many organisms, in almost all cases rDNA repeat sequences are lacking, primarily due to problems in assembling these intriguing regions during whole genome assemblies. Here, we present a method to obtain complete rDNA repeat unit sequences from whole genome assemblies. Limitations of next generation sequencing (NGS) data make them unsuitable for assembling complete rDNA unit sequences; therefore, the method we present relies on the use of Sanger whole genome sequence data. Our method makes use of the Arachne assembler, which can assemble highly repetitive regions such as the rDNA in a memory-efficient way. We provide a detailed step-by-step protocol for generating rDNA sequences from whole genome Sanger sequence data using Arachne, for refining complete rDNA unit sequences, and for validating the sequences obtained. In principle, our method will work for any species where the rDNA is organized into tandem repeats. This will help researchers working on species without a complete rDNA sequence, those working on evolutionary aspects of the rDNA, and those interested in conducting phylogenetic footprinting studies with the rDNA. PMID:27576718

  7. B chromosomes showing active ribosomal RNA genes contribute insignificant amounts of rRNA in the grasshopper Eyprepocnemis plorans.

    PubMed

    Ruiz-Estévez, Mercedes; Badisco, Liesbeth; Broeck, Jozef Vanden; Perfectti, Francisco; López-León, María Dolores; Cabrero, Josefa; Camacho, Juan Pedro M

    2014-12-01

    The genetic inertness of supernumerary (B) chromosomes has recently been called into question after finding several cases of gene activity on them. The grasshopper Eyprepocnemis plorans harbors B chromosomes containing large amounts of ribosomal DNA (rDNA) units, some of which are eventually active, but the amount of rRNA transcripts contributed by B chromosomes, compared to those of the standard (A) chromosomes, is unknown. Here, we address this question by means of quantitative PCR (qPCR) for two different ITS2 amplicons, one coming from rDNA units located in both A and B chromosomes (ITS2(A+B)) and the other being specific to B chromosomes (ITS2(B)). We analyzed six body parts in nine males showing rDNA expression in their B chromosomes in the testis. Amplification of the ITS2(B) amplicon was successful in RNA extracted from all six body parts analyzed, but showed relative quantification (RQ) values four orders of magnitude lower than those obtained for the ITS(A+B) amplicon. RQ values differed significantly between body parts for the two amplicons, with testis, accessory gland and wing muscle showing threefold higher values than head, gastric cecum and hind leg. We conclude that the level of B-specific rDNA expression is extremely low even in individuals where B chromosome rDNA is not completely silenced. Bearing in mind that B chromosomes carry the largest rDNA cluster in the E. plorans genome, we also infer that the relative contribution of B chromosome rRNA genes to ribosome biogenesis is insignificant, at least in the body parts analyzed. PMID:24997085

  8. ASSOCIATION OF ERYTHROMYCIN SUSCEPTIBILITY AND ABSENCE OF INTERVENING SEQUENCES IN 23S RIBOSOMAL RNA GENES OF CAMPYLOBACTER COLI ISOLATED FROM TURKEYS.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Certain Campylobacter strains have been found to harbor a transcribed intervening sequence (IVS) in at least one copy of the 23S ribosomal RNA gene. Following transcription, the IVS is excised, leading to fragmentation of the 23S rRNA. The origin and possible functions of the IVS are unknown. Furthe...

  9. Role of horizontal gene transfer as a control on the coevolution of ribosomal proteins and the genetic code

    SciTech Connect

    Woese, Carl R.; Goldenfeld, Nigel; Luthey-Schulten, Zaida

    2011-03-31

    Our main goal is to develop the conceptual and computational tools necessary to understand the evolution of the universal processes of translation and replication and to identify events of horizontal gene transfer that occurred within the components. We will attempt to uncover the major evolutionary transitions that accompanied the development of protein synthesis by the ribosome and associated components of the translation apparatus. Our project goes beyond standard genomic approaches to explore homologs that are represented at both the structure and sequence level. Accordingly, use of structural phylogenetic analysis allows us to probe further back into deep evolutionary time than competing approaches, permitting greater resolution of primitive folds and structures. Specifically, our work focuses on the elements of translation, ranging from the emergence of the canonical genetic code to the evolution of specific protein folds, mediated by the predominance of horizontal gene transfer in early life. A unique element of this study is the explicit accounting for the impact of phenotype selection on translation, through a coevolutionary control mechanism. Our work contributes to DOE mission objectives through: (1) sophisticated computer simulation of protein dynamics and evolution, and the further refinement of techniques for structural phylogeny, which complement sequence information, leading to improved annotation of genomic databases; (2) development of evolutionary approaches to exploring cellular function and machinery in an integrated way; and (3) documentation of the phenotype interaction with translation over evolutionary time, reflecting the system response to changing selection pressures through horizontal gene transfer.

  10. Expression of ribosomal RNA genes in lines of barley with a standard karyotype and with a translocated nucleolar organizer

    SciTech Connect

    Karag'ozov, L.K.; Ananiev, E.D.; Mateeva, Z.E.; Khadzhiolov, A.A.

    1986-10-01

    The authors have investigated the rRNA synthesis and the sensitivity of rRNA genes to the action of DNAase I in developing embryos of two forms of barley. The Frigga variety has a standard karyotype and the T/sub 506/ line is characterized by translocation of the nucleolar organizer, which leads to a reduction in the number of nucleoli observed in the telophase. The results of the investigation of rRNA synthesis in vivo and of the activity of RNA polymerase I in isolated nuclei revealed the absence of differences between the two barley forms. They have established that the genes of ribosomal RNAs possess greater sensitivity to digestion by DNAase the authors compared to that of the total nuclear DNA. They conclude that the translocation of one of the nucleolar organizers causes a delay in the appearance of its activity during the telophase, this not changing the expression of the rRNA genes in the subsequent stages of cell development.

  11. Organization of a large gene cluster encoding ribosomal proteins in the cyanobacterium Synechococcus sp. strain PCC 6301: comparison of gene clusters among cyanobacteria, eubacteria and chloroplast genomes.

    PubMed

    Sugita, M; Sugishita, H; Fujishiro, T; Tsuboi, M; Sugita, C; Endo, T; Sugiura, M

    1997-08-11

    The structure of a large gene cluster containing 22 ribosomal protein (r-protein) genes of the cyanobacterium Synechococcus sp. strain PCC6301 is presented. Based on DNA and protein sequence analyses, genes encoding r-proteins L3, L4, L23, L2, S19, L22, S3, L16, L29, S17, L14, L24, L5, S8, L6, L18, S5, L15, L36, S13, S11, L17, SecY, adenylate kinase (AK) and the alpha subunit of RNA polymerase were identified. The gene order is similar to that of the E. coli S10, spc and alpha operons. Unlike the corresponding E. coli operons, the genes for r-proteins S4, S10, S14 and L30 are not present in this cluster. The organization of Synechococcus r-protein genes also resembles that of chloroplast (cp) r-protein genes of red and brown algal species. This strongly supports the endosymbiotic theory that the cp genome evolved from an ancient photosynthetic bacterium. PMID:9300823

  12. Molecular phylogeny of the assassin bugs (Hemiptera: Reduviidae), based on mitochondrial and nuclear ribosomal genes.

    PubMed

    Weirauch, Christiane; Munro, James B

    2009-10-01

    The first comprehensive cladistic analysis of Reduviidae, the assassin bugs, based on molecular data is presented and discussed in the context of a recently-published morphological analysis. Assassin bugs are essential components of ecosystems, but also important in agriculture and medicine. Sampling included 94 taxa (89 Reduviidae, 5 outgroups) in 15 subfamilies and 24 tribes of Reduviidae and is based on approximately 3300 base pairs of mitochondrial (16S) and nuclear (18S, 28SD2, 28SD3-5) ribosomal DNA. Partitions of the dataset were aligned using different algorithms implemented in MAFFT and the combined dataset was analyzed using parsimony, partitioned maximum likelihood and partitioned Bayesian criteria. Clades recovered in all analyses, independent of alignment and analytical method, comprise: Cimicomorpha and Reduviidae; Hammacerinae; Harpactorinae; Apiomerini; Peiratinae; Phymatinae; Salyavatinae; Triatominae; Phymatinae+Holoptilinae; the higher Reduviidae (Reduviidae excluding Hammacerinae and the Phymatine Complex); Ectrichodiinae+Tribelocephalinae; (Triatominae+Zelurus)+Stenopodainae. Hammacerinae are rejected as sister group to all remaining Reduviidae in all analyses, as is the monophyly of Reduviinae, Emesinae and Harpactorini. High support values for Triatominae imply that blood-feeding has evolved only once within Reduviidae. Stenopodainae and part of Reduviinae are discussed as close relatives to Triatominae. PMID:19531379

  13. Basic Mechanisms in RNA Polymerase I Transcription of the Ribosomal RNA Genes

    PubMed Central

    Goodfellow, Sarah J.; Zomerdijk, Joost C. B. M.

    2013-01-01

    RNA Polymerase (Pol) I produces ribosomal (r)RNA, an essential component of the cellular protein synthetic machinery that drives cell growth, underlying many fundamental cellular processes. Extensive research into the mechanisms governing transcription by Pol I has revealed an intricate set of control mechanisms impinging upon rRNA production. Pol I-specific transcription factors guide Pol I to the rDNA promoter and contribute to multiple rounds of transcription initiation, promoter escape, elongation and termination. In addition, many accessory factors are now known to assist at each stage of this transcription cycle, some of which allow the integration of transcriptional activity with metabolic demands. The organisation and accessibility of rDNA chromatin also impinge upon Pol I output, and complex mechanisms ensure the appropriate maintenance of the epigenetic state of the nucleolar genome and its effective transcription by Pol I. The following review presents our current understanding of the components of the Pol I transcription machinery, their functions and regulation by associated factors, and the mechanisms operating to ensure the proper transcription of rDNA chromatin. The importance of such stringent control is demonstrated by the fact that deregulated Pol I transcription is a feature of cancer and other disorders characterised by abnormal translational capacity. PMID:23150253

  14. Cytogenetic analysis of Astylus antis (Perty, 1830) (Coleoptera, Melyridae): Karyotype, heterochromatin and location of ribosomal genes

    PubMed Central

    2010-01-01

    Cytogenetic analysis of Astylus antis using mitotic and meiotic cells was performed to characterize the haploid and diploid numbers, sex determination system, chromosome morphology, constitutive heterochromatin distribution pattern and chromosomes carrying nucleolus organizer regions (NORs). Analysis of spermatogonial metaphase cells revealed the diploid number 2n = 18, with mostly metacentric chromosomes. Metaphase I cells exhibited 2n = 8II+Xyp and a parachute configuration of the sex chromosomes. Spermatogonial metaphase cells submitted to C-banding showed the presence of small dots of constitutive heterochromatin in the centromeric regions of nearly all the autosomes and on the short arm of the X chromosome (Xp), as well as an additional band on one of the arms of pair 1. Mitotic cells submitted to double staining with base-specific fluorochromes (DAPI-CMA3 ) revealed no regions rich in A+T or G+C sequences. Analysis of spermatogonial mitotic cells after sequential Giemsa/AgNO 3 staining did not reveal any specific mark on the chromosomes. Meiotic metaphase I cells stained with silver nitrate revealed a strong impregnation associated to the sex chromosomes, and in situ hybridization with an 18S rDNA probe showed ribosomal cistrons in an autosomal bivalent. PMID:21637476

  15. Comparison of quantitative PCR assays for Escherichia coli targeting ribosomal RNA and single copy genes

    EPA Science Inventory

    Aims: Compare specificity and sensitivity of quantitative PCR (qPCR) assays targeting single and multi-copy gene regions of Escherichia coli. Methods and Results: A previously reported assay targeting the uidA gene (uidA405) was used as the basis for comparing the taxono...

  16. Recognition of chimeric small-subunit ribosomal DNAs composed of genes from uncultivated microorganisms

    NASA Technical Reports Server (NTRS)

    Kopczynski, E. D.; Bateson, M. M.; Ward, D. M.

    1994-01-01

    When PCR was used to recover small-subunit (SSU) rRNA genes from a hot spring cyanobacterial mat community, chimeric SSU rRNA sequences which exhibited little or no secondary structural abnormality were recovered. They were revealed as chimeras of SSU rRNA genes of uncultivated species through separate phylogenetic analysis of short sequence domains.

  17. Conserved codon composition of ribosomal protein coding genes in Escherichia coli, Mycobacterium tuberculosis and Saccharomyces cerevisiae: lessons from supervised machine learning in functional genomics.

    PubMed

    Lin, Kui; Kuang, Yuyu; Joseph, Jeremiah S; Kolatkar, Prasanna R

    2002-06-01

    Genomics projects have resulted in a flood of sequence data. Functional annotation currently relies almost exclusively on inter-species sequence comparison and is restricted in cases of limited data from related species and widely divergent sequences with no known homologs. Here, we demonstrate that codon composition, a fusion of codon usage bias and amino acid composition signals, can accurately discriminate, in the absence of sequence homology information, cytoplasmic ribosomal protein genes from all other genes of known function in Saccharomyces cerevisiae, Escherichia coli and Mycobacterium tuberculosis using an implementation of support vector machines, SVM(light). Analysis of these codon composition signals is instructive in determining features that confer individuality to ribosomal protein genes. Each of the sets of positively charged, negatively charged and small hydrophobic residues, as well as codon bias, contribute to their distinctive codon composition profile. The representation of all these signals is sensitively detected, combined and augmented by the SVMs to perform an accurate classification. Of special mention is an obvious outlier, yeast gene RPL22B, highly homologous to RPL22A but employing very different codon usage, perhaps indicating a non-ribosomal function. Finally, we propose that codon composition be used in combination with other attributes in gene/protein classification by supervised machine learning algorithms. PMID:12034849

  18. Exploring Ribosome Positioning on Translating Transcripts with Ribosome Profiling.

    PubMed

    Spealman, Pieter; Wang, Hao; May, Gemma; Kingsford, Carl; McManus, C Joel

    2016-01-01

    Recent technological advances (e.g., microarrays and massively parallel sequencing) have facilitated genome-wide measurement of many aspects of gene regulation. Ribosome profiling is a high-throughput sequencing method used to measure gene expression at the level of translation. This is accomplished by quantifying both the number of translating ribosomes and their locations on mRNA transcripts. The inventors of this approach have published several methods papers detailing its implementation and addressing the basics of ribosome profiling data analysis. Here we describe our lab's procedure, which differs in some respects from those published previously. In addition, we describe a data analysis pipeline, Ribomap, for ribosome profiling data. Ribomap allocates sequence reads to alternative mRNA isoforms, normalizes sequencing bias along transcripts using RNA-seq data, and outputs count vectors of per-codon ribosome occupancy for each transcript. PMID:26463378

  19. Revealing pancrustacean relationships: Phylogenetic analysis of ribosomal protein genes places Collembola (springtails) in a monophyletic Hexapoda and reinforces the discrepancy between mitochondrial and nuclear DNA markers

    PubMed Central

    2008-01-01

    Background In recent years, several new hypotheses on phylogenetic relations among arthropods have been proposed on the basis of DNA sequences. One of the challenged hypotheses is the monophyly of hexapods. This discussion originated from analyses based on mitochondrial DNA datasets that, due to an unusual positioning of Collembola, suggested that the hexapod body plan evolved at least twice. Here, we re-evaluate the position of Collembola using ribosomal protein gene sequences. Results In total 48 ribosomal proteins were obtained for the collembolan Folsomia candida. These 48 sequences were aligned with sequence data on 35 other ecdysozoans. Each ribosomal protein gene was available for 25% to 86% of the taxa. However, the total sequence information was unequally distributed over the taxa and ranged between 4% and 100%. A concatenated dataset was constructed (5034 inferred amino acids in length), of which ~66% of the positions were filled. Phylogenetic tree reconstructions, using Maximum Likelihood, Maximum Parsimony, and Bayesian methods, resulted in a topology that supports monophyly of Hexapoda. Conclusion Although ribosomal proteins in general may not evolve independently, they once more appear highly valuable for phylogenetic reconstruction. Our analyses clearly suggest that Hexapoda is monophyletic. This underpins the inconsistency between nuclear and mitochondrial datasets when analyzing pancrustacean relationships. Caution is needed when applying mitochondrial markers in deep phylogeny. PMID:18366624

  20. An intron within the 16S ribosomal RNA gene of the archaeon Pyrobaculum aerophilum

    NASA Technical Reports Server (NTRS)

    Burggraf, S.; Larsen, N.; Woese, C. R.; Stetter, K. O.

    1993-01-01

    The 16S rRNA genes of Pyrobaculum aerophilum and Pyrobaculum islandicum were amplified by the polymerase chain reaction, and the resulting products were sequenced directly. The two organisms are closely related by this measure (over 98% similar). However, they differ in that the (lone) 16S rRNA gene of Pyrobaculum aerophilum contains a 713-bp intron not seen in the corresponding gene of Pyrobaculum islandicum. To our knowledge, this is the only intron so far reported in the small subunit rRNA gene of a prokaryote. Upon excision the intron is circularized. A secondary structure model of the intron-containing rRNA suggests a splicing mechanism of the same type as that invoked for the tRNA introns of the Archaea and Eucarya and 23S rRNAs of the Archaea. The intron contains an open reading frame whose protein translation shows no certain homology with any known protein sequence.

  1. Ribosome-inactivating proteins: from plant defense to tumor attack.

    PubMed

    de Virgilio, Maddalena; Lombardi, Alessio; Caliandro, Rocco; Fabbrini, Maria Serena

    2010-11-01

    Ribosome-inactivating proteins (RIPs) are EC3.2.32.22 N-glycosidases that recognize a universally conserved stem-loop structure in 23S/25S/28S rRNA, depurinating a single adenine (A4324 in rat) and irreversibly blocking protein translation, leading finally to cell death of intoxicated mammalian cells. Ricin, the plant RIP prototype that comprises a catalytic A subunit linked to a galactose-binding lectin B subunit to allow cell surface binding and toxin entry in most mammalian cells, shows a potency in the picomolar range. The most promising way to exploit plant RIPs as weapons against cancer cells is either by designing molecules in which the toxic domains are linked to selective tumor targeting domains or directly delivered as suicide genes for cancer gene therapy. Here, we will provide a comprehensive picture of plant RIPs and discuss successful designs and features of chimeric molecules having therapeutic potential. PMID:22069572

  2. Ribosome-Inactivating Proteins: From Plant Defense to Tumor Attack

    PubMed Central

    de Virgilio, Maddalena; Lombardi, Alessio; Caliandro, Rocco; Fabbrini, Maria Serena

    2010-01-01

    Ribosome-inactivating proteins (RIPs) are EC3.2.32.22 N-glycosidases that recognize a universally conserved stem-loop structure in 23S/25S/28S rRNA, depurinating a single adenine (A4324 in rat) and irreversibly blocking protein translation, leading finally to cell death of intoxicated mammalian cells. Ricin, the plant RIP prototype that comprises a catalytic A subunit linked to a galactose-binding lectin B subunit to allow cell surface binding and toxin entry in most mammalian cells, shows a potency in the picomolar range. The most promising way to exploit plant RIPs as weapons against cancer cells is either by designing molecules in which the toxic domains are linked to selective tumor targeting domains or directly delivered as suicide genes for cancer gene therapy. Here, we will provide a comprehensive picture of plant RIPs and discuss successful designs and features of chimeric molecules having therapeutic potential. PMID:22069572

  3. Directed gene copy number amplification in Pichia pastoris by vector integration into the ribosomal DNA locus.

    PubMed

    Marx, Hans; Mecklenbräuker, Astrid; Gasser, Brigitte; Sauer, Michael; Mattanovich, Diethard

    2009-12-01

    The yeast Pichia pastoris is a widely used host organism for heterologous protein production. One of the basic steps for strain improvement is to ensure a sufficient level of transcription of the heterologous gene, based on promoter strength and gene copy number. To date, high-copy-number integrants of P. pastoris are achievable only by screening of random events or by cloning of gene concatemers. Methods for rapid and reliable multicopy integration of the expression cassette are therefore desirable. Here we present such a method based on vector integration into the rDNA locus and post-transformational vector amplification by repeated selection on increased antibiotic concentrations. Data are presented for two exemplary products: human serum albumin, which is secreted into the supernatant, and human superoxide dismutase, which is accumulated in the cytoplasm of the cells. The striking picture evolving is that intracellular protein production is tightly correlated with gene copy number, while use of the secretory pathway introduces a high clonal variability and the correlation with gene copy number is valid only for low gene copy numbers. PMID:19799640

  4. Organization and nucleotide sequences of the Spiroplasma citri genes for ribosomal protein S2, elongation factor Ts, spiralin, phosphofructokinase, pyruvate kinase, and an unidentified protein.

    PubMed Central

    Chevalier, C; Saillard, C; Bové, J M

    1990-01-01

    The gene for spiralin, the major membrane protein of the helical mollicute Spiroplasma citri, was cloned in Escherichia coli as a 5-kilobase-pair (kbp) DNA fragment. The complete nucleotide sequence of the 5.0-kbp spiroplasmal DNA fragment was determined (GenBank accession no. M31161). The spiralin gene was identified by the size and amino acid composition of its translational product. Besides the spiralin gene, the spiroplasmal DNA fragment was found to contain five additional open reading frames (ORFs). The translational products of four of these ORFs were identified by their amino acid sequence homologies with known proteins: ribosomal protein S2, elongation factor Ts, phosphofructokinase, and pyruvate kinase, respectively encoded by the genes rpsB, tsf, pfk, and pyk. The product of the fifth ORF remains to be identified and was named protein X (X gene). The order of the above genes was tsf--X--spiralin gene--pfk--pyk. These genes were transcribed in one direction, while the gene for ribosomal protein S2 (rpsB) was transcribed in the opposite direction. Images PMID:2139649

  5. Ribosome-omics of the human ribosome

    PubMed Central

    Gupta, Varun; Warner, Jonathan R.

    2014-01-01

    The torrent of RNA-seq data becoming available not only furnishes an overview of the entire transcriptome but also provides tools to focus on specific areas of interest. Our focus on the synthesis of ribosomes asked whether the abundance of mRNAs encoding ribosomal proteins (RPs) matched the equimolar need for the RPs in the assembly of ribosomes. We were at first surprised to find, in the mapping data of ENCODE and other sources, that there were nearly 100-fold differences in the level of the mRNAs encoding the different RPs. However, after correcting for the mapping ambiguities introduced by the presence of more than 2000 pseudogenes derived from RP mRNAs, we show that for 80%–90% of the RP genes, the molar ratio of mRNAs varies less than threefold, with little tissue specificity. Nevertheless, since the RPs are needed in equimolar amounts, there must be sluggish or regulated translation of the more abundant RP mRNAs and/or substantial turnover of unused RPs. In addition, seven of the RPs have subsidiary genes, three of which are pseudogenes that have been “rescued” by the introduction of promoters and/or upstream introns. Several of these are transcribed in a tissue-specific manner, e.g., RPL10L in testis and RPL3L in muscle, leading to potential variation in ribosome structure from one tissue to another. Of the 376 introns in the RP genes, a single one is alternatively spliced in a tissue-specific manner. PMID:24860015

  6. Identification and characterization of rhizospheric microbial diversity by 16S ribosomal RNA gene sequencing.

    PubMed

    Naveed, Muhammad; Mubeen, Samavia; Khan, SamiUllah; Ahmed, Iftikhar; Khalid, Nauman; Suleria, Hafiz Ansar Rasul; Bano, Asghari; Mumtaz, Abdul Samad

    2014-01-01

    In the present study, samples of rhizosphere and root nodules were collected from different areas of Pakistan to isolate plant growth promoting rhizobacteria. Identification of bacterial isolates was made by 16S rRNA gene sequence analysis and taxonomical confirmation on EzTaxon Server. The identified bacterial strains were belonged to 5 genera i.e. Ensifer, Bacillus, Pseudomona, Leclercia and Rhizobium. Phylogenetic analysis inferred from 16S rRNA gene sequences showed the evolutionary relationship of bacterial strains with the respective genera. Based on phylogenetic analysis, some candidate novel species were also identified. The bacterial strains were also characterized for morphological, physiological, biochemical tests and glucose dehydrogenase (gdh) gene that involved in the phosphate solublization using cofactor pyrroloquinolone quinone (PQQ). Seven rhizoshperic and 3 root nodulating stains are positive for gdh gene. Furthermore, this study confirms a novel association between microbes and their hosts like field grown crops, leguminous and non-leguminous plants. It was concluded that a diverse group of bacterial population exist in the rhizosphere and root nodules that might be useful in evaluating the mechanisms behind plant microbial interactions and strains QAU-63 and QAU-68 have sequence similarity of 97 and 95% which might be declared as novel after further taxonomic characterization. PMID:25477935

  7. Identification and characterization of rhizospheric microbial diversity by 16S ribosomal RNA gene sequencing

    PubMed Central

    Naveed, Muhammad; Mubeen, Samavia; khan, SamiUllah; Ahmed, Iftikhar; Khalid, Nauman; Suleria, Hafiz Ansar Rasul; Bano, Asghari; Mumtaz, Abdul Samad

    2014-01-01

    In the present study, samples of rhizosphere and root nodules were collected from different areas of Pakistan to isolate plant growth promoting rhizobacteria. Identification of bacterial isolates was made by 16S rRNA gene sequence analysis and taxonomical confirmation on EzTaxon Server. The identified bacterial strains were belonged to 5 genera i.e. Ensifer, Bacillus, Pseudomona, Leclercia and Rhizobium. Phylogenetic analysis inferred from 16S rRNA gene sequences showed the evolutionary relationship of bacterial strains with the respective genera. Based on phylogenetic analysis, some candidate novel species were also identified. The bacterial strains were also characterized for morphological, physiological, biochemical tests and glucose dehydrogenase (gdh) gene that involved in the phosphate solublization using cofactor pyrroloquinolone quinone (PQQ). Seven rhizoshperic and 3 root nodulating stains are positive for gdh gene. Furthermore, this study confirms a novel association between microbes and their hosts like field grown crops, leguminous and non-leguminous plants. It was concluded that a diverse group of bacterial population exist in the rhizosphere and root nodules that might be useful in evaluating the mechanisms behind plant microbial interactions and strains QAU-63 and QAU-68 have sequence similarity of 97 and 95% which might be declared as novel after further taxonomic characterization. PMID:25477935

  8. Histone and ribosomal RNA repetitive gene clusters linked in tandem array

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Histones are the major protein component of chromatin structure. The histone family is made up of a quintet of proteins, four core histones (H2A, H2B, H3 & H4) and the linker histones (H1). Spacers are found between the coding regions. Among insects this quintet of genes is usually clustered and ...

  9. Alternating tandem array of histone and ribosomal RNA gene blocks in the boll weevil

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Histones are the major protein component of the ncleosome. The histone family is made up of a quintet of proteins, four core histones (H2A, H2B, H3 & H4) and the linker histones (H1). Spacers are found between the coding regions. Among insects this quintet of genes is usually clustered and the clust...

  10. Prevalent Ciliate Symbiosis on Copepods: High Genetic Diversity and Wide Distribution Detected Using Small Subunit Ribosomal RNA Gene

    PubMed Central

    Guo, Zhiling; Liu, Sheng; Hu, Simin; Li, Tao; Huang, Yousong; Liu, Guangxing; Zhang, Huan; Lin, Senjie

    2012-01-01

    Toward understanding the genetic diversity and distribution of copepod-associated symbiotic ciliates and the evolutionary relationships with their hosts in the marine environment, we developed a small subunit ribosomal RNA gene (18S rDNA)-based molecular method and investigated the genetic diversity and genotype distribution of the symbiotic ciliates on copepods. Of the 10 copepod species representing six families collected from six locations of Pacific and Atlantic Oceans, 9 were found to harbor ciliate symbionts. Phylogenetic analysis of the 391 ciliate 18S rDNA sequences obtained revealed seven groups (ribogroups), six (containing 99% of all the sequences) belonging to subclass Apostomatida, the other clustered with peritrich ciliate Vorticella gracilis. Among the Apostomatida groups, Group III were essentially identical to Vampyrophrya pelagica, and the other five groups represented the undocumented ciliates that were close to Vampyrophrya/Gymnodinioides/Hyalophysa. Group VI ciliates were found in all copepod species but one (Calanus sinicus), and were most abundant among all ciliate sequences obtained, indicating that they are the dominant symbiotic ciliates universally associated with copepods. In contrast, some ciliate sequences were found only in some of the copepods examined, suggesting the host selectivity and geographic differentiation of ciliates, which requires further verification by more extensive sampling. Our results reveal the wide occurrence and high genetic diversity of symbiotic ciliates on marine copepods and highlight the need to systematically investigate the host- and geography-based genetic differentiation and ecological roles of these ciliates globally. PMID:23024768

  11. Ribosomal protein genes of holometabolan insects reject the Halteria, instead revealing a close affinity of Strepsiptera with Coleoptera.

    PubMed

    Longhorn, Stuart J; Pohl, Hans W; Vogler, Alfried P

    2010-06-01

    The phylogenetic relationships among holometabolan insect orders remain poorly known, despite a wealth of previous studies. In particular, past attempts to clarify the sister-group of the enigmatic order Strepsiptera with rRNA genes have led to intense debate about long-branch attraction (the 'Strepsiptera problem'), without resolving the taxonomic question at hand. Here, we appealed to alternative nuclear sequences of 27 ribosomal proteins (RPs) to generate a data matrix of 10,731 nucleotides for 22 holometabolan taxa, including two strepsipteran species. Phylogenetic relationships among holometabolan insects were analyzed under several nucleotide-coding schemes to explore differences in signal and systematic biases. Saturation and compositional bias particularly affected third positions, which greatly differed in AT content (18-72%). Such confounding factors were best reduced by R-Y coding and removal of third codon positions, resulting in more strongly supported topologies, whereas amino acid coding gave poor resolution. The placement of Strepsiptera with Coleoptera (the Coleopterida) was recovered under most coding schemes and analytical methods, if often with modest support and ambiguity. In contrast, an alternative sister-group with Diptera (the Halteria) was only found in one analysis using parsimony, and weakly supported. The topologies here generally support a Coleoptera+Strepsiptera as sister-group to Mecopterida (Siphonaptera+Mecoptera+Diptera+Lepidoptera+Trichoptera), while Hymenoptera were always recovered as sister-group to the remaining Holometabola. PMID:20348001

  12. Evolutionary relationships of a plant-pathogenic mycoplasmalike organism and Acholeplasma laidlawii deduced from two ribosomal protein gene sequences.

    PubMed Central

    Lim, P O; Sears, B B

    1992-01-01

    The families within the class Mollicutes are distinguished by their morphologies, nutritional requirements, and abilities to metabolize certain compounds. Biosystematic classification of the plant-pathogenic mycoplasmalike organisms (MLOs) has been difficult because these organisms have not been cultured in vitro, and hence their nutritional requirements have not been determined nor have physiological characterizations been possible. To investigate the evolutionary relationship of the MLOs to other members of the class Mollicutes, a segment of a ribosomal protein operon was cloned and sequenced from an aster yellows-type MLO which is pathogenic for members of the genus Oenothera and from Acholeplasma laidlawii. The deduced amino acid sequence data from the rpl22 and rps3 genes indicate that the MLOs are more closely related to A. laidlawii than to animal mycoplasmas, confirming previous results from 16S rRNA sequence comparisons. This conclusion is also supported by the finding that the UGA codon is not read as a tryptophan codon in the MLO and A. laidlawii, in contrast to its usage in Mycoplasma capricolum. PMID:1556079

  13. Yeast ribosomal proteins: XII. YS11 of Saccharomyces cerevisiae is a homologue to E. coli S4 according to the gene analysis.

    PubMed Central

    Mizuta, K; Hashimoto, T; Suzuki, K; Otaka, E

    1991-01-01

    We isolated and sequenced a gene, YS11A, encoding ribosomal protein YS11 of Saccharomyces cerevisiae. YS11A is one of two functional copies of the YS11 gene, located on chromosome XVI and transcribed in a lower amount than the other copy which is located on chromosome II. The disruption of YS11A has no effect on the growth of yeast. The 5'-flanking region contains a similar sequence to consensus UASrpg and the T-rich region. The open reading frame is interrupted with an intron located near the 5'-end. The predicted amino acid sequence reveals that yeast YS11 is a homologue to E. coli S4, one of the ram proteins, three chloroplast S4s and others out of the ribosomal protein sequences currently available. Images PMID:2041737

  14. Optimized P2A for reporter gene insertion into Nipah virus results in efficient ribosomal skipping and wild-type lethality.

    PubMed

    Park, Arnold; Yun, Tatyana; Hill, Terence E; Ikegami, Tetsuro; Juelich, Terry L; Smith, Jennifer K; Zhang, Lihong; Freiberg, Alexander N; Lee, Benhur

    2016-04-01

    Incorporation of reporter genes within virus genomes is an indispensable tool for interrogation of virus biology and pathogenesis. In previous work, we incorporated a fluorophore into a viral ORF by attaching it to the viral gene via a P2A ribosomal skipping sequence. This recombinant Nipah virus, however, was attenuated in vitro relative to WT virus. In this work, we determined that inefficient ribosomal skipping was a major contributing factor to this attenuation. Inserting a GSG linker before the P2A sequence resulted in essentially complete skipping, significantly improved growth in vitro, and WT lethality in vivo. To the best of our knowledge, this represents the first time a recombinant virus of Mononegavirales with integration of a reporter into a viral ORF has been compared with the WT virus in vivo. Incorporating the GSG linker for improved skipping efficiency whenever functionally important is a critical consideration for recombinant virus design. PMID:26781134

  15. New Hosts of Simplicimonas similis and Trichomitus batrachorum Identified by 18S Ribosomal RNA Gene Sequences

    PubMed Central

    Dimasuay, Kris Genelyn B.; Lavilla, Orlie John Y.; Rivera, Windell L.

    2013-01-01

    Trichomonads are obligate anaerobes generally found in the digestive and genitourinary tract of domestic animals. In this study, four trichomonad isolates were obtained from carabao, dog, and pig hosts using rectal swab. Genomic DNA was extracted using Chelex method and the 18S rRNA gene was successfully amplified through novel sets of primers and undergone DNA sequencing. Aligned isolate sequences together with retrieved 18S rRNA gene sequences of known trichomonads were utilized to generate phylogenetic trees using maximum likelihood and neighbor-joining analyses. Two isolates from carabao were identified as Simplicimonas similis while each isolate from dog and pig was identified as Pentatrichomonas hominis and Trichomitus batrachorum, respectively. This is the first report of S. similis in carabao and the identification of T. batrachorum in pig using 18S rRNA gene sequence analysis. The generated phylogenetic tree yielded three distinct groups mostly with relatively moderate to high bootstrap support and in agreement with the most recent classification. Pathogenic potential of the trichomonads in these hosts still needs further investigation. PMID:23936631

  16. A characterization of the elements comprising the promoter of the mouse ribosomal protein gene RPS16.

    PubMed

    Hariharan, N; Perry, R P

    1989-07-11

    The elements comprising the mouse rpS16 promoter were characterized by transfection experiments with mutant genes in which various portions of the 5' flanking region and exon I were removed or substituted with extraneous DNA sequence. These experiments were carried out with otherwise intact rpS16 genes transfected into monkey kidney (COS) cells and also with chimeric rpS16-CAT gene constructs transfected into mouse plasmacytoma cells and COS cells. The locations of the functionally important elements were generally correlated with the locations of binding sites for specific nuclear factors, which were identified by gel-mobility shift analyses and methylation interference footprints. The most upstream element, which is located approximately 165 bp from the cap site, binds the Sp1 transcription factor and augments the promoter activity by 2 to 2.5-fold. In addition, there is a complex bipartite element in the -83 to -59 region, an element in the -37 to -12 region and an element in the +9 to +29 region of exon I, all of which are essential for rpS16 expression. The rpS16 promoter has a general architecture that resembles other mouse rp promoters; however, it also possesses some distinctive characteristics. PMID:2762128

  17. Mitochondrial 16S ribosomal RNA gene for forensic identification of crocodile species.

    PubMed

    Naga Jogayya, K; Meganathan, P R; Dubey, Bhawna; Haque, I

    2013-05-01

    All crocodilians are under various threats due to over exploitation and these species have been listed in Appendix I or II of CITES. Lack of molecular techniques for the forensic identification of confiscated samples makes it difficult to enforce the law. Therefore, we herein present a molecular method developed on the basis on 16S rRNA gene of mitochondrial DNA for identification of crocodile species. We have developed a set of 16S rRNA primers for PCR based identification of crocodilian species. These novel primers amplify partial 16S rRNA sequences of six crocodile species which can be later combined to obtain a larger region (1290 bp) of 16S rRNA gene. This 16S rRNA gene could be used as an effective tool for forensic authentication of crocodiles. The described primers hold great promise in forensic identification of crocodile species, which can aid in the effective enforcement of law and conservation of these species. PMID:23622485

  18. Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5' end of the large-subunit (26S) ribosomal DNA gene.

    PubMed Central

    Kurtzman, C P; Robnett, C J

    1997-01-01

    Clinically important species of Candida and related organisms were compared for extent of nucleotide divergence in the 5' end of the large-subunit (26S) ribosomal DNA (rDNA) gene. This rDNA region is sufficiently variable to allow reliable separation of all known clinically significant yeast species. Of the 204 described species examined, 21 appeared to be synonyms of previously described organisms. Phylogenetic relationships among the species are presented. PMID:9114410

  19. Structure and regulation of a nuclear gene in Saccharomyces cerevisiae that specifies MRP7, a protein of the large subunit of the mitochondrial ribosome.

    PubMed Central

    Fearon, K; Mason, T L

    1988-01-01

    The gene for MRP7, a 40-kilodalton protein of the large subunit of the yeast mitochondrial ribosome, was identified in a lambda gt11 expression library by immunological screening with a monoclonal antibody to MRP7. An intact copy of MRP7 was then isolated from a yeast genomic library by colony hybridization. Gene disruption showed that MRP7 protein was essential for ribosomal function. Sequencing of MRP7 revealed a coding region for a basic (pI 10.6), 43.2-kilodalton protein containing 371 amino acid residues. Amino acid residues 28 to 112 of the deduced MRP7 sequence aligned with the 84 residues of the Escherichia coli ribosomal protein L27, but no significant similarity was detected between the carboxy-terminal 259 amino acids of MRP7 and other protein sequences in existing computer data bases. Within the aligned region, there was 49% amino acid identity between MRP7 and L27, compared with the 57% identity observed between L27 and its homolog in Bacillus stearothermophilus. The steady-state levels of the MRP7 protein and its mRNA were monitored in response to catabolite repression and to increased dosage of the MRP7 gene. The response to catabolite repression was characterized by a ninefold change in the level of the protein and little, if any, change in the level of the mRNA. In cells carrying the MRP7 gene on a high-copy-number plasmid, the mRNA was increased 20-fold, but there was no significant increase in MRP7 protein. Furthermore, MRP7 mRNA and protein accumulated at normal levels in [rho0] cells, which are devoid of 21S rRNA, indicating that the protein is relatively stable in the absence of ribosome assembly. Together, these results suggest that MRP7 is regulated posttranscriptionally, probably at the level of protein synthesis rather than protein turnover. Images PMID:2851722

  20. The nucleotide sequence of the large ribosomal RNA gene and the adjacent tRNA genes from rat mitochondria.

    PubMed Central

    Saccone, C; Cantatore, P; Gadaleta, G; Gallerani, R; Lanave, C; Pepe, G; Kroon, A M

    1981-01-01

    We have sequenced the Eco R(1) fragment D from rat mitochondrial DNA. It contains one third of the tRNA (Val) gene (the remaining part has been sequenced from the 3' end of the Eco R(1) fragment A) the complete gene for the large mt 16S rRNA, the tRNA (Leu) gene and the 5' end of an unidentified reading frame. The mt gene for the large rRNA from rat has been aligned with the homologous genes from mouse and human using graphic computer programs. Hypervariable regions at the center of the molecule and highly conserved regions toward the 3' end have been detected. The mt gene for tRNA Leu is of the conventional type and its primary structure is highly conserved among mammals. The mt gene for tRNA(Val) shows characteristics similar to those of other mt tRNA genes but the degree of homology is lower. Comparative studies confirm that AGA and AGG are read as stop codons in mammalian mitochondria. PMID:6913863

  1. Frequent Changes in the Number of Reiterated Ribosomal RNA Genes Throughout the Life Cycle of the Basidiomycete Coprinus Cinereus

    PubMed Central

    Pukkila, P. J.; Skrzynia, C.

    1993-01-01

    We have examined the stability of the tandemly repeated genes that encode the ribosomal RNA in Coprinus cinereus. These genes are contained within two linked HindIII fragments in a 3.0-Mb chromosome. We monitored the size of these fragments in both mitotic and meiotic segregants using the contour-clamped homogeneous electric field (CHEF) method. No length changes were observed in the smaller HindIII fragment (100 kb; 10 repeats) among the DNAs prepared from 46 asexual spore derivatives (oidia) or 128 meiotic segregants (basidiospores from 32 tetrads). However, the larger HindIII fragment (1100 kb; 120 repeats) did exhibit variability. Substantial changes, involving up to 40% of the larger HindIII fragment were recorded in 7 of 46 oidial isolates (including 4 of 22 transformed derivatives). To learn if the changes were confined to the vegetative portion of the life cycle, we examined transmission of HindIII variants through three crosses. In the first two crosses (16 tetrads total), no changes were observed in the large HindIII fragment. However, in the third cross (16 tetrads), each tetrad showed at least one alteration. In half of the tetrads from the third cross, the altered patterns segregated 2:2, suggesting that the changes occurred after mating but prior to premeiotic DNA replication. We conclude that breakage and rejoining reactions within the rDNA are frequent and are not confined to any particular stage of the life cycle. It also appears that certain repeats are sheltered from these events. Finally, marked differences in rDNA stability were observed in the crosses analyzed. PMID:8436270

  2. 16S partial gene mitochondrial DNA and internal transcribed spacers ribosomal DNA as differential markers of Trichuris discolor populations.

    PubMed

    Callejón, R; Halajian, A; de Rojas, M; Marrugal, A; Guevara, D; Cutillas, C

    2012-05-25

    Comparative morphological, biometrical and molecular studies of Trichuris discolor isolated from Bos taurus from Spain and Iran was carried out. Furthermore, Trichuris ovis isolated from B. taurus and Capra hircus from Spain has been, molecularly, analyzed. Morphological studies revealed clear differences between T. ovis and T. discolor isolated from B. taurus but differences were not observed between populations of T. discolor isolated from different geographical regions. Nevertheless, the molecular studies based on the amplification and sequencing of the internal transcribed spacers 1 and 2 ribosomal DNA and 16S partial gene mitochondrial DNA showed clear differences between both populations of T. discolor from Spain and Iran suggesting two cryptic species. Phylogenetic studies corroborated these data. Thus, phylogenetic trees based on ITS1, ITS2 and 16S partial gene sequences showed that individuals of T. discolor from B. taurus from Iran clustered together and separated, with high bootstrap values, of T. discolor isolated from B. taurus from Spain, while populations of T. ovis from B. taurus and C. hircus from Spain clustered together but separated with high bootstrap values of both populations of T. discolor. Furthermore, a comparative phylogenetic study has been carried out with the ITS1and ITS2 sequences of Trichuris species from different hosts. Three clades were observed: the first clustered all the species of Trichuris parasitizing herbivores (T. discolor, T. ovis, Trichuris leporis and Trichuris skrjabini), the second clustered all the species of Trichuris parasitizing omnivores (Trichuris trichiura and Trichuris suis) and finally, the third clustered species of Trichuris parasitizing carnivores (Trichuris muris, Trichuris arvicolae and Trichuris vulpis). PMID:22136768

  3. Programmed Ribosomal Frameshift Alters Expression of West Nile Virus Genes and Facilitates Virus Replication in Birds and Mosquitoes

    PubMed Central

    Du, Fangyao; Owens, Nick; Bosco-Lauth, Angela M.; Nagasaki, Tomoko; Rudd, Stephen; Brault, Aaron C.; Bowen, Richard A.; Hall, Roy A.; van den Hurk, Andrew F.; Khromykh, Alexander A.

    2014-01-01

    West Nile virus (WNV) is a human pathogen of significant medical importance with close to 40,000 cases of encephalitis and more than 1,600 deaths reported in the US alone since its first emergence in New York in 1999. Previous studies identified a motif in the beginning of non-structural gene NS2A of encephalitic flaviviruses including WNV which induces programmed −1 ribosomal frameshift (PRF) resulting in production of an additional NS protein NS1′. We have previously demonstrated that mutant WNV with abolished PRF was attenuated in mice. Here we have extended our previous observations by showing that PRF does not appear to have a significant role in virus replication, virion formation, and viral spread in several cell lines in vitro. However, we have also shown that PRF induces an over production of structural proteins over non-structural proteins in virus-infected cells and that mutation abolishing PRF is present in ∼11% of the wild type virus population. In vivo experiments in house sparrows using wild type and PRF mutant of New York 99 strain of WNV viruses showed some attenuation for the PRF mutant virus. Moreover, PRF mutant of Kunjin strain of WNV showed significant decrease compared to wild type virus infection in dissemination of the virus from the midgut through the haemocoel, and ultimately the capacity of infected mosquitoes to transmit virus. Thus our results demonstrate an important role for PRF in regulating expression of viral genes and consequently virus replication in avian and mosquito hosts. PMID:25375107

  4. Ribosomal proteins: functions beyond the ribosome

    PubMed Central

    Zhou, Xiang; Liao, Wen-Juan; Liao, Jun-Ming; Liao, Peng; Lu, Hua

    2015-01-01

    Although ribosomal proteins are known for playing an essential role in ribosome assembly and protein translation, their ribosome-independent functions have also been greatly appreciated. Over the past decade, more than a dozen of ribosomal proteins have been found to activate the tumor suppressor p53 pathway in response to ribosomal stress. In addition, these ribosomal proteins are involved in various physiological and pathological processes. This review is composed to overview the current understanding of how ribosomal stress provokes the accumulation of ribosome-free ribosomal proteins, as well as the ribosome-independent functions of ribosomal proteins in tumorigenesis, immune signaling, and development. We also propose the potential of applying these pieces of knowledge to the development of ribosomal stress-based cancer therapeutics. PMID:25735597

  5. NINJA-OPS: Fast Accurate Marker Gene Alignment Using Concatenated Ribosomes

    PubMed Central

    Al-Ghalith, Gabriel A.; Montassier, Emmanuel; Ward, Henry N.; Knights, Dan

    2016-01-01

    The explosion of bioinformatics technologies in the form of next generation sequencing (NGS) has facilitated a massive influx of genomics data in the form of short reads. Short read mapping is therefore a fundamental component of next generation sequencing pipelines which routinely match these short reads against reference genomes for contig assembly. However, such techniques have seldom been applied to microbial marker gene sequencing studies, which have mostly relied on novel heuristic approaches. We propose NINJA Is Not Just Another OTU-Picking Solution (NINJA-OPS, or NINJA for short), a fast and highly accurate novel method enabling reference-based marker gene matching (picking Operational Taxonomic Units, or OTUs). NINJA takes advantage of the Burrows-Wheeler (BW) alignment using an artificial reference chromosome composed of concatenated reference sequences, the “concatesome,” as the BW input. Other features include automatic support for paired-end reads with arbitrary insert sizes. NINJA is also free and open source and implements several pre-filtering methods that elicit substantial speedup when coupled with existing tools. We applied NINJA to several published microbiome studies, obtaining accuracy similar to or better than previous reference-based OTU-picking methods while achieving an order of magnitude or more speedup and using a fraction of the memory footprint. NINJA is a complete pipeline that takes a FASTA-formatted input file and outputs a QIIME-formatted taxonomy-annotated BIOM file for an entire MiSeq run of human gut microbiome 16S genes in under 10 minutes on a dual-core laptop. PMID:26820746

  6. Cytoskeletal protein filamin A is a nucleolar protein that suppresses ribosomal RNA gene transcription.

    PubMed

    Deng, Wensheng; Lopez-Camacho, Cesar; Tang, Jen-Yang; Mendoza-Villanueva, Daniel; Maya-Mendoza, Apolinar; Jackson, Dean A; Shore, Paul

    2012-01-31

    Filamin A (FLNA) is an actin-binding protein with a well-established role in the cytoskeleton, where it determines cell shape and locomotion by cross-linking actin filaments. Mutations in FLNA are associated with a wide range of genetic disorders. Here we demonstrate a unique role for FLNA as a nucleolar protein that associates with the RNA polymerase I (Pol I) transcription machinery to suppress rRNA gene transcription. We show that depletion of FLNA by siRNAs increased rRNA expression, rDNA promoter activity and cell proliferation. Immunodepletion of FLNA from nuclear extracts resulted in a decrease in rDNA promoter-driven transcription in vitro. FLNA coimmunoprecipitated with the Pol I components actin, TIF-IA, and RPA40, and their occupancy of the rDNA promoter was increased in the absence of FLNA in vivo. The FLNA actin-binding domain is essential for the suppression of rRNA expression and for inhibiting recruitment of the Pol I machinery to the rDNA promoter. These findings reveal an additional role for FLNA as a regulator of rRNA gene expression and have important implications for our understanding of the role of FLNA in human disease. PMID:22307607

  7. Zfrp8/PDCD2 Interacts with RpS2 Connecting Ribosome Maturation and Gene-Specific Translation.

    PubMed

    Minakhina, Svetlana; Naryshkina, Tatyana; Changela, Neha; Tan, William; Steward, Ruth

    2016-01-01

    Zfrp8/PDCD2 is a highly conserved protein essential for stem cell maintenance in both flies and mammals. It is also required in fast proliferating cells such as cancer cells. Our previous studies suggested that Zfrp8 functions in the formation of mRNP (mRNA ribonucleoprotein) complexes and also controls RNA of select Transposable Elements (TEs). Here we show that in Zfrp8/PDCD2 knock down (KD) ovaries, specific mRNAs and TE transcripts show increased nuclear accumulation. We also show that Zfrp8/PDCD2 interacts with the (40S) small ribosomal subunit through direct interaction with RpS2 (uS5). By studying the distribution of endogenous and transgenic fluorescently tagged ribosomal proteins we demonstrate that Zfrp8/PDCD2 regulates the cytoplasmic levels of components of the small (40S) ribosomal subunit, but does not control nuclear/nucleolar localization of ribosomal proteins. Our results suggest that Zfrp8/PDCD2 functions at late stages of ribosome assembly and may regulate the binding of specific mRNA-RNPs to the small ribosomal subunit ultimately controlling their cytoplasmic localization and translation. PMID:26807849

  8. Zfrp8/PDCD2 Interacts with RpS2 Connecting Ribosome Maturation and Gene-Specific Translation

    PubMed Central

    Minakhina, Svetlana; Naryshkina, Tatyana; Changela, Neha; Tan, William; Steward, Ruth

    2016-01-01

    Zfrp8/PDCD2 is a highly conserved protein essential for stem cell maintenance in both flies and mammals. It is also required in fast proliferating cells such as cancer cells. Our previous studies suggested that Zfrp8 functions in the formation of mRNP (mRNA ribonucleoprotein) complexes and also controls RNA of select Transposable Elements (TEs). Here we show that in Zfrp8/PDCD2 knock down (KD) ovaries, specific mRNAs and TE transcripts show increased nuclear accumulation. We also show that Zfrp8/PDCD2 interacts with the (40S) small ribosomal subunit through direct interaction with RpS2 (uS5). By studying the distribution of endogenous and transgenic fluorescently tagged ribosomal proteins we demonstrate that Zfrp8/PDCD2 regulates the cytoplasmic levels of components of the small (40S) ribosomal subunit, but does not control nuclear/nucleolar localization of ribosomal proteins. Our results suggest that Zfrp8/PDCD2 functions at late stages of ribosome assembly and may regulate the binding of specific mRNA-RNPs to the small ribosomal subunit ultimately controlling their cytoplasmic localization and translation. PMID:26807849

  9. Unusual dispersion of histone repeats on the whole chromosomal complement and their colocalization with ribosomal genes in Rachycentron canadum (Rachycentridae, Perciformes).

    PubMed

    Costa, Gideão W W F; Cioffi, Marcelo B; Bertollo, Luiz A C; Molina, Wagner F

    2014-01-01

    Rachycentron canadum, the only representative of the family Rachycentridae, has been the focus of biotechnological interest due to its significant potential in marine fish farming. The chromosome set of this species has been widely investigated with respect to the location of genes and multigene families. A FISH analysis was performed using 4 multigene families as probes, represented by 5S and 18S ribosomal genes and histones H2B-H2A and H3. Earlier data suggested that differential replication of heterochromatin could be partially associated with functional genes. Indeed, our results showed that the DNA contained in heterochromatic regions of R. canadum contains 5S and 18S ribosomal genes as well as the gene sequences of histones H2B-H2A and H3, which were colocalized. The distribution of H3 sequences in all heterochromatic regions, except in 13q, could indicate an important evolutionary role for this class of repetitive sequences. Besides, the presence of chromosome regions bearing multifunctional repetitive sequences formed by H2B-H2A/H3/18S rDNA and H2B-H2A/H3/5S rDNA clusters was demonstrated for the first time in fishes. The implications of differential histone gene extension and its functionality in the karyotype of R. canadum remain unknown. PMID:25341625

  10. Mutation in P0, a dual function ribosomal protein/apurinic/apyrimidinic endonuclease, modifies gene expression and position effect variegation in Drosophila.

    PubMed Central

    Frolov, M V; Birchler, J A

    1998-01-01

    In a search for modifiers of gene expression with the white eye color gene as a target, a third chromosomal P-element insertion mutant l(3)01544 has been identified that exhibits a strong pigment increase in a white-apricot background. Molecular analysis shows that the P-element insertion is found in the first intron of the gene surrounding the insertion site. Sequencing both the cDNA and genomic fragments revealed that the identified gene is identical to one encoding ribosomal protein P0/apurinic/apyrimidinic endonuclease. The P-element-induced mutation, l(3)01544, affects the steady-state level of white transcripts and transcripts of some other genes. In addition, l(3)01544 suppresses the variegated phenotypes of In(1)wm4h and In(1)y3P, suggesting a potential involvement of the P0 protein in modifying position effect variegation. The revertant generated by the precise excision of the P element has lost all mutant phenotypes. Recent work revealed that Drosophila ribosomal protein P0 contains an apurinic/apyrimidinic endonuclease activity. Our results suggest that this multifunctional protein is also involved in regulation of gene expression in Drosophila. PMID:9832526

  11. 16Stimator: statistical estimation of ribosomal gene copy numbers from draft genome assemblies.

    PubMed

    Perisin, Matthew; Vetter, Madlen; Gilbert, Jack A; Bergelson, Joy

    2016-04-01

    The 16S rRNA gene (16S) is an accepted marker of bacterial taxonomic diversity, even though differences in copy number obscure the relationship between amplicon and organismal abundances. Ancestral state reconstruction methods can predict 16S copy numbers through comparisons with closely related reference genomes; however, the database of closed genomes is limited. Here, we extend the reference database of 16S copy numbers to de novo assembled draft genomes by developing 16Stimator, a method to estimate 16S copy numbers when these repetitive regions collapse during assembly. Using a read depth approach, we estimate 16S copy numbers for 12 endophytic isolates from Arabidopsis thaliana and confirm estimates by qPCR. We further apply this approach to draft genomes deposited in NCBI and demonstrate accurate copy number estimation regardless of sequencing platform, with an overall median deviation of 14%. The expanded database of isolates with 16S copy number estimates increases the power of phylogenetic correction methods for determining organismal abundances from 16S amplicon surveys. PMID:26359911

  12. Important Genes in the Pathogenesis of 5q- Syndrome and Their Connection with Ribosomal Stress and the Innate Immune System Pathway

    PubMed Central

    Fuchs, Ota

    2012-01-01

    Myelodysplastic syndrome (MDS) with interstitial deletion of a segment of the long arm of chromosome 5q [del(5q)] is characterized by bone marrow erythroid hyperplasia, atypical megakaryocytes, thrombocythemia, refractory anemia, and low risk of progression to acute myeloid leukemia (AML) compared with other types of MDS. The long arm of chromosome 5 contains two distinct commonly deleted regions (CDRs). The more distal CDR lies in 5q33.1 and contains 40 protein-coding genes and genes coding microRNAs (miR-143, miR-145). In 5q-syndrome one allele is deleted that accounts for haploinsufficiency of these genes. The mechanism of erythroid failure appears to involve the decreased expression of the ribosomal protein S14 (RPS14) gene and the upregulation of the p53 pathway by ribosomal stress. Friend leukemia virus integration 1 (Fli1) is one of the target genes of miR145. Increased Fli1 expression enables effective megakaryopoiesis in 5q-syndrome. PMID:23213547

  13. Translational machinery of the chaetognath Spadella cephaloptera: a transcriptomic approach to the analysis of cytosolic ribosomal protein genes and their expression

    PubMed Central

    Barthélémy, Roxane M; Chenuil, Anne; Blanquart, Samuel; Casanova, Jean-Paul; Faure, Eric

    2007-01-01

    Background Chaetognaths, or arrow worms, are small marine, bilaterally symmetrical metazoans. The objective of this study was to analyse ribosomal protein (RP) coding sequences from a published collection of expressed sequence tags (ESTs) from a chaetognath (Spadella cephaloptera) and to use them in phylogenetic studies. Results This analysis has allowed us to determine the complete primary structures of 23 out of 32 RPs from the small ribosomal subunit (SSU) and 32 out of 47 RPs from the large ribosomal subunit (LSU). Ten proteins are partially determined and 14 proteins are missing. Phylogenetic analyses of concatenated RPs from six animals (chaetognath, echinoderm, mammalian, insect, mollusc and sponge) and one fungal taxa do not resolve the chaetognath phylogenetic position, although each mega-sequence comprises approximately 5,000 amino acid residues. This is probably due to the extremely biased base composition and to the high evolutionary rates in chaetognaths. However, the analysis of chaetognath RP genes revealed three unique features in the animal Kingdom. First, whereas generally in animals one RP appeared to have a single type of mRNA, two or more genes are generally transcribed for one RP type in chaetognath. Second, cDNAs with complete 5'-ends encoding a given protein sequence can be divided in two sub-groups according to a short region in their 5'-ends: two novel and highly conserved elements have been identified (5'-TAATTGAGTAGTTT-3' and 5'-TATTAAGTACTAC-3') which could correspond to different transcription factor binding sites on paralog RP genes. And, third, the overall number of deduced paralogous RPs is very high compared to those published for other animals. Conclusion These results suggest that in chaetognaths the deleterious effects of the presence of paralogous RPs, such as apoptosis or cancer are avoided, and also that in each protein family, some of the members could have tissue-specific and extra-ribosomal functions. These results are

  14. Co-expression of a modified maize ribosome-inactivating protein and a rice basic chitinase gene in transgenic rice plants confers enhanced resistance to sheath blight.

    PubMed

    Kim, Ju-Kon; Jang, In-Cheol; Wu, Ray; Zuo, Wei-Neng; Boston, Rebecca S; Lee, Yong-Hwan; Ahn, Il-Pyung; Nahm, Baek Hie

    2003-08-01

    Chitinases, beta-1,3-glucanases, and ribosome-inactivating proteins are reported to have antifungal activity in plants. With the aim of producing fungus-resistant transgenic plants, we co-expressed a modified maize ribosome-inactivating protein gene, MOD1, and a rice basic chitinase gene, RCH10, in transgenic rice plants. A construct containing MOD1 and RCH10 under the control of the rice rbcS and Act1 promoters, respectively, was co-transformed with a plasmid containing the herbicide-resistance gene bar as a selection marker into rice by particle bombardment. Several transformants analyzed by genomic Southern-blot hybridization demonstrated integration of multiple copies of the foreign gene into rice chromosomes. Immunoblot experiments showed that MOD1 formed approximately 0.5% of the total soluble protein in transgenic leaves. RCH10 expression was examined using the native polyacrylamide-overlay gel method, and high RCH10 activity was observed in leaf tissues where endogenous RCH10 is not expressed. R1 plants were analyzed in a similar way, and the Southern-blot patterns and levels of transgene expression remained the same as in the parental line. Analysis of the response of R2 plants to three fungal pathogens of rice, Rhizoctonia solani, Bipolaris oryzae, and Magnaporthe grisea, indicated statistically significant symptom reduction only in the case of R. solani (sheath blight). The increased resistance co-segregated with herbicide tolerance, reflecting a correlation between the resistance phenotype and transgene expression. PMID:12885168

  15. The trp RNA-binding attenuation protein of Bacillus subtilis regulates translation of the tryptophan transport gene trpP (yhaG) by blocking ribosome binding.

    PubMed

    Yakhnin, Helen; Zhang, Hong; Yakhnin, Alexander V; Babitzke, Paul

    2004-01-01

    Expression of the Bacillus subtilis tryptophan biosynthetic genes (trpEDCFBA and pabA [trpG]) is regulated in response to tryptophan by TRAP, the trp RNA-binding attenuation protein. TRAP-mediated regulation of the tryptophan biosynthetic genes includes a transcription attenuation and two distinct translation control mechanisms. TRAP also regulates translation of trpP (yhaG), a single-gene operon that encodes a putative tryptophan transporter. Its translation initiation region contains triplet repeats typical of TRAP-regulated mRNAs. We found that regulation of trpP and pabA is unaltered in a rho mutant strain. Results from filter binding and gel mobility shift assays demonstrated that TRAP binds specifically to a segment of the trpP transcript that includes the untranslated leader and translation initiation region. While the affinities of TRAP for the trpP and pabA transcripts are similar, TRAP-mediated translation control of trpP is much more extensive than for pabA. RNA footprinting revealed that the trpP TRAP binding site consists of nine triplet repeats (five GAG, three UAG, and one AAG) that surround and overlap the trpP Shine-Dalgarno (S-D) sequence and translation start codon. Results from toeprint and RNA-directed cell-free translation experiments indicated that tryptophan-activated TRAP inhibits TrpP synthesis by preventing binding of a 30S ribosomal subunit. Taken together, our results establish that TRAP regulates translation of trpP by blocking ribosome binding. Thus, TRAP coordinately regulates tryptophan synthesis and transport by three distinct mechanisms: attenuation transcription of the trpEDCFBA operon, promoting formation of the trpE S-D blocking hairpin, and blocking ribosome binding to the pabA and trpP transcripts. PMID:14702295

  16. Synthesis of ribosomes in Saccharomyces cerevisiae.

    PubMed Central

    Warner, J R

    1989-01-01

    The assembly of a eucaryotic ribosome requires the synthesis of four ribosomal ribonucleic acid (RNA) molecules and more than 75 ribosomal proteins. It utilizes all three RNA polymerases; it requires the cooperation of the nucleus and the cytoplasm, the processing of RNA, and the specific interaction of RNA and protein molecules. It is carried out efficiently and is exquisitely sensitive to the needs of the cell. Our current understanding of this process in the genetically tractable yeast Saccharomyces cerevisiae is reviewed. The ribosomal RNA genes are arranged in a tandem array of 100 to 200 copies. This tandem array has led to unique ways of carrying out a number of functions. Replication is asymmetric and does not initiate from every autonomously replicating sequence. Recombination is suppressed. Transcription of the major ribosomal RNA appears to involve coupling between adjacent transcription units, which are separated by the 5S RNA transcription unit. Genes for many ribosomal proteins have been cloned and sequenced. Few are linked; most are duplicated; most have an intron. There is extensive homology between yeast ribosomal proteins and those of other species. Most, but not all, of the ribosomal protein genes have one or two sites that are essential for their transcription and that bind a common transcription factor. This factor binds also to many other places in the genome, including the telomeres. There is coordinated transcription of the ribosomal protein genes under a variety of conditions. However, the cell seems to possess no mechanism for regulating the transcription of individual ribosomal protein genes in response either to a deficiency or an excess of a particular ribosomal protein. A deficiency causes slow growth. Any excess ribosomal protein is degraded very rapidly, with a half-life of 1 to 5 min. Unlike most types of cells, yeast cells appear not to regulate the translation of ribosomal proteins. However, in the case of ribosomal protein L32

  17. A Case of Sepsis in a 92-Year-Old Korean Woman Caused by Aerococcus urinae and Identified by Sequencing the 16S Ribosomal RNA Gene.

    PubMed

    Lee, Min Young; Kim, Myeong Hee; Lee, Woo In; Kang, So Young; Jeon, You La

    2016-05-01

    Aerococcus urinaeis an uncommon pathogen that was first identified in 1992. Herein, we report a case of bloodstream infection caused byA. urinae, which occurred in a 92-year-old Korean female patient with an underlying urologic infection who had altered consciousness. The blood culture yielded positive results forA. urinae; however, identifyingA. urinaewas challenging. Ultimately, we used 16S ribosomal RNA (rRNA) gene sequencing to identify the organism. The patient recovered after being treated with ertapenem and meropenem. To our knowledge, this is the first report of a case ofA. urinaesepsis in South Korea. PMID:26868516

  18. Systematics of Chaetognatha under the light of molecular data, using duplicated ribosomal 18S DNA sequences.

    PubMed

    Papillon, Daniel; Perez, Yvan; Caubit, Xavier; Le Parco, Yannick

    2006-03-01

    While the phylogenetic position of Chaetognatha has became central to the question of early bilaterian evolution, the internal systematics of the phylum are still not clear. The phylogenetic relationships of the chaetognaths were investigated using newly obtained small subunit ribosomal RNA nuclear 18S (SSU rRNA) sequences from 16 species together with 3 sequences available in GenBank. As previously shown with the large subunit ribosomal RNA 28S gene, two classes of Chaetognatha SSU rRNA gene can be identified, suggesting a duplication of the whole ribosomal cluster; allowing the rooting of one class of genes by another in phylogenetic analyses. Maximum Parsimony, Maximum Likelihood and Bayesian analyses of the molecular data, and statistical tests showed (1) that there are three main monophyletic groups: Sagittidae/Krohnittidae, Spadellidae/Pterosagittidae, and Eukrohniidae/Heterokrohniidae, (2) that the group of Aphragmophora without Pterosagittidae (Sagittidae/Krohnittidae) is monophyletic, (3) the Spadellidae/Pterosagittidae and Eukrohniidae/Heterokrohniidae families are very likely clustered, (4) the Krohnittidae and Pterosagittidae groups should no longer be considered as families as they are included in other groups designated as families, (5) suborder Ctenodontina is not monophyletic and the Flabellodontina should no longer be considered as a suborder, and (6) the Syngonata/Chorismogonata and the Monophragmophora/Biphragmophora hypotheses are rejected. Such conclusions are considered in the light of morphological characters, several of which are shown to be prone to homoplasy. PMID:16434216

  19. The yeast NOP4 gene product is an essential nucleolar protein required for pre-rRNA processing and accumulation of 60S ribosomal subunits.

    PubMed Central

    Sun, C; Woolford, J L

    1994-01-01

    The Saccharomyces cerevisiae NOP4 gene was isolated by screening a lambda gt11 yeast genomic DNA library with a monoclonal antibody against a yeast nucleolar protein. NOP4 encodes a 78 kDa protein that contains two prototypical RNA recognition motifs (RRMs) flanking an imperfect RRM lacking characteristic RNP1 and RNP2 motifs. In addition, there is a fourth incomplete RRM. NOP4 is a single copy essential gene present on chromosome XVI, between RAD1 and PEP4. To examine the function of Nop4p, we constructed a conditional null allele of NOP4 by placing this gene under the control of the glucose-repressible GAL1 promoter. When cells are shifted from galactose-containing medium to glucose-containing medium, NOP4 transcription is terminated, Nop4 protein is depleted and cell growth is impaired. Nop4 protein depletion results in diminished accumulation of 60S ribosomal subunits, assignable to a defect in ribosome biogenesis arising from a lack of production of mature 25S rRNA from 27S precursor rRNA. Images PMID:8039505

  20. Increased Expression of X-Linked Genes in Mammals Is Associated with a Higher Stability of Transcripts and an Increased Ribosome Density

    PubMed Central

    Faucillion, Marie-Line; Larsson, Jan

    2015-01-01

    Mammalian sex chromosomes evolved from the degeneration of one homolog of a pair of ancestral autosomes, the proto-Y. This resulted in a gene dose imbalance that is believed to be restored (partially or fully) through upregulation of gene expression from the single active X-chromosome in both sexes by a dosage compensatory mechanism. We analyzed multiple genome-wide RNA stability data sets and found significantly longer average half-lives for X-chromosome transcripts than for autosomal transcripts in various human cell lines, both male and female, and in mice. Analysis of ribosome profiling data shows that ribosome density is higher on X-chromosome transcripts than on autosomal transcripts in both humans and mice, suggesting that the higher stability is causally linked to a higher translation rate. Our results and observations are in accordance with a dosage compensatory upregulation of expressed X-linked genes. We therefore propose that differential mRNA stability and translation rates of the autosomes and sex chromosomes contribute to an evolutionarily conserved dosage compensation mechanism in mammals. PMID:25786432

  1. Evidence for the ability of L10 ribosomal proteins of Salmonella typhimurium and Klebsiella pneumoniae to regulate rplJL gene expression in Escherichia coli.

    PubMed

    Paton, E B; Woodmaska, M I; Kroupskaya, I V; Zhyvoloup, A N; Matsuka, G Kh

    1990-06-01

    Genes rplJ, coding for ribosomal protein L10 of Salmonella typhimurium and Klebsiella pneumoniae, have been cloned on pUC plasmid. The resultant multicopy recombinant plasmids were detrimental for the growth of normal JM101 E. coli host cells and harmless for the mutant JF3029 host. This negative effect is the evidence for the ability of heterologous L10 proteins to regulate expression of rplJL genes in E. coli. Nucleotide sequence was determined completely for S. typhimurium rplJL' DNA portion and partially for rplJL' genes of K. pneumoniae. According to the nucleotide sequence data obtained three amino acid substitutions differ L10 proteins of S. typhimurium and E. coli and the long range, providing for the coupled translations of L10 and L7/L12 cistrons in E. coli mRNA is also valid for S. typhimurium and K. pneumoniae. PMID:2194828

  2. Structure of the DNA distal to the gene for ribosomal protein S20 in Escherichia coli K12: presence of a strong terminator and an IS1 element.

    PubMed Central

    Mackie, G A

    1986-01-01

    The sequence of nucleotides extending over 2.3 kb distal to the gene for ribosomal protein S20 of E. coli has been determined. Included in the sequence is an efficient rho-independent terminator 50 b.p. distal to the coding sequence for S20, a complete copy of IS1 which lacks, however, flanking direct repeats, and finally, an open reading frame capable of encoding a 28 kDa polypeptide of unknown function. Several lines of evidence suggest that the IS1 sequence described here must represent one of the copies resident in the bacterial chromosome rather than a newly transposed copy. Northern blotting experiments show that the gene for S20 is functionally monocistronic under all conditions tested in several genetic backgrounds. Thus it seems unlikely that the distal copy of IS1 plays any role in the termination or stability of mRNA transcribed from the gene for S20. Images PMID:2429258

  3. Promoter architectures in the yeast ribosomal expression program

    PubMed Central

    Bosio, Maria Cristina; Negri, Rodolfo

    2011-01-01

    Ribosome biogenesis begins with the orchestrated expression of hundreds of genes, including the three large classes of ribosomal protein, ribosome biogenesis and snoRNA genes. Current knowledge about the corresponding promoters suggests the existence of novel class-specific transcriptional strategies and crosstalk between telomere length and cell growth control. PMID:21468232

  4. Ribosomal Database Project II

    DOE Data Explorer

    The Ribosomal Database Project (RDP) provides ribosome related data and services to the scientific community, including online data analysis and aligned and annotated Bacterial small-subunit 16S rRNA sequences. As of March 2008, RDP Release 10 is available and currently (August 2009) contains 1,074,075 aligned 16S rRNA sequences. Data that can be downloaded include zipped GenBank and FASTA alignment files, a histogram (in Excel) of the number of RDP sequences spanning each base position, data in the Functional Gene Pipeline Repository, and various user submitted data. The RDP-II website also provides numerous analysis tools.[From the RDP-II home page at http://rdp.cme.msu.edu/index.jsp

  5. Characterizing inactive ribosomes in translational profiling.

    PubMed

    Liu, Botao; Qian, Shu-Bing

    2016-01-01

    The broad impact of translational regulation has emerged explosively in the last few years in part due to the technological advance in genome-wide interrogation of gene expression. During mRNA translation, the majority of actively translating ribosomes exist as polysomes in cells with multiple ribosomes loaded on a single transcript. The importance of the monosome, however, has been less appreciated in translational profiling analysis. Here we report that the monosome fraction isolated by sucrose sedimentation contains a large quantity of inactive ribosomes that do not engage on mRNAs to direct translation. We found that the elongation factor eEF2, but not eEF1A, stably resides in these non-translating ribosomes. This unique feature permits direct evaluation of ribosome status under various stress conditions and in the presence of translation inhibitors. Ribosome profiling reveals that the monosome has a similar but not identical pattern of ribosome footprints compared to the polysome. We show that the association of free ribosomal subunits minimally contributes to ribosome occupancy outside of the coding region. Our results not only offer a quantitative method to monitor ribosome availability, but also uncover additional layers of ribosome status needed to be considered in translational profiling analysis. PMID:27335722

  6. Characterization of the L11, L1, L10 and L12 equivalent ribosomal protein gene cluster of the halophilic archaebacterium Halobacterium cutirubrum.

    PubMed Central

    Shimmin, L C; Dennis, P P

    1989-01-01

    We have cloned and characterized a 5.2 kb fragment of genomic Halobacterium cutirubrum DNA encoding two potential proteins of unknown function (ORF and NAB) and four proteins which are equivalent to the L11, L1, L10 and L12 ribosomal proteins of Escherichia coli (L11e, L1e, L10e and L12e). The ribosomal protein genes are clustered in the same order as that in E. coli although the transcription pattern differs. Transcripts characterized include (i) abundant monocistronic L11e and tricistronic L1e-L10e-L12e transcripts; (ii) less abundant bicistronic NAB-L11e and monocistronic NAB transcripts and (iii) a very rare ORF monocistronic transcript. The consensus sequence in the promoter region is TTCGA ... 4-10 nucleotides ... TTAA ... 25-26 nucleotides ... initiation site; termination generally occurs on poly(T) tracts following GC-rich regions. Poly(T) tracts in the sense strands within coding regions are notably absent; this is probably related to their participation in transcription termination and to the fact that these ribosomal protein genes are highly expressed and stoichiometrically balanced. In the third position of the codons G or C is utilized 87% of the time. The 74 nt long untranslated leader of the L1e-L10e-L12e transcript contains a region that has a sequence and structure almost identical to a region within the binding domain for the L1e protein in 23S rRNA and highly similar to the E. coli L11-L1 mRNA leader sequence that has been implicated in autogenous translational regulation. Other transcripts are initiated at or adjacent to the ATG translation initiation codon. Images PMID:2743981

  7. Mice with a Mutation in the Mdm2 Gene That Interferes with MDM2/Ribosomal Protein Binding Develop a Defect in Erythropoiesis

    PubMed Central

    Kamio, Takuya; Gu, Bai-wei; Olson, Timothy S.; Zhang, Yanping; Mason, Philip J.; Bessler, Monica

    2016-01-01

    MDM2, an E3 ubiquitin ligase, is an important negative regulator of tumor suppressor p53. In turn the Mdm2 gene is a transcriptional target of p53, forming a negative feedback loop that is important in cell cycle control. It has recently become apparent that the ubiquitination of p53 by MDM2 can be inhibited when certain ribosomal proteins, including RPL5 and RPL11, bind to MDM2. This inhibition, and the resulting increase in p53 levels has been proposed to be responsible for the red cell aplasia seen in Diamond-Blackfan anemia (DBA) and in 5q- myelodysplastic syndrome (MDS). DBA and 5q- MDS are associated with inherited (DBA) or acquired (5q- MDS) haploinsufficiency of ribosomal proteins. A mutation in Mdm2 causing a C305F amino acid substitution blocks the binding of ribosomal proteins. Mice harboring this mutation (Mdm2C305F), retain a normal p53 response to DNA damage, but lack the p53 response to perturbations in ribosome biogenesis. While studying the interaction between RP haploinsufficiency and the Mdm2C305F mutation we noticed that Mdm2C305F homozygous mice had altered hematopoiesis. These mice developed a mild macrocytic anemia with reticulocytosis. In the bone marrow (BM), these mice showed a significant decrease in Ter119hi cells compared to wild type (WT) littermates, while no decrease in the number of mature erythroid cells (Ter119hiCD71low) was found in the spleen, which showed compensated bone marrow hematopoiesis. In methylcellulose cultures, BFU-E colonies from the mutant mice were slightly reduced in number and there was a significant reduction in CFU-E colony numbers in mutant mice compared with WT controls (p < 0.01). This erythropoietic defect was abrogated by concomitant p53 deficiency (Trp53ko/ko). Further investigation revealed that in Mdm2C305F animals, there was a decrease in Lin-Sca-1+c-Kit+ (LSK) cells, accompanied by significant decreases in multipotent progenitor (MPP) cells (p < 0.01). Competitive BM repopulation experiments showed

  8. Mice with a Mutation in the Mdm2 Gene That Interferes with MDM2/Ribosomal Protein Binding Develop a Defect in Erythropoiesis.

    PubMed

    Kamio, Takuya; Gu, Bai-Wei; Olson, Timothy S; Zhang, Yanping; Mason, Philip J; Bessler, Monica

    2016-01-01

    MDM2, an E3 ubiquitin ligase, is an important negative regulator of tumor suppressor p53. In turn the Mdm2 gene is a transcriptional target of p53, forming a negative feedback loop that is important in cell cycle control. It has recently become apparent that the ubiquitination of p53 by MDM2 can be inhibited when certain ribosomal proteins, including RPL5 and RPL11, bind to MDM2. This inhibition, and the resulting increase in p53 levels has been proposed to be responsible for the red cell aplasia seen in Diamond-Blackfan anemia (DBA) and in 5q- myelodysplastic syndrome (MDS). DBA and 5q- MDS are associated with inherited (DBA) or acquired (5q- MDS) haploinsufficiency of ribosomal proteins. A mutation in Mdm2 causing a C305F amino acid substitution blocks the binding of ribosomal proteins. Mice harboring this mutation (Mdm2C305F), retain a normal p53 response to DNA damage, but lack the p53 response to perturbations in ribosome biogenesis. While studying the interaction between RP haploinsufficiency and the Mdm2C305F mutation we noticed that Mdm2C305F homozygous mice had altered hematopoiesis. These mice developed a mild macrocytic anemia with reticulocytosis. In the bone marrow (BM), these mice showed a significant decrease in Ter119hi cells compared to wild type (WT) littermates, while no decrease in the number of mature erythroid cells (Ter119hiCD71low) was found in the spleen, which showed compensated bone marrow hematopoiesis. In methylcellulose cultures, BFU-E colonies from the mutant mice were slightly reduced in number and there was a significant reduction in CFU-E colony numbers in mutant mice compared with WT controls (p < 0.01). This erythropoietic defect was abrogated by concomitant p53 deficiency (Trp53ko/ko). Further investigation revealed that in Mdm2C305F animals, there was a decrease in Lin-Sca-1+c-Kit+ (LSK) cells, accompanied by significant decreases in multipotent progenitor (MPP) cells (p < 0.01). Competitive BM repopulation experiments showed

  9. The economics of ribosome biosynthesis in yeast.

    PubMed

    Warner, J R

    1999-11-01

    In a rapidly growing yeast cell, 60% of total transcription is devoted to ribosomal RNA, and 50% of RNA polymerase II transcription and 90% of mRNA splicing are devoted to ribosomal proteins (RPs). Coordinate regulation of the approximately 150 rRNA genes and 137 RP genes that make such prodigious use of resources is essential for the economy of the cell. This is entrusted to a number of signal transduction pathways that can abruptly induce or silence the ribosomal genes, leading to major implications for the expression of other genes as well. PMID:10542411

  10. 28s rDNA group-I introns: a powerful tool for identifying strains of Beauveria brongniartii.

    PubMed

    Neuvéglise, C; Brygoo, Y; Riba, G

    1997-04-01

    The nuclear ribosomal DNA of the entomopathogenic fungus Beauveria brongniartii is polymorphic in terms of both restriction site and length. Insertions of 350-450 bp long, identified as group-I introns, were detected in the 28s rDNA. A panel of 47 strains of B. brongniartii, two B. bassiana and one Metarhizium anisopliae of various geographical and biological origins were found to contain 14 variant forms of intron differing in size and restriction pattern, at four different positions. Twelve types of ribosomal large subunit were defined on the basis of variant distribution and compared with strain clustering based on internal transcribed spacers analysis. There was a correlation between the characteristic introns and isolates collected from the sugar cane pest Hoplochelus marginalis. Primers for polymerase chain reaction amplification were chosen from these variants, and used to develop a specific method for detecting strains pathogenic towards Hoplochelus. PMID:9131812

  11. Microsporidian Encephalitozoon cuniculi, a unicellular eukaryote with an unusual chromosomal dispersion of ribosomal genes and a LSU rRNA reduced to the universal core.

    PubMed Central

    Peyretaillade, E; Biderre, C; Peyret, P; Duffieux, F; Méténier, G; Gouy, M; Michot, B; Vivarès, C P

    1998-01-01

    Microsporidia are eukaryotic parasites lacking mitochondria, the ribosomes of which present prokaryote-like features. In order to better understand the structural evolution of rRNA molecules in microsporidia, the 5S and rDNA genes were investigated in Encephalitozoon cuniculi . The genes are not in close proximity. Non-tandemly arranged rDNA units are on every one of the 11 chromosomes. Such a dispersion is also shown in two other Encephalitozoon species. Sequencing of the 5S rRNA coding region reveals a 120 nt long RNA which folds according to the eukaryotic consensus structural shape. In contrast, the LSU rRNA molecule is greatly reduced in length (2487 nt). This dramatic shortening is essentially due to truncation of divergent domains, most of them being removed. Most variable stems of the conserved core are also deleted, reducing the LSU rRNA to only those structural features preserved in all living cells. This suggests that the E.cuniculi LSU rRNA performs only the basic mechanisms of translation. LSU rRNA phylogenetic analysis with the BASEML program favours a relatively recent origin of the fast evolving microsporidian lineage. Therefore, the prokaryote-like ribosomal features, such as the absence of ITS2, may be derived rather than primitive characters. PMID:9671812

  12. Genotyping of a miso and soy sauce fermentation yeast, Zygosaccharomyces rouxii, based on sequence analysis of the partial 26S ribosomal RNA gene and two internal transcribed spacers.

    PubMed

    Suezawa, Yasuhiko; Suzuki, Motofumi; Mori, Haruhiko

    2008-09-01

    We analyzed sequences of the D1D2 domain of the 26S ribosomal RNA gene (26S rDNA sequence), the internal transcribed spacer 1, the 5.8S ribosomal RNA gene, and the internal transcribed spacer 2 (the ITS sequence) from 46 strains of miso and soy sauce fermentation yeast, Zygosaccharomyces rouxii and a closely related species, Z. mellis, for typing. Based on the 26S rDNA sequence analysis, the Z. rouxii strains were of two types, and the extent of sequence divergence between them was 2.6%. Based on the ITS sequence analysis, they were divided into seven types (I-VII). Between the type strain (type I) and type VI, in particular, a 12% difference was detected. The occurrence of these nine genotypes with a divergence of more than 1% in these two sequences suggests that Z. rouxii is a species complex including novel species and hybrids. Z. mellis strains were of two types (type alpha and type beta) based on the ITS sequence. Z. rouxii could clearly be distinguished from Z. mellis by 26S rDNA and ITS sequence analyses, but not by the 16% NaCl tolerance, when used as the sole key characteristic for differentiation between the two species. PMID:18776675

  13. Functional characterization of transcriptional regulatory elements in the upstream region and intron 1 of the human S6 ribosomal protein gene.

    PubMed Central

    Antoine, M; Kiefer, P

    1998-01-01

    Expression of housekeeping genes involves regulation at comparable levels in a wide spectrum of cells. To define the cis-regulatory elements in the human S6 ribosomal protein (rpS6) gene, we made a series of deletions of the upstream non-transcribed region, including or excluding exon 1 or intron 1 sequences. The mutated rpS6 gene regulatory regions were fused to the chloramphenicol acetyltransferase reporter gene and transfected into HeLa and COS-1 cells. The results have identified three parts of the rpS6 gene that are required for efficient and specific transcription. The core promoter includes only a 40 bp region upstream of the transcription start site and initiation region. Both upstream and intronic elements enhance transcription from the core promoter. Furthermore, mutation of the splice donor site of intron 1 almost completely abolished the enhancing activity of the intronic transcriptional modulator. We used gel retardation assays to identify sequence-specific binding sites in the upstream region and in the proximal half of intron 1. Both common and different nuclear factors that bind the rpS6 gene promoter were identified in extracts from HeLa and COS-1 cells, suggesting that different transcription factors may bind specifically to the same binding region and might be interchangeable in their function to ensure high-level expression of housekeeping genes independently of the cell type. PMID:9820808

  14. The Zinc Finger Protein ZNF658 Regulates the Transcription of Genes Involved in Zinc Homeostasis and Affects Ribosome Biogenesis through the Zinc Transcriptional Regulatory Element

    PubMed Central

    Ogo, Ogo A.; Tyson, John; Cockell, Simon J.; Howard, Alison; Valentine, Ruth A.

    2015-01-01

    We previously identified the ZTRE (zinc transcriptional regulatory element) in genes involved in zinc homeostasis and showed that it mediates transcriptional repression in response to zinc. We now report that ZNF658 acts at the ZTRE. ZNF658 was identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry of a band excised after electrophoretic mobility shift assay using a ZTRE probe. The protein contains a KRAB domain and 21 zinc fingers. It has similarity with ZAP1 from Saccharomyces cerevisiae, which regulates the response to zinc restriction, including a conserved DNA binding region we show to be functional also in ZNF658. Small interfering RNA (siRNA) targeted to ZNF658 abrogated the zinc-induced, ZTRE-dependent reduction in SLC30A5 (ZnT5 gene), SLC30A10 (ZnT10 gene), and CBWD transcripts in human Caco-2 cells and the ability of zinc to repress reporter gene expression from corresponding promoter-reporter constructs. Microarray analysis of the effect of reducing ZNF658 expression by siRNA uncovered a large decrease in rRNA. We find that ZTREs are clustered within the 45S rRNA precursor. We also saw effects on expression of multiple ribosomal proteins. ZNF658 thus links zinc homeostasis with ribosome biogenesis, the most active transcriptional, and hence zinc-demanding, process in the cell. ZNF658 is thus a novel transcriptional regulator that plays a fundamental role in the orchestrated cellular response to zinc availability. PMID:25582195

  15. Development of a Newcastle disease virus vector expressing a foreign gene through an internal ribosomal entry site provides direct proof for a sequential transcription mechanism.

    PubMed

    Zhang, Zhenyu; Zhao, Wei; Li, Deshan; Yang, Jinlong; Zsak, Laszlo; Yu, Qingzhong

    2015-08-01

    In the present study, we developed a novel approach for foreign gene expression by Newcastle disease virus (NDV) from a second ORF through an internal ribosomal entry site (IRES). Six NDV LaSota strain-based recombinant viruses vectoring the IRES and a red fluorescence protein (RFP) gene behind the nucleocapsid (NP), phosphoprotein (P), matrix (M), fusion (F), haemagglutinin-neuraminidase (HN) or large polymerase (L) gene ORF were generated using reverse genetics technology. The insertion of the second ORF slightly attenuated virus pathogenicity, but did not affect ability of the virus to grow. Quantitative measurements of RFP expression in virus-infected DF-1 cells revealed that the abundance of viral mRNAs and red fluorescence intensity were positively correlated with the gene order of NDV, 3'-NP-P-M-F-HN-L-5', proving the sequential transcription mechanism for NDV. The results herein suggest that the level of foreign gene expression could be regulated by selecting the second ORF insertion site to maximize the efficacy of vaccine and gene therapy. PMID:25872740

  16. The structure of the human intron-containing S8 ribosomal protein gene and determination of its chromosomal location at 1p32-p32. 4

    SciTech Connect

    Davies, B.; Fried, M. )

    1993-01-01

    The intron-containing gene encoding human ribosomal protein SS (RPS8) has been cloned and characterized, and its chromosomal position determined. Using a PCR-based cloning strategy, we have isolated the intron-containing gene in the presence of its many processed pseudogenes and determined the DNA sequence of the entire gene and its upstream and downstream flanking regions. The human RPS8 gene is 3161 bp in length and comprises six exons. Despite lacking a consensus TATA box, primer extension analysis indicates that the start of transcription is precisely located at a C residue within an 11-bp oligopyrimidine tract. The first exon, which contains the ATG start codon, is just 27 bp in length. The DNA sequence 5[prime] to the RPS8 gene and within the first exon and intron shows several features of a CpG island. A combination of Southern blotting, PCR, and fluorescence in situ hybridization analyses has enabled the chromosomal location of the human RPSS gene to be determined as lp32-p34.1. 51 refs., 5 figs.

  17. Ribosome engineering to promote new crystal forms

    SciTech Connect

    Selmer, Maria; Gao, Yong-Gui; Weixlbaumer, Albert; Ramakrishnan, V.

    2012-05-01

    Truncation of ribosomal protein L9 in T. thermophilus allows the generation of new crystal forms and the crystallization of ribosome–GTPase complexes. Crystallographic studies of the ribosome have provided molecular details of protein synthesis. However, the crystallization of functional complexes of ribosomes with GTPase translation factors proved to be elusive for a decade after the first ribosome structures were determined. Analysis of the packing in different 70S ribosome crystal forms revealed that regardless of the species or space group, a contact between ribosomal protein L9 from the large subunit and 16S rRNA in the shoulder of a neighbouring small subunit in the crystal lattice competes with the binding of GTPase elongation factors to this region of 16S rRNA. To prevent the formation of this preferred crystal contact, a mutant strain of Thermus thermophilus, HB8-MRCMSAW1, in which the ribosomal protein L9 gene has been truncated was constructed by homologous recombination. Mutant 70S ribosomes were used to crystallize and solve the structure of the ribosome with EF-G, GDP and fusidic acid in a previously unobserved crystal form. Subsequent work has shown the usefulness of this strain for crystallization of the ribosome with other GTPase factors.

  18. Deficiency of the ribosome biogenesis gene Sbds in hematopoietic stem and progenitor cells causes neutropenia in mice by attenuating lineage progression in myelocytes.

    PubMed

    Zambetti, Noemi A; Bindels, Eric M J; Van Strien, Paulina M H; Valkhof, Marijke G; Adisty, Maria N; Hoogenboezem, Remco M; Sanders, Mathijs A; Rommens, Johanna M; Touw, Ivo P; Raaijmakers, Marc H G P

    2015-10-01

    Shwachman-Diamond syndrome is a congenital bone marrow failure disorder characterized by debilitating neutropenia. The disease is associated with loss-of-function mutations in the SBDS gene, implicated in ribosome biogenesis, but the cellular and molecular events driving cell specific phenotypes in ribosomopathies remain poorly defined. Here, we established what is to our knowledge the first mammalian model of neutropenia in Shwachman-Diamond syndrome through targeted downregulation of Sbds in hematopoietic stem and progenitor cells expressing the myeloid transcription factor CCAAT/enhancer binding protein α (Cebpa). Sbds deficiency in the myeloid lineage specifically affected myelocytes and their downstream progeny while, unexpectedly, it was well tolerated by rapidly cycling hematopoietic progenitor cells. Molecular insights provided by massive parallel sequencing supported cellular observations of impaired cell cycle exit and formation of secondary granules associated with the defect of myeloid lineage progression in myelocytes. Mechanistically, Sbds deficiency activated the p53 tumor suppressor pathway and induced apoptosis in these cells. Collectively, the data reveal a previously unanticipated, selective dependency of myelocytes and downstream progeny, but not rapidly cycling progenitors, on this ubiquitous ribosome biogenesis protein, thus providing a cellular basis for the understanding of myeloid lineage biased defects in Shwachman-Diamond syndrome. PMID:26185170

  19. Deficiency of the ribosome biogenesis gene Sbds in hematopoietic stem and progenitor cells causes neutropenia in mice by attenuating lineage progression in myelocytes

    PubMed Central

    Zambetti, Noemi A.; Bindels, Eric M. J.; Van Strien, Paulina M. H.; Valkhof, Marijke G.; Adisty, Maria N.; Hoogenboezem, Remco M.; Sanders, Mathijs A.; Rommens, Johanna M.; Touw, Ivo P.; Raaijmakers, Marc H. G. P.

    2015-01-01

    Shwachman-Diamond syndrome is a congenital bone marrow failure disorder characterized by debilitating neutropenia. The disease is associated with loss-of-function mutations in the SBDS gene, implicated in ribosome biogenesis, but the cellular and molecular events driving cell specific phenotypes in ribosomopathies remain poorly defined. Here, we established what is to our knowledge the first mammalian model of neutropenia in Shwachman-Diamond syndrome through targeted downregulation of Sbds in hematopoietic stem and progenitor cells expressing the myeloid transcription factor CCAAT/enhancer binding protein α (Cebpa). Sbds deficiency in the myeloid lineage specifically affected myelocytes and their downstream progeny while, unexpectedly, it was well tolerated by rapidly cycling hematopoietic progenitor cells. Molecular insights provided by massive parallel sequencing supported cellular observations of impaired cell cycle exit and formation of secondary granules associated with the defect of myeloid lineage progression in myelocytes. Mechanistically, Sbds deficiency activated the p53 tumor suppressor pathway and induced apoptosis in these cells. Collectively, the data reveal a previously unanticipated, selective dependency of myelocytes and downstream progeny, but not rapidly cycling progenitors, on this ubiquitous ribosome biogenesis protein, thus providing a cellular basis for the understanding of myeloid lineage biased defects in Shwachman-Diamond syndrome. PMID:26185170

  20. The leukemogenic t(8;21) fusion protein AML1-ETO controls ribosomal RNA genes and associates with nucleolar organizing regions at mitotic chromosomes

    PubMed Central

    Bakshi, Rachit; Zaidi, Sayyed K.; Pande, Sandhya; Hassan, Mohammad Q.; Young, Daniel W.; Lian, Jane B.; van Wijnen, Andre J.; Stein, Janet L.; Stein, Gary S.

    2010-01-01

    SUMMARY RUNX1/AML1 is required for definitive hematopoiesis and is frequently targeted by chromosomal translocation in acute myeloid leukemias (AML). The t(8;21) related AML1-ETO fusion protein blocks differentiation of myeloid progenitors. Here, we show by immunofluorescence microscopy that during interphase, endogenous AML1-ETO localizes to nuclear microenvironments distinct from those containing native RUNX1/AML1 protein. At mitosis, we clearly detect binding of AML1-ETO to nucleolar organizing regions (NORs) in AML derived Kasumi-1 cells and binding of RUNX1/AML1 to NORs in Jurkat cells. Both RUNX1/AML1 and AML1-ETO occupy ribosomal DNA repeats during interphase, as well as interact with the endogenous RNA Pol I transcription factor UBF-1. Promoter cytosine methylation analysis indicates that RUNX1/AML1 binds to rDNA repeats that are more highly CpG methylated than those bound by AML1-ETO. Down-regulation by RNA interference reveals that RUNX1/AML1 negatively regulates rDNA transcription, while AML1-ETO is a positive regulator in Kasumi-1 cells. Taken together, our findings identify a novel role for the leukemia-related AML1-ETO protein in epigenetic control of cell growth through upregulation of RNA Pol I-mediated ribosomal gene transcription, consistent with the hyper-proliferative phenotype of myeloid cells in AML patients. PMID:19001502

  1. New arrangements on several species subcomplexes of Triatoma genus based on the chromosomal position of ribosomal genes (Hemiptera - Triatominae).

    PubMed

    Pita, Sebastián; Lorite, Pedro; Nattero, Julieta; Galvão, Cleber; Alevi, Kaio C C; Teves, Simone C; Azeredo-Oliveira, Maria T V; Panzera, Francisco

    2016-09-01

    The hemipteran subfamily Triatominae includes 150 blood-sucking species, vectors of Chagas disease. By far the most specious genus is Triatoma, assembled in groups, complexes and subcomplexes based on morphological similarities, geographic distribution and genetic data. However, many molecular studies questioned the species integration of several subcomplexes as monophyletic units. In triatomines, chromosomal position of major ribosomal DNA (rDNA) loci is extremely variable but seems to be species-specific and an evolutionary conserved genetic trait, so that closely related species tend to have ribosomal clusters in the same chromosomal location. Considering that the autosomal position as the ancestral character for all heteropteran species, including triatomines, we suggest that the movement of rDNA loci from autosomes to sex chromosomes rapidly established reproductive barriers between divergent lineages. We proposed that the rDNA translocation from the autosomes to the sex chromosomes restrict reproductive compatibility and eventually promote speciation processes. We analyzed the chromosomal position of 45S rDNA clusters in almost all species of the matogrossensis, rubrovaria, maculata and sordida subcomplexes. The fluorescent in situ hybridization results are discussed considering the available genetic data and we proposed new arrangements in the species that constitute each one of these subcomplexes. PMID:27245153

  2. Intraspecific Variation in Ribosomal DNA in Populations of the Potato Cyst Nematode Globodera pallida.

    PubMed

    Blok, V C; Malloch, G; Harrower, B; Phillips, M S; Vrain, T C

    1998-06-01

    The relationships among a number of populations of Globodera pallida from Britian, the Netherlands, Germany, Switzerland, and South America were examined using PCR amplification of the ribosomal cistron between the 18S and 28S genes that include the two intergenic spacer regions (ITS1 and ITS2) and the 5.8S gene. Amplifications produced a similar-sized product of 1150 bp from all populations. Digestion of the amplified fragment with a number of restriction enzymes showed differences among the populations. The restriction enzyme RsaI distinguished the most populations. The RFLP patterns revealed by this enzyme were complex and could have arisen from heterogeneity between individuals within populations and from differences between the repeats of an individual. Sequence analysis from six of the populations, together with RFLP analysis of PCR products, shows that there is intraspecific variation in the rDNA of G. pallida. PMID:19274220

  3. Intraspecific Variation in Ribosomal DNA in Populations of the Potato Cyst Nematode Globodera pallida

    PubMed Central

    Blok, V. C.; Malloch, G.; Harrower, B.; Phillips, M. S.; Vrain, T. C.

    1998-01-01

    The relationships among a number of populations of Globodera pallida from Britian, the Netherlands, Germany, Switzerland, and South America were examined using PCR amplification of the ribosomal cistron between the 18S and 28S genes that include the two intergenic spacer regions (ITS1 and ITS2) and the 5.8S gene. Amplifications produced a similar-sized product of 1150 bp from all populations. Digestion of the amplified fragment with a number of restriction enzymes showed differences among the populations. The restriction enzyme RsaI distinguished the most populations. The RFLP patterns revealed by this enzyme were complex and could have arisen from heterogeneity between individuals within populations and from differences between the repeats of an individual. Sequence analysis from six of the populations, together with RFLP analysis of PCR products, shows that there is intraspecific variation in the rDNA of G. pallida. PMID:19274220

  4. Phylogenetic Analysis of the Hoplolaiminae Inferred from Combined D2 and D3 Expansion Segments of 28S rDNA.

    PubMed

    Bae, C H; Szalanski, A L; Robbins, R T

    2009-03-01

    DNA sequences of the D2-D3 expansion segments of the 28S gene of ribosomal DNA from 23 taxa of the subfamily Hoplolaiminae were obtained and aligned to infer phylogenetic relationships. The D2 and D3 expansion regions are G-C rich (59.2%), with up to 20.7% genetic divergence between Scutellonema brachyurum and Hoplolaimus concaudajuvencus. Molecular phylogenetic analysis using maximum likelihood and maximum parsimony was conducted using the D2-D3 sequence data. Of 558 characters, 254 characters (45.5%) were variable and 198 characters (35.4%) were parsimony informative. All phylogenetic methods produced a similar topology with two distinct clades: One clade consists of all Hoplolaimus species while the other clade consists of the rest of the studied Hoplolaiminae genera. This result suggests that Hoplolaimus is monophyletic. Another clade consisted of Aorolaimus, Helicotylenchus, Rotylenchus, and Scutellonema species. Phylogenetic analysis using the outgroup species Globodera rostocheinsis suggests that Hoplolaiminae is paraphyletic. In this study, the D2-D3 region had levels of DNA sequence divergence sufficient for phylogenetic analysis and delimiting species of Hoplolaiminae. PMID:22661775

  5. 16S-23S ribosomal RNA spacer regions of Acetobacter europaeus and A. xylinum, tRNA genes and antitermination sequences.

    PubMed

    Sievers, M; Alonso, L; Gianotti, S; Boesch, C; Teuber, M

    1996-08-15

    The 16S-23S ribosomal RNA spacer regions of Acetobacter europaeus DSM 6160, A. xylinum NCIB 11664 and A. xylinum CL27 were amplified by PCR. Specific PCR products were obtained from each strain and their nucleotide sequences determined. The spacer region of A. europaeus comprises 768 nucleotides (nt), that of A. xylinum 778 nt and that of A. xylinum CL27 759 nt. Genes encoding tRNAIle and tRNAAla were identified. Putative antitermination sequences were found between the tRNAAla sequence and the 5'-terminus of the 23S rRNA coding sequence. The boxA element has the nucleotide sequence TGCTCTTTGATA. Based on hybridization data of digested chromosomal DNA with spacer-specific probes, the copy number of the rrn operons on the chromosome of Acetobacter strains is estimated to be four. PMID:8759788

  6. The tails of ubiquitin precursors are ribosomal proteins whose fusion to ubiquitin facilitates ribosome biogenesis

    NASA Astrophysics Data System (ADS)

    Finley, Daniel; Bartel, Bonnie; Varshavsky, Alexander

    1989-03-01

    Three of the four yeast ubiquitin genes encode hybrid proteins which are cleaved to yield ubiquitin and previously unidentified ribosomal proteins. The transient association between ubiquitin and these proteins promotes their incorporation into nascent ribosomes and is required for efficient ribosome biogenesis. These results suggest a novel 'chaperone' function for ubiquitin, in which its covalent association with other proteins promotes the formation of specific cellular structures.

  7. Chromosome Mapping of 18S Ribosomal RNA Genes in Eleven Hypostomus Species (Siluriformes, Loricariidae): Diversity Analysis of the Sites.

    PubMed

    Rubert, Marceléia; da Rosa, Renata; Zawadzki, Claudio H; Mariotto, Sandra; Moreira-Filho, Orlando; Giuliano-Caetano, Lucia

    2016-08-01

    We investigated the chromosomal distribution of 18S ribosomal DNA (rDNA) in different populations of 11 species of Hypostomus collected in important Brazilian basins, namely South Atlantic, Upper Paraná, and Paraguay applying the fluorescence in situ hybridization (FISH). Hypostomus cochliodon, Hypostomus commersoni, Hypostomus hermanni, Hypostomus regani, Hypostomus albopunctatus, Hypostomus paulinus, Hypostomus aff. paulinus, Hypostomus iheringii, and Hypostomus mutucae presented multiple 18S rDNA sites while Hypostomus strigaticeps and Hypostomus nigromaculatus exhibited a single pair of chromosomes with 18S rDNA sites. The studied species presented variations in the number and position of these sites. The results accomplished were similar to those obtained by the analysis of AgNORs, revealing the same interspecific variability. Each species exhibited distinctive patterns of AgNOR and 18S rDNA distribution, which can be considered cytogenetic markers in each species of the genus and help improve the discussions on the phylogeny of the group. PMID:27192329

  8. Mutation detection analysis of a region of 16S-like ribosomal RNA gene of Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii

    PubMed Central

    Parija, Subhash Chandra; Khairnar, Krishna

    2008-01-01

    Background The level of intra-species genetic variation in Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii populations in a localized geographic area, like Puducherry, India, remains unknown. Methods In the present study the existence of genetic variation in the nested multiplex polymerase chain reaction (NM-PCR) amplified region of the 16S-like ribosomal RNA genes of E. histolytica, E. dispar and E. moshkovskii was investigated by riboprinting and single strand conformation polymorphism (SSCP) analysis. Results We found that 70 stool specimens were positive for E. histolytica, 171 stool specimens were positive for E. dispar, and 37 stool specimens were positive for E. moshkovskii by NM-PCR. Ninety liver abscess pus specimens, 21 urine specimens, and 8 saliva specimens were positive for E. histolytica by NM-PCR. Riboprinting analysis detected a mutation in the PCR product of only one E. histolytica isolate from a stool specimen. However, SSCP analysis detected mutations in the PCR products of five E. histolytica isolates and three E. moshkovskii isolates from stool specimens, and one E. histolytica isolate from a saliva specimen. The mutations detected by riboprinting and SSCP analysis were confirmed by sequencing. All the nucleotide sequences showing mutations in this study have already been deposited into the NCBI GenBank database under accession numbers [GenBank: EF682200 to GenBank: EF682208]. Conclusion The present study has revealed the subsistence of mutations in the ribosomal RNA genes of E. histolytica and E. moshkovskii, which points towards the existence of intra-species genetic variation in E. histolytica and E. moshkovskii isolates infecting humans. PMID:18822136

  9. Expression of Ribosomal RNA and Protein Genes in Human Embryonic Stem Cells Is Associated With the Activating H3K4me3 Histone Mark.

    PubMed

    Zaidi, Sayyed K; Boyd, Joseph R; Grandy, Rodrigo A; Medina, Ricardo; Lian, Jane B; Stein, Gary S; Stein, Janet L

    2016-09-01

    Embryonic stem cells (ESCs) exhibit unrestricted and indefinite, but stringently controlled, proliferation, and can differentiate into any lineage in the body. In the current study, we test the hypothesis that expression of ribosomal RNA (rRNA) and ribosomal protein genes (RPGs) contribute to the ability of hESCs to proliferate indefinitely. Consistent with the accelerated growth rate of hESCs, we find that hESC lines H1 and H9 both exhibit significantly higher levels of rRNA when compared to a panel of normal and cancer human cell lines. Although many RPGs are expressed at levels that comparable to other human cell lines, a few RPGs also exhibit higher expression levels. In situ nuclear run-on assays reveal that both nucleoli in hESCs actively transcribe nascent rRNA. Employing genome-wide chromatin immunoprecipitation-deep sequencing and bioinformatics approaches, we discovered that, RPGs are dominantly marked by the activating H3K4me3 histone mark in the G1, M, and G2 phases of the cell cycle. Interestingly, the rDNA repeats are marked by the activating H3K4me3 only in the M phase, and repressive H3K27me3 histone mark in all three cell cycle phases. Bioinformatics analyses also reveal that Myc, a known regulator of cell growth and proliferation, occupies both the rRNA genes and RPGs. Functionally, down-regulation of Myc expression by siRNA results in a concomitant decrease in rRNA levels. Together, our results show that expression of rRNA, which is regulated by the Myc pluripotency transcription factor, and of RPGs in hESCs is associated with the activating H3K4me3 modification. J. Cell. Physiol. 231: 2007-2013, 2016. © 2016 Wiley Periodicals, Inc. PMID:26755341

  10. deaD, a new Escherichia coli gene encoding a presumed ATP-dependent RNA helicase, can suppress a mutation in rpsB, the gene encoding ribosomal protein S2.

    PubMed Central

    Toone, W M; Rudd, K E; Friesen, J D

    1991-01-01

    We have cloned and sequenced a new gene from Escherichia coli which encodes a 64-kDa protein. The inferred amino acid sequence of the protein shows remarkable similarity to eIF4A, a murine translation initiation factor that has an ATP-dependent RNA helicase activity and is a founding member of the D-E-A-D family of proteins (characterized by a conserved Asp-Glu-Ala-Asp motif). Our new gene, called deaD, was cloned as a gene dosage-dependent suppressor of temperature-sensitive mutations in rpsB, the gene encoding ribosomal protein S2. We suggest that the DeaD protein plays a hitherto unknown role in translation in E. coli. Images PMID:2045359

  11. Modification of ribosomal RNA by ribosome-inactivating proteins from plants.

    PubMed Central

    Stirpe, F; Bailey, S; Miller, S P; Bodley, J W

    1988-01-01

    We have surveyed 14 different toxic and nontoxic ribosome-inactivating proteins from plants for the ability to act on the RNA of the eucaryotic 60 S ribosomal subunit. All of these proteins act to introduce a specific modification into 26-28 S RNA which renders the RNA sensitive to cleavage by aniline. Sequence analysis of the 5'-termini of the fragments produced by ricin and saporin following aniline cleavage indicate that both proteins possess identical specificity. Our observations support the conclusion of Endo and Tsurugi (J. Biol. Chem. 262, 8128-8130, 1987) that ricin is a specific N-glycosidase and we have located the site of this cleavage by direct sequence analysis. Our results further suggest that all plant ribosome-inactivating proteins function as specific N-glycosidases with the same specificity. Images PMID:3347493

  12. Structures of Eukaryotic Ribosomal Stalk Proteins and Its Complex with Trichosanthin, and Their Implications in Recruiting Ribosome-Inactivating Proteins to the Ribosomes

    PubMed Central

    Choi, Andrew K. H.; Wong, Eddie C. K.; Lee, Ka-Ming; Wong, Kam-Bo

    2015-01-01

    Ribosome-inactivating proteins (RIP) are RNA N-glycosidases that inactivate ribosomes by specifically depurinating a conserved adenine residue at the α-sarcin/ricin loop of 28S rRNA. Recent studies have pointed to the involvement of the C-terminal domain of the eukaryotic stalk proteins in facilitating the toxic action of RIPs. This review highlights how structural studies of eukaryotic stalk proteins provide insights into the recruitment of RIPs to the ribosomes. Since the C-terminal domain of eukaryotic stalk proteins is involved in specific recognition of elongation factors and some eukaryote-specific RIPs (e.g., trichosanthin and ricin), we postulate that these RIPs may have evolved to hijack the translation-factor-recruiting function of ribosomal stalk in reaching their target site of rRNA. PMID:25723321

  13. Profiling of Mycoplasma gallisepticum Ribosomes.

    PubMed

    Fisunov, G Y; Evsyutina, D V; Arzamasov, A A; Butenko, I O; Govorun, V M

    2015-01-01

    The development of high-throughput technologies is increasingly resulting in identification of numerous cases of low correlation between mRNA and the protein level in cells. These controversial observations were made on various bacteria, such as E. coli, Desulfovibrio vulgaris, and Lactococcus lactis. Thus, it is important to develop technologies, including high-throughput techniques, aimed at studying gene expression regulation at the level of translation. In the current study, we performed proteomic profiling of M. gallisepticum ribosomes and identified high abundant noncanonical proteins. We found that binding of mRNAs to ribosomes is mainly determined by two parameters: (1) abundance of mRNA itself and (2) complimentary interactions between the 3' end of 16S rRNA and the ribosome binding site in the 5'-untranslated region of mRNA. PMID:26798497

  14. Profiling of Mycoplasma gallisepticum Ribosomes

    PubMed Central

    Fisunov, G. Y.; Evsyutina, D. V.; Arzamasov, A. A.; Butenko, I. O.; Govorun, V. M.

    2015-01-01

    The development of high-throughput technologies is increasingly resulting in identification of numerous cases of low correlation between mRNA and the protein level in cells. These controversial observations were made on various bacteria, such as E. coli, Desulfovibrio vulgaris, and Lactococcus lactis. Thus, it is important to develop technologies, including high-throughput techniques, aimed at studying gene expression regulation at the level of translation. In the current study, we performed proteomic profiling of M. gallisepticum ribosomes and identified high abundant noncanonical proteins. We found that binding of mRNAs to ribosomes is mainly determined by two parameters: (1) abundance of mRNA itself and (2) complimentary interactions between the 3’ end of 16S rRNA and the ribosome binding site in the 5’-untranslated region of mRNA. PMID:26798497

  15. Chloroplast ribosomes and protein synthesis.

    PubMed Central

    Harris, E H; Boynton, J E; Gillham, N W

    1994-01-01

    Consistent with their postulated origin from endosymbiotic cyanobacteria, chloroplasts of plants and algae have ribosomes whose component RNAs and proteins are strikingly similar to those of eubacteria. Comparison of the secondary structures of 16S rRNAs of chloroplasts and bacteria has been particularly useful in identifying highly conserved regions likely to have essential functions. Comparative analysis of ribosomal protein sequences may likewise prove valuable in determining their roles in protein synthesis. This review is concerned primarily with the RNAs and proteins that constitute the chloroplast ribosome, the genes that encode these components, and their expression. It begins with an overview of chloroplast genome structure in land plants and algae and then presents a brief comparison of chloroplast and prokaryotic protein-synthesizing systems and a more detailed analysis of chloroplast rRNAs and ribosomal proteins. A description of the synthesis and assembly of chloroplast ribosomes follows. The review concludes with discussion of whether chloroplast protein synthesis is essential for cell survival. PMID:7854253

  16. Development of a Newcastle disease virus vector expressing a foreign gene through an internal ribosomal entry site

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Newcastle disease virus (NDV) has been developed as a vector to express foreign genes for vaccine and gene therapy purposes. The foreign genes are usually inserted into a non-coding region of the NDV genome as an independent transcription unit (ITU). Based on the well-accepted “stop-start” transcr...

  17. A Method for Studying Protistan Diversity Using Massively Parallel Sequencing of V9 Hypervariable Regions of Small-Subunit Ribosomal RNA Genes

    PubMed Central

    Amaral-Zettler, Linda A.; McCliment, Elizabeth A.; Ducklow, Hugh W.; Huse, Susan M.

    2009-01-01

    Background Massively parallel pyrosequencing of amplicons from the V6 hypervariable regions of small-subunit (SSU) ribosomal RNA (rRNA) genes is commonly used to assess diversity and richness in bacterial and archaeal populations. Recent advances in pyrosequencing technology provide read lengths of up to 240 nucleotides. Amplicon pyrosequencing can now be applied to longer variable regions of the SSU rRNA gene including the V9 region in eukaryotes. Methodology/Principal Findings We present a protocol for the amplicon pyrosequencing of V9 regions for eukaryotic environmental samples for biodiversity inventories and species richness estimation. The International Census of Marine Microbes (ICoMM) and the Microbial Inventory Research Across Diverse Aquatic Long Term Ecological Research Sites (MIRADA-LTERs) projects are already employing this protocol for tag sequencing of eukaryotic samples in a wide diversity of both marine and freshwater environments. Conclusions/Significance Massively parallel pyrosequencing of eukaryotic V9 hypervariable regions of SSU rRNA genes provides a means of estimating species richness from deeply-sampled populations and for discovering novel species from the environment. PMID:19633714

  18. Discrimination of Burkholderia mallei/pseudomallei from Burkholderia thailandensis by sequence comparison of a fragment of the ribosomal protein S21 (rpsU) gene

    PubMed Central

    Frickmann, H.; Chantratita, N.; Gauthier, Y. P.; Neubauer, H.; Hagen, R. M.

    2012-01-01

    Discrimination of Burkholderia (B.) pseudomallei and B. mallei from environmental B. thailandensis is challenging. We describe a discrimination method based on sequence comparison of the ribosomal protein S21 (rpsU) gene. The rpsU gene was sequenced in ten B. pseudomallei, six B. mallei, one B. thailandensis reference strains, six isolates of B. pseudomallei, and 37 of B. thailandensis. Further rpsU sequences of six B. pseudomallei, three B. mallei, and one B. thailandensis were identified via NCBI GenBank. Three to four variable base-positions were identified within a 120-base-pair fragment, allowing discrimination of the B. pseudomallei/mallei-cluster from B. thailandensis, whose sequences clustered identically. All B. mallei and three B. pseudomallei sequences were identical, while 17/22 B. pseudomallei strains differed in one nucleotide (78A>C). Sequences of the rpsU fragment of ‘out-stander’ reference strains of B. cepacia, B. gladioli, B. plantarii, and B. vietnamensis clustered differently. Sequence comparison of the described rpsU gene fragment can be used as a supplementary diagnostic procedure for the discrimination of B. mallei/pseudomallei from B. thailandensis as well as from other species of the genus Burkholderia, keeping in mind that it does not allow for a differentiation between B. mallei and B. pseudomallei. PMID:23227305

  19. Cotranscription of two genes necessary for ribosomal protein L11 methylation (prmA) and pantothenate transport (panF) in Escherichia coli K-12.

    PubMed

    Vanet, A; Plumbridge, J A; Alix, J H

    1993-11-01

    Genetic complementation and enzyme assays have shown that the DNA region between panF, which encodes pantothenate permease, and orf1, the first gene of the fis operon, encodes prmA, the genetic determinant for the ribosomal protein L11 methyltransferase. Sequencing of this region identified one long open reading frame that encodes a protein of 31,830 Da and corresponds to the prmA gene. We found, both in vivo and in vitro, that prmA is expressed from promoters located upstream of panF and thus that the panF and prmA genes constitute a bifunctional operon. We located the major 3' end of prmA transcripts 90 nucleotides downstream of the stop codon of prmA in the DNA region upstream of the fis operon, a region implicated in the control of the expression of the fis operon. Although no promoter activity was detected immediately upstream of prmA, S1 mapping detected 5' ends of mRNA in this region, implying that some mRNA processing occurs within the bicistronic panF-prmA mRNA. PMID:8226664

  20. Characterization of the multigene family encoding the mouse S16 ribosomal protein: strategy for distinguishing an expressed gene from its processed pseudogene counterparts by an analysis of total genomic DNA.

    PubMed Central

    Wagner, M; Perry, R P

    1985-01-01

    Two genes from the family encoding mouse ribosomal protein S16 were cloned, sequenced, and analyzed. One gene was found to be a processed pseudogene, i.e., a nonfunctional gene presumably derived from an mRNA intermediate. The other S16 gene contained introns and had exonic sequences identical to those of a cloned S16 cDNA. The expression of this gene was demonstrated by Northern blot analysis of nuclear poly(A)+ RNA with cDNA and unique sequence intron probes. Each S16 intron contains a well-preserved remnant of the TACTAAC motif, which is ubiquitous in yeast introns and known to play a critical role in intron splicing. A sequence comparison with two other mouse ribosomal protein genes analyzed in our laboratory, L30 and L32, revealed common structural features which might be involved in the control and coordination of ribosomal protein gene expression. These include the lack of a canonical TATA box in the -20 to -30 region and a remarkably similar 12-nucleotide pyrimidine sequence (CTTCCYTYYTC) that spans the cap site and is flanked by C + G-rich sequences. The nature of the other members of the S16 family was evaluated by three types of experiment: a DNase I sensitivity analysis to measure the extent of chromatin condensation; an analysis of the thermal stability of cDNA-gene hybrids to estimate the extent of divergence of each gene sequence from that of the expressed gene; and a restriction fragment analysis which distinguishes intron-containing genes from intronless processed genes. The results of these analyses show that all genes except the expressed S16 gene are in a condensed chromatin configuration associated with transcriptional quiescence; that most of the genes within the S16 family have sequences greater than 7% divergent from the expressed S16 gene; and that at least 7 of the 10 S16 genes lack introns. We conclude that the ribosomal protein S16 multigene family contains one expressed intron-containing gene and nine inactive pseudogenes, most or all

  1. The NMR solution structure of the 30S ribosomal protein S27e encoded in gene RS27_ARCFU of Archaeoglobus fulgidis reveals a novel protein fold

    PubMed Central

    Herve du Penhoat, Catherine; Atreya, Hanudatta S.; Shen, Yang; Liu, Gaohua; Acton, Thomas B.; Xiao, Rong; Li, Zhaohui; Murray, Diana; Montelione, Gaetano T.; Szyperski, Thomas

    2004-01-01

    The Archaeoglobus fulgidis gene RS27_ARCFU encodes the 30S ribosomal protein S27e. Here, we present the high-quality NMR solution structure of this archaeal protein, which comprises a C4 zinc finger motif of the CX2CX14-16CX2C class. S27e was selected as a target of the Northeast Structural Genomics Consortium (target ID: GR2), and its three-dimensional structure is the first representative of a family of more than 116 homologous proteins occurring in eukaryotic and archaeal cells. As a salient feature of its molecular architecture, S27e exhibits a β-sandwich consisting of two three-stranded sheets with topology B(↓), A(↑), F(↓), and C(↑), D(↓), E(↑). Due to the uniqueness of the arrangement of the strands, the resulting fold was found to be novel. Residues that are highly conserved among the S27 proteins allowed identification of a structural motif of putative functional importance; a conserved hydrophobic patch may well play a pivotal role for functioning of S27 proteins, be it in archaeal or eukaryotic cells. The structure of human S27, which possesses a 26-residue amino-terminal extension when compared with the archaeal S27e, was modeled on the basis of two structural templates, S27e for the carboxy-terminal core and the amino-terminal segment of the archaeal ribosomal protein L37Ae for the extension. Remarkably, the electrostatic surface properties of archaeal and human proteins are predicted to be entirely different, pointing at either functional variations among archaeal and eukaryotic S27 proteins, or, assuming that the function remained invariant, to a concerted evolutionary change of the surface potential of proteins interacting with S27. PMID:15096641

  2. The ribosome filter redux.

    PubMed

    Mauro, Vincent P; Edelman, Gerald M

    2007-09-15

    The ribosome filter hypothesis postulates that ribosomes are not simply translation machines but also function as regulatory elements that differentially affect or filter the translation of particular mRNAs. On the basis of new information, we take the opportunity here to review the ribosome filter hypothesis, suggest specific mechanisms of action, and discuss recent examples from the literature that support it. PMID:17890902

  3. Differential Stoichiometry among Core Ribosomal Proteins

    PubMed Central

    Slavov, Nikolai; Semrau, Stefan; Airoldi, Edoardo; Budnik, Bogdan; van Oudenaarden, Alexander

    2015-01-01

    Summary Understanding the regulation and structure of ribosomes is essential to understanding protein synthesis and its dysregulation in disease. While ribosomes are believed to have a fixed stoichiometry among their core ribosomal proteins (RPs), some experiments suggest a more variable composition. Testing such variability requires direct and precise quantification of RPs. We used mass spectrometry to directly quantify RPs across monosomes and polysomes of mouse embryonic stem cells (ESC) and budding yeast. Our data show that the stoichiometry among core RPs in wild-type yeast cells and ESC depends both on the growth conditions and on the number of ribosomes bound per mRNA. Furthermore, we find that the fitness of cells with a deleted RP-gene is inversely proportional to the enrichment of the corresponding RP in polysomes. Together, our findings support the existence of ribosomes with distinct protein composition and physiological function. PMID:26565899

  4. Differential Stoichiometry among Core Ribosomal Proteins.

    PubMed

    Slavov, Nikolai; Semrau, Stefan; Airoldi, Edoardo; Budnik, Bogdan; van Oudenaarden, Alexander

    2015-11-01

    Understanding the regulation and structure of ribosomes is essential to understanding protein synthesis and its dysregulation in disease. While ribosomes are believed to have a fixed stoichiometry among their core ribosomal proteins (RPs), some experiments suggest a more variable composition. Testing such variability requires direct and precise quantification of RPs. We used mass spectrometry to directly quantify RPs across monosomes and polysomes of mouse embryonic stem cells (ESC) and budding yeast. Our data show that the stoichiometry among core RPs in wild-type yeast cells and ESC depends both on the growth conditions and on the number of ribosomes bound per mRNA. Furthermore, we find that the fitness of cells with a deleted RP-gene is inversely proportional to the enrichment of the corresponding RP in polysomes. Together, our findings support the existence of ribosomes with distinct protein composition and physiological function. PMID:26565899

  5. Repeated reunions and splits feature the highly dynamic evolution of 5S and 35S ribosomal RNA genes (rDNA) in the Asteraceae family

    PubMed Central

    2010-01-01

    Background In flowering plants and animals the most common ribosomal RNA genes (rDNA) organisation is that in which 35S (encoding 18S-5.8S-26S rRNA) and 5S genes are physically separated occupying different chromosomal loci. However, recent observations established that both genes have been unified to a single 35S-5S unit in the genus Artemisia (Asteraceae), a genomic arrangement typical of primitive eukaryotes such as yeast, among others. Here we aim to reveal the origin, distribution and mechanisms leading to the linked organisation of rDNA in the Asteraceae by analysing unit structure (PCR, Southern blot, sequencing), gene copy number (quantitative PCR) and chromosomal position (FISH) of 5S and 35S rRNA genes in ~200 species representing the family diversity and other closely related groups. Results Dominant linked rDNA genotype was found within three large groups in subfamily Asteroideae: tribe Anthemideae (93% of the studied cases), tribe Gnaphalieae (100%) and in the "Heliantheae alliance" (23%). The remaining five tribes of the Asteroideae displayed canonical non linked arrangement of rDNA, as did the other groups in the Asteraceae. Nevertheless, low copy linked genes were identified among several species that amplified unlinked units. The conserved position of functional 5S insertions downstream from the 26S gene suggests a unique, perhaps retrotransposon-mediated integration event at the base of subfamily Asteroideae. Further evolution likely involved divergence of 26S-5S intergenic spacers, amplification and homogenisation of units across the chromosomes and concomitant elimination of unlinked arrays. However, the opposite trend, from linked towards unlinked arrangement was also surmised in few species indicating possible reversibility of these processes. Conclusions Our results indicate that nearly 25% of Asteraceae species may have evolved unusual linked arrangement of rRNA genes. Thus, in plants, fundamental changes in intrinsic structure of rDNA units

  6. Identification and characterization of an intervening sequence within the 23S ribosomal RNA genes of Edwardsiella ictaluri

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Comparison of the 23S rRNA gene sequences of Edwardsiella tarda and Edwardsiella ictaluri confirmed a close phylogenetic relationship between these two fish pathogen species and a distant relation with the 'core' members of the Enterobacteriaceae family. Analysis of the rrl gene for 23S rRNA in E. i...

  7. Locations and contexts of sequences that hybridize to poly(dG-dT).(dC-dA) in mammalian ribosomal DNAs and two X-linked genes.

    PubMed Central

    Braaten, D C; Thomas, J R; Little, R D; Dickson, K R; Goldberg, I; Schlessinger, D; Ciccodicola, A; D'Urso, M

    1988-01-01

    Sequences located several kilobases both 5' and 3' of the stably transcribed portion of several genes hybridize to radio-labeled pure fragments of the alternating sequence poly (dG-dT) (dC-dA) ["poly(GT)"]. The genes include the ribosomal DNA of mouse, rat, and human, and also human glucose-6-phosphate dehydrogenase (G6PD) and mouse hypoxanthine-guanine phosphoribosyl transferase (HPRT). HPRT has additional hybridizing sequences in introns. Fragments that include the hybridizing sequences and up to 300 bp of adjoining DNA show perfect runs of poly(GT) (greater than 30bp) in all but the human 5' region of rDNA, which shows a somewhat different alternating purine:pyrimidine sequence, poly(GTAT) (36bp). Within 150 bp of these sequences in various instances are found a number of other sequences reported to affect DNA conformation in model systems. Most marked is an enhancement of sequences matching at least 67% to the consensus binding sequence for topoisomerase II. Two to ten-fold less of such sequences were found in other sequenced portions of the nontranscribed spacer or in the transcribed portion of rDNA. The conservation of the locations of tracts of alternating purine:pyrimidine between evolutionarily diverse species is consistent with a possible functional role for these sequences. Images PMID:3267216

  8. Riproximin: A type II ribosome inactivating protein with anti-neoplastic potential induces IL24/MDA-7 and GADD genes in colorectal cancer cell lines.

    PubMed

    Pervaiz, Asim; Adwan, Hassan; Berger, Martin R

    2015-09-01

    Riproximin (Rpx) is a type II ribosome inactivating protein, which was extracted and purified from the seeds of Ximenia americana. Previous studies demonstrated cytotoxicity of Rpx against a variety of cell lines originating from solid and non-solid cancers. In this study, we investigated the mechanistic aspects of Rpx in selected human and rat colorectal cancer (CRC) cell lines. Cytotoxic levels of Rpx were determined by MTT assay, while cytostatic and apoptotic effects were investigated by flow cytometry and nuclear staining procedures. Effects of Rpx exposure on colony formation/migration of CRC cells and expressional modulations in anticancer/stress-related genes were also studied. Rpx showed significant and comparable levels of cytotoxicity in CRC cells as determined by inhibitory concentration (IC) values. Similar inhibitory effects were found for clonogenicity, while more pronounced inhibition of migration was observed in response to Rpx exposure. Profound arrest in S phases of the cell cycle was noted especially in primary CRC cells. Apoptotic effects were more prominent in rat CRC cells as indicated by Annexin V-FITC assay and Hoechst 33342 nuclear staining. Rpx exposure induced significantly increased levels of the IL24/MDA-7, a well characterized anticancer gene, in all CRC cells. In addition, following Rpx treatment, high expression levels of growth arrest and DNA damage (GADD family) genes were also observed. Increased expression of two additional GADD genes (34 and 153) only in rat CRC cells (CC531) conferred higher sensitivity towards Rpx and subsequent anti-proliferative/apoptotic effects as compared to human CRC cells (SW480 and SW620). The present investigation indicates the anticancer potential of Rpx in CRC and favor further evaluation of this natural compound as therapeutic agent. PMID:26151662

  9. Deconstructing ribosome construction

    PubMed Central

    Connolly, Keith; Culver, Gloria

    2013-01-01

    The ribosome is an essential ribonucleoprotein enzyme, and its biogenesis is a fundamental process in all living cells. Recent X-ray crystal structures of the bacterial ribosome and new technologies have allowed a greater interrogation of in vitro ribosome assembly; however, substantially less is known about ribosome biogenesis in vivo. Ongoing investigations are focused on elucidating the cellular processes that facilitate biogenesis of the ribosomal subunits, and many extraribosomal factors, including modification enzymes, remodeling enzymes and GTPases, are being uncovered. Moreover, specific roles for ribosome biogenesis factors in subunit maturation are now being elaborated. Ultimately, such studies will reveal a more complete understanding of processes at work in in vivo ribosome biogenesis. PMID:19376708

  10. Ribosomopathies: human disorders of ribosome dysfunction.

    PubMed

    Narla, Anupama; Ebert, Benjamin L

    2010-04-22

    Ribosomopathies compose a collection of disorders in which genetic abnormalities cause impaired ribosome biogenesis and function, resulting in specific clinical phenotypes. Congenital mutations in RPS19 and other genes encoding ribosomal proteins cause Diamond-Blackfan anemia, a disorder characterized by hypoplastic, macrocytic anemia. Mutations in other genes required for normal ribosome biogenesis have been implicated in other rare congenital syndromes, Schwachman-Diamond syndrome, dyskeratosis congenita, cartilage hair hypoplasia, and Treacher Collins syndrome. In addition, the 5q- syndrome, a subtype of myelodysplastic syndrome, is caused by a somatically acquired deletion of chromosome 5q, which leads to haploinsufficiency of the ribosomal protein RPS14 and an erythroid phenotype highly similar to Diamond-Blackfan anemia. Acquired abnormalities in ribosome function have been implicated more broadly in human malignancies. The p53 pathway provides a surveillance mechanism for protein translation as well as genome integrity and is activated by defects in ribosome biogenesis; this pathway appears to be a critical mediator of many of the clinical features of ribosomopathies. Elucidation of the mechanisms whereby selective abnormalities in ribosome biogenesis cause specific clinical syndromes will hopefully lead to novel therapeutic strategies for these diseases. PMID:20194897

  11. Towards a classification of E. coli ribosomal proteins: A hypothetical `small ribosome' as a primitive protein-synthesizing apparatus

    NASA Astrophysics Data System (ADS)

    Ohnishi, Koji

    1984-12-01

    Homologies were searched among the published primary sequences of 51 E. coli ribosomal proteins, partly by ‘eye’ and partly by computer-assisted methods. By employing Moore and Goodman's alignment statistics for evaluating homology levels, 33 out of these 51 ribosomal proteins has been classified into 9 homology groups, some of which being yet tentative and remaining to be further analyzed. Taking it into consideration that most ribosomal protein genes are clustered at str- stc region, rif region and several other regions, these results strongly suggest that most or all of the contemporary ribosomal proteins must have evolved by repeated gene duplications of very few (or only one) primitive ancestral ribosomal protein gene(s). Thus it is most reasonable to propose that a ‘ small ribosome’ consisting of very few (or only one) ribosomal protein(s) must have existed as a primitive protein-synthesizing apparatus.

  12. The ribosomal protein L34 gene from the mosquito, Aedes albopictus: exon-intron organization, copy number, and potential regulatory elements.

    PubMed

    Niu, L L; Fallon, A M

    1999-12-01

    We describe the structural analysis of genomic DNA encoding ribosomal protein (rp) L34 from the mosquito, Aedes albopictus. Comparison of genomic DNA sequences encompassing approximately 8 kb with the rpL34 cDNA sequence showed that the gene contains three exons and two introns, encoding a primary transcript with a deduced size of 6196 nucleotides from the transcription start site to the polyadenylation site. Exon 1, which is not translated, measures only 45 bp, and is separated from Exon 2 by a 359 bp intron. Exon 2 measures 78 bp, and contains the AUG translation initiation codon 14 nucleotides downstream of its 5'-end. Downstream of Exon 2 is a 5270 bp intron, followed by the remainder of the coding sequence in Exon 3, which measures 444 bp including the polyadenylation signal. We used a novel PCR-based procedure to obtain 1.7 kb of DNA upstream of the rpL34 gene. Like the previously described Ae. albopictus rpL8 gene and various mammalian rp genes, the DNA immediately upstream of the rpL34 gene lacks the TATA box, and the rpL34 transcription initiation site is embedded in a characteristic polypyrimidine tract. The 5'-flanking DNA contained a number of cis-acting elements that potentially interact with transcription factors characterized by basic domains, zinc-coordinating DNA binding domains, helix-turn-helix motifs, and beta scaffold factors with minor groove contacts. Particularly striking was the conservation of an AP-4 binding site within 100 nucleotides upstream of the transcription initiation site in both Aal-rpL34 and Aal-rpL8 genes. Comparison of Southern hybridization signals using probes from the 5' and 3'-ends of the 5.3 kb second intron and the cDNA suggested that the Ae. albopictus rpL34 gene most likely occurs as a single expressed copy per haploid genome with restriction enzyme polymorphisms in the upstream flanking DNA and the likely presence of one or more pseudogenes. PMID:10612044

  13. Regulation of gene expression by internal ribosome entry sites or cryptic promoters: the eIF4G story.

    PubMed

    Han, Baoguang; Zhang, Jian-Ting

    2002-11-01

    As an alternative to the scanning mechanism of initiation, the direct-internal-initiation mechanism postulates that the translational machinery assembles at the AUG start codon without traversing the entire 5' untranslated region (5'-UTR) of the mRNA. Although the existence of internal ribosome entry sites (IRESs) in viral mRNAs is considered to be well established, the existence of IRESs in cellular mRNAs has recently been challenged, in part because when testing is carried out using a conventional dicistronic vector, Northern blot analyses might not be sensitive enough to detect low levels of monocistronic transcripts derived via a cryptic promoter or splice site. To address this concern, we created a new promoterless dicistronic vector to test the putative IRES derived from the 5'-UTR of an mRNA that encodes the translation initiation factor eIF4G. Our analysis of this 5'-UTR sequence unexpectedly revealed a strong promoter. The activity of the internal promoter relies on the integrity of a polypyrimidine tract (PPT) sequence that had been identified as an essential component of the IRES. The PPT sequence overlaps with a binding site for transcription factor C/EBPbeta. Two other transcription factors, Sp1 and Ets, were also found to bind to and mediate expression from the promoter in the 5'-UTR of eIF4G mRNA. The biological significance of the internal promoter in the eIF4G mRNA might lie in the production of an N-terminally truncated form of the protein. Consistent with the idea that the cryptic promoter we identified underlies the previously reported IRES activity, we found no evidence of IRES function when a dicistronic mRNA containing the eIF4G sequence was translated in vitro or in vivo. Using the promoterless dicistronic vector, we also found promoter activities in the long 5'-UTRs of human Sno and mouse Bad mRNAs although monocistronic transcripts were not detectable on Northern blot analyses. The promoterless dicistronic vector might therefore prove

  14. Illuminating Parasite Protein Production by Ribosome Profiling.

    PubMed

    Parsons, Marilyn; Myler, Peter J

    2016-06-01

    While technologies for global enumeration of transcript abundance are well-developed, those that assess protein abundance require tailoring to penetrate to low-abundance proteins. Ribosome profiling circumvents this challenge by measuring global protein production via sequencing small mRNA fragments protected by the assembled ribosome. This powerful approach is now being applied to protozoan parasites including trypanosomes and Plasmodium. It has been used to identify new protein-coding sequences (CDSs) and clarify the boundaries of previously annotated CDSs in Trypanosoma brucei. Ribosome profiling has demonstrated that translation efficiencies vary widely between genes and, for trypanosomes at least, for the same gene across stages. The ribosomal proteins are themselves subjected to translational control, suggesting a means of reinforcing global translational regulation. PMID:27061497

  15. The Ribosome Biogenesis Protein Nol9 Is Essential for Definitive Hematopoiesis and Pancreas Morphogenesis in Zebrafish.

    PubMed

    Bielczyk-Maczyńska, Ewa; Lam Hung, Laure; Ferreira, Lauren; Fleischmann, Tobias; Weis, Félix; Fernández-Pevida, Antonio; Harvey, Steven A; Wali, Neha; Warren, Alan J; Barroso, Inês; Stemple, Derek L; Cvejic, Ana

    2015-12-01

    Ribosome biogenesis is a ubiquitous and essential process in cells. Defects in ribosome biogenesis and function result in a group of human disorders, collectively known as ribosomopathies. In this study, we describe a zebrafish mutant with a loss-of-function mutation in nol9, a gene that encodes a non-ribosomal protein involved in rRNA processing. nol9sa1022/sa1022 mutants have a defect in 28S rRNA processing. The nol9sa1022/sa1022 larvae display hypoplastic pancreas, liver and intestine and have decreased numbers of hematopoietic stem and progenitor cells (HSPCs), as well as definitive erythrocytes and lymphocytes. In addition, ultrastructural analysis revealed signs of pathological processes occurring in endothelial cells of the caudal vein, emphasizing the complexity of the phenotype observed in nol9sa1022/sa1022 larvae. We further show that both the pancreatic and hematopoietic deficiencies in nol9sa1022/sa1022 embryos were due to impaired cell proliferation of respective progenitor cells. Interestingly, genetic loss of Tp53 rescued the HSPCs but not the pancreatic defects. In contrast, activation of mRNA translation via the mTOR pathway by L-Leucine treatment did not revert the erythroid or pancreatic defects. Together, we present the nol9sa1022/sa1022 mutant, a novel zebrafish ribosomopathy model, which recapitulates key human disease characteristics. The use of this genetically tractable model will enhance our understanding of the tissue-specific mechanisms following impaired ribosome biogenesis in the context of an intact vertebrate. PMID:26624285

  16. The Ribosome Biogenesis Protein Nol9 Is Essential for Definitive Hematopoiesis and Pancreas Morphogenesis in Zebrafish

    PubMed Central

    Ferreira, Lauren; Fleischmann, Tobias; Weis, Félix; Fernández-Pevida, Antonio; Harvey, Steven A.; Wali, Neha; Warren, Alan J.; Barroso, Inês; Stemple, Derek L.; Cvejic, Ana

    2015-01-01

    Ribosome biogenesis is a ubiquitous and essential process in cells. Defects in ribosome biogenesis and function result in a group of human disorders, collectively known as ribosomopathies. In this study, we describe a zebrafish mutant with a loss-of-function mutation in nol9, a gene that encodes a non-ribosomal protein involved in rRNA processing. nol9 sa1022/sa1022 mutants have a defect in 28S rRNA processing. The nol9 sa1022/sa1022 larvae display hypoplastic pancreas, liver and intestine and have decreased numbers of hematopoietic stem and progenitor cells (HSPCs), as well as definitive erythrocytes and lymphocytes. In addition, ultrastructural analysis revealed signs of pathological processes occurring in endothelial cells of the caudal vein, emphasizing the complexity of the phenotype observed in nol9 sa1022/sa1022 larvae. We further show that both the pancreatic and hematopoietic deficiencies in nol9 sa1022/sa1022 embryos were due to impaired cell proliferation of respective progenitor cells. Interestingly, genetic loss of Tp53 rescued the HSPCs but not the pancreatic defects. In contrast, activation of mRNA translation via the mTOR pathway by L-Leucine treatment did not revert the erythroid or pancreatic defects. Together, we present the nol9 sa1022/sa1022 mutant, a novel zebrafish ribosomopathy model, which recapitulates key human disease characteristics. The use of this genetically tractable model will enhance our understanding of the tissue-specific mechanisms following impaired ribosome biogenesis in the context of an intact vertebrate. PMID:26624285

  17. Single-step co-integration of multiple expressible heterologous genes into the ribosomal DNA of the methylotrophic yeast Hansenula polymorpha.

    PubMed

    Klabunde, J; Diesel, A; Waschk, D; Gellissen, G; Hollenberg, C P; Suckow, M

    2002-05-01

    We have investigated the methylotrophic yeast Hansenula polymorpha as a host for the co-integration and expression of multiple heterologous genes using an rDNA integration approach. The ribosomal DNA (rDNA) of H. polymorpha was found to consist of a single rDNA cluster of about 50-60 repeats of an 8-kb unit located on chromosome II. A 2.4-kb segment of H. polymorpha rDNA encompassing parts of the 25S, the complete 5S and the non-transcribed spacer region between 25S and 18S rDNA was isolated and inserted into conventional integrative H. polymorpha plasmids harboring the Saccharomyces- cerevisiae-derived URA3 gene for selection. These rDNA plasmids integrated homologously into the rDNA repeats of a H. polymorpha (odc1) host as several independent clusters. Anticipating that this mode of multiple-cluster integration could be used for the simultaneous integration of several distinct rDNA plasmids, the host strain was co-transformed with a mixture of up to three different plasmids, all bearing the same URA3 selection marker. Transformations indeed resulted in mitotically stable strains harboring one, two, or all three plasmids integrated into the rDNA. The overall copy number of the plasmids integrated did not exceed the number of rDNA repeats present in the untransformed host strain, irrespective of the number of different plasmids involved. Strains harboring different plasmids co-expressed the introduced genes, resulting in functional proteins. Thus, this approach provides a new and attractive tool for the rapid generation of recombinant strains that simultaneously co-produce several proteins in desired stoichiometric ratios. PMID:12021801

  18. Allele-specific germ cell epimutation in the spacer promoter of the 45S ribosomal RNA gene after Cr(III) exposure

    SciTech Connect

    Shiao, Y.-H. . E-mail: shiao@mail.ncifrcf.gov; Crawford, Erik B.; Anderson, Lucy M.; Patel, Pritesh; Ko, Kinarm

    2005-06-15

    Paternal exposure of mice to Cr(III) causes increased tumor risk in offspring; an epigenetic mechanism has been hypothesized. Representational difference analysis of gene methylation in sperm revealed hypomethylation in the 45S ribosomal RNA (rRNA) gene after Cr(III) exposure, compared with controls. The most striking effects were seen in the rRNA spacer promoter, a region in the intergenic region of rRNA gene clusters that can influence transcription. Methylation of the rRNA spacer promoter has not been studied heretofore. Sperm DNAs from Cr(III)-treated and control mice were modified by the bisulfite method followed by PCR amplification of the spacer promoter, including 27 CpG sites. Cloning and dideoxy sequencing identified sequence variants (T or G at base -2214) in the spacer promoter. The T allele had less DNA methylation than the G allele in control mice (17 of 17 clones vs. 42 of 72 clones, P = 0.0004). In spite of diversity of sperm DNA methylation patterns, the DNA clones from Cr(III)-exposed mice had fewer methylated CpG sites, by an average of 19% (P < 0.0001). This difference was limited to the G allele. The pyrosequencing technique was applied to quantify the percentage of methylation directly from amplified PCR products. Strikingly, for nine CpG sites including the spacer promoter core region, hypomethylation was highly significant in the Cr(III)-treated group (paired T test, P < 0.0001). Thus, one allele of the 45S rRNA spacer promoter is hypomethylated in sperm germ cells after Cr(III) exposure. This epimutation may lead to increase of tumor risk in the offspring.

  19. Flow Cytometry-assisted Cloning of Specific Sequence Motifs fromComplex 16S ribosomal RNA Gene Libraries.

    SciTech Connect

    Nielsen, J.L.; Schramm, A.; Bernhard, A.E.; van den Engh, G.J.; Stahl, D.A.

    2004-07-21

    A flow cytometry method was developed for rapid screeningand recovery of cloned DNA containing common sequence motifs. Thisapproach, termed fluorescence-activated cell sorting-assisted cloning,was used to recover sequences affiliated with a unique lineage within theBacteroidetes not abundant in a clone library of environmental 16S rRNAgenes. Retrieval and sequence analysis of phylogenetically informativegenes has become a standard cultivation-independent technique toinvestigate microbial diversity in nature (7, 18). Genes encoding the 16SrRNA, because of the relative ease of their selective amplification, havebeen most frequently employed for general diversity surveys (16).Environmental studies have also focused on specific subpopulationsaffiliated with a phylogenetic group or identified by genes encodingspecific metabolic functions (e.g., ammonia oxidation, sulfaterespiration, and nitrate reduction) (8,15,20). However, specificpopulations may be of low abundance (1,23), or the genes encodingspecific metabolic functions may be insufficiently conserved to providepriming sites for general PCR amplification. Three general approacheshave been used to obtain 16S rRNA sequence information from low-abundancepopulations: screening hundreds to thousands of clones in a general 16SrRNA gene library (21), flow cytometric sorting of a subpopulation ofenvironmentally derived cells labeled by fluorescent in situhybridization (FISH) (27), or selective PCR amplification using primersspecific for the subpopulation (2,23). While the first approach is simplytime-consuming and tedious, the second has been restricted to fairlylarge and strongly fluorescent cells from aquatic samples (5, 27). Thethird approach often generates fragments of only a few hundred bases dueto the limited number of specific priming sites. Partial sequenceinformation often degrades analysis, obscuring or distorting thephylogenetic placement of the new sequences (11, 20). A more robustcharacterization of environ

  20. Phylogenetic Analysis of Myobia musculi (Schranck, 1781) by Using the 18S Small Ribosomal Subunit Sequence

    PubMed Central

    Feldman, Sanford H; Ntenda, Abraham M

    2011-01-01

    We used high-fidelity PCR to amplify 2 overlapping regions of the ribosomal gene complex from the rodent fur mite Myobia musculi. The amplicons encompassed a large portion of the mite's ribosomal gene complex spanning 3128 nucleotides containing the entire 18S rRNA, internal transcribed spacer (ITS) 1, 5.8S rRNA, ITS2, and a portion of the 5′-end of the 28S rRNA. M. musculi’s 179-nucleotide 5.8S rRNA nucleotide sequence was not conserved, so this region was identified by conservation of rRNA secondary structure. Maximum likelihood and Bayesian inference phylogenetic analyses were performed by using multiple sequence alignment consisting of 1524 nucleotides of M. musculi 18S rRNA and homologous sequences from 42 prostigmatid mites and the tick Dermacentor andersoni. The phylograms produced by both methods were in agreement regarding terminal, secondary, and some tertiary phylogenetic relationships among mites. Bayesian inference discriminated most infraordinal relationships between Eleutherengona and Parasitengona mites in the suborder Anystina. Basal relationships between suborders Anystina and Eupodina historically determined by comparing differences in anatomic characteristics were less well-supported by our molecular analysis. Our results recapitulated similar 18S rRNA sequence analyses recently reported. Our study supports M. musculi as belonging to the suborder Anystina, infraorder Eleutherenona, and superfamily Cheyletoidea. PMID:22330574

  1. Selection of Reference Genes for Expression Analysis Using Quantitative Real-Time PCR in the Pea Aphid, Acyrthosiphon pisum (Harris) (Hemiptera, Aphidiae)

    PubMed Central

    Liu, Yong; Zhou, Xuguo

    2014-01-01

    To facilitate gene expression study and obtain accurate qRT-PCR analysis, normalization relative to stable expressed housekeeping genes is required. In this study, expression profiles of 11 candidate reference genes, including actin (Actin), elongation factor 1 α (EF1A), TATA-box-binding protein (TATA), ribosomal protein L12 (RPL12), β-tubulin (Tubulin), NADH dehydrogenase (NADH), vacuolar-type H+-ATPase (v-ATPase), succinate dehydrogenase B (SDHB), 28S ribosomal RNA (28S), 16S ribosomal RNA (16S), and 18S ribosomal RNA (18S) from the pea aphid Acyrthosiphon pisum, under different developmental stages and temperature conditions, were investigated. A total of four analytical tools, geNorm, Normfinder, BestKeeper, and the ΔCt method, were used to evaluate the suitability of these genes as endogenous controls. According to RefFinder, a web-based software tool which integrates all four above-mentioned algorithms to compare and rank the reference genes, SDHB, 16S, and NADH were the three most stable house-keeping genes under different developmental stages and temperatures. This work is intended to establish a standardized qRT-PCR protocol in pea aphid and serves as a starting point for the genomics and functional genomics research in this emerging insect model. PMID:25423476

  2. Histone and Ribosomal RNA Repetitive Gene Clusters of the Boll Weevil are Linked in a Tandem Array

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Histones are the major protein component of chromatin structure. The histone family is made up of a quintet of proteins, four core histones (H2A, H2B, H3 & H4) and the linker histones (H1). Spacers are found between the coding regions. Among insects this quintet of genes is usually clustered and ...

  3. Ribosomal RNA analysis in the diagnosis of Diamond-Blackfan Anaemia.

    PubMed

    Quarello, Paola; Garelli, Emanuela; Carando, Adriana; Mancini, Cecilia; Foglia, Luiselda; Botto, Carlotta; Farruggia, Piero; De Keersmaecker, Kim; Aspesi, Anna; Ellis, Steve R; Dianzani, Irma; Ramenghi, Ugo

    2016-03-01

    Diamond-Blackfan anaemia (DBA) is an inherited disease characterized by pure erythroid aplasia that has been tagged as a 'ribosomopathy'. We report a multi-centre study focused on the analysis of rRNA processing of 53 Italian DBA patients using capillary electrophoresis analysis of rRNA maturation of the 40S and 60S ribosomal subunits. The ratio of 28S/18S rRNA was higher in patients with mutated ribosomal proteins (RPs) of the small ribosomal subunit. In contrast, patients with mutated RPs of the large ribosomal subunit (RPLs) had a lower 28S/18S ratio. The assay reported here would be amenable for development as a diagnostic tool. PMID:26763766

  4. First description of the karyotype and localization of major and minor ribosomal genes in Rhoadsiaaltipinna Fowler, 1911 (Characiformes, Characidae) from Ecuador.

    PubMed

    Sánchez-Romero, Omar; Abad, César Quezada; Cordero, Patricio Quizhpe; de Sene, Viviani França; Nirchio, Mauro; Oliveira, Claudio

    2015-01-01

    Karyotypic features of Rhoadsiaaltipinna Fowler, 1911 from Ecuador were investigated by examining metaphase chromosomes through Giemsa staining, C-banding, Ag-NOR, and two-color-fluorescence in situ hybridization (FISH) for mapping of 18S and 5S ribosomal genes. The species exhibit a karyotype with 2n = 50, composed of 10 metacentric, 26 submetacentric and 14 subtelocentric elements, with a fundamental number FN=86 and is characterized by the presence of a larger metacentric pair (number 1), which is about 2/3 longer than the average length of the rest of the metacentric series. Sex chromosomes were not observed. Heterochromatin is identifiable on 44 chromosomes, distributed in paracentromeric position near the centromere. The first metacentric pair presents two well-defined heterochromatic blocks in paracentromeric position, near the centromere. Impregnation with silver nitrate showed a single pair of Ag-positive NORs localized at terminal regions of the short arms of the subtelocentric chromosome pair number 12. FISH assay confirmed these localization of NORs and revealed that minor rDNA clusters occur interstitially on the larger metacentric pair number 1. Comparison of results here reported with those available on other Characidae permit to hypothesize that the presence of a very large metacentric pair might represent a unique and derived condition that characterize one of four major lineages molecularly identified in this family. PMID:26140168

  5. First description of the karyotype and localization of major and minor ribosomal genes in Rhoadsia altipinna Fowler, 1911 (Characiformes, Characidae) from Ecuador

    PubMed Central

    Sánchez-Romero, Omar; Abad, César Quezada; Cordero, Patricio Quizhpe; de Sene, Viviani França; Nirchio, Mauro; Oliveira, Claudio

    2015-01-01

    Abstract Karyotypic features of Rhoadsia altipinna Fowler, 1911 from Ecuador were investigated by examining metaphase chromosomes through Giemsa staining, C-banding, Ag-NOR, and two-color-fluorescence in situ hybridization (FISH) for mapping of 18S and 5S ribosomal genes. The species exhibit a karyotype with 2n = 50, composed of 10 metacentric, 26 submetacentric and 14 subtelocentric elements, with a fundamental number FN=86 and is characterized by the presence of a larger metacentric pair (number 1), which is about 2/3 longer than the average length of the rest of the metacentric series. Sex chromosomes were not observed. Heterochromatin is identifiable on 44 chromosomes, distributed in paracentromeric position near the centromere. The first metacentric pair presents two well-defined heterochromatic blocks in paracentromeric position, near the centromere. Impregnation with silver nitrate showed a single pair of Ag-positive NORs localized at terminal regions of the short arms of the subtelocentric chromosome pair number 12. FISH assay confirmed these localization of NORs and revealed that minor rDNA clusters occur interstitially on the larger metacentric pair number 1. Comparison of results here reported with those available on other Characidae permit to hypothesize that the presence of a very large metacentric pair might represent a unique and derived condition that characterize one of four major lineages molecularly identified in this family. PMID:26140168

  6. Ribosome. The complete structure of the 55S mammalian mitochondrial ribosome.

    PubMed

    Greber, Basil J; Bieri, Philipp; Leibundgut, Marc; Leitner, Alexander; Aebersold, Ruedi; Boehringer, Daniel; Ban, Nenad

    2015-04-17

    Mammalian mitochondrial ribosomes (mitoribosomes) synthesize mitochondrially encoded membrane proteins that are critical for mitochondrial function. Here we present the complete atomic structure of the porcine 55S mitoribosome at 3.8 angstrom resolution by cryo-electron microscopy and chemical cross-linking/mass spectrometry. The structure of the 28S subunit in the complex was resolved at 3.6 angstrom resolution by focused alignment, which allowed building of a detailed atomic structure including all of its 15 mitoribosomal-specific proteins. The structure reveals the intersubunit contacts in the 55S mitoribosome, the molecular architecture of the mitoribosomal messenger RNA (mRNA) binding channel and its interaction with transfer RNAs, and provides insight into the highly specialized mechanism of mRNA recruitment to the 28S subunit. Furthermore, the structure contributes to a mechanistic understanding of aminoglycoside ototoxicity. PMID:25837512

  7. Effect of alpha-sarcin and ribosome-inactivating proteins on the interaction of elongation factors with ribosomes.

    PubMed

    Brigotti, M; Rambelli, F; Zamboni, M; Montanaro, L; Sperti, S

    1989-02-01

    alpha-Sarcin from Aspergillus giganteus and the ribosome-inactivating proteins (RIPs) from higher plants inactivate the 60 S ribosomal subunit. The former is an RNAase, whereas RIPs are N-glycosidases. The site of cleavage of RNA and that of N-glycosidic depurinization are at one nucleotide distance in 28 S rRNA [Endo & Tsurugi (1987) J. Biol. Chem. 262, 8128-8130]. The effect of alpha-sarcin and that of RIPs on the interaction of elongation factors with Artemia salina (brine shrimp) ribosomes have been investigated. alpha-Sarcin inhibits both the EF1 (elongation factor 1)-dependent binding of aminoacyl-tRNA and the GTP-dependent binding of EF2 (elongation factor 2) to ribosomes, whereas two of the RIPs tested, ricin from Ricinus communis (castor bean) and volkensin from Adenia volkensii (kilyambiti), inhibit only the latter reaction. EF2 protects ribosomes from inactivation by both alpha-sarcin and ricin. The EF1-binding site is affected only by alpha-sarcin. The sensitivity of this site to alpha-sarcin is increased by pretreatment of ribosomes with ricin. A. salina ribosomes were highly resistant to the third RIP tested, namely gelonin from Gelonium multiflorum. All four proteins tested have, however, a comparable activity on the rabbit reticulocyte-lysate system. PMID:2930482

  8. Identification of probiotic lactobacilli used for animal feeds on the basis of 16S ribosomal RNA gene sequence.

    PubMed

    Higuchi, Wataru; Muramatsu, Mineo; Dohmae, Soshi; Takano, Tomomi; Isobe, Hirokazu; Yabe, Shizuka; Da, Shi; Baranovich, Tatiana; Yamamoto, Tatsuo

    2008-11-01

    The use of probiotics such as Lactobacillus in animal feeds has gained popularity in recent years. In this study the 16S rRNA gene sequence of L. acidophilus in two commercial agents which have been used in animal feeds, LAB-MOS and Ghenisson 22, was determined. Phylogenetic tree analysis revealed that the two agents, strain MNFLM01 in LAB-MOS and strain GAL-2 in Ghenisson 22, belonged to L. rhamnosus (a member of the L. casei group) and L. johnsonii (a member of the L. acidophilus group), respectively. Biochemical tests assigned the two as L. rhamnosus and ambiguously as L. acidophilus. The data suggest that 16S rRNA gene sequence analysis provides more accurate identification of Lactobacillus species than biochemical tests and would allow quality assurance of relevant commercial products. The 16S rRNA gene sequences of strains MNFLM01 and GAL-2 determined in this study have been submitted to the DDBJ/EMBL/GenBank accession numbers under accession numbers AB288235 and AB295648, respectively. PMID:19090836

  9. The ribosomal database project.

    PubMed Central

    Larsen, N; Olsen, G J; Maidak, B L; McCaughey, M J; Overbeek, R; Macke, T J; Marsh, T L; Woese, C R

    1993-01-01

    The Ribosomal Database Project (RDP) is a curated database that offers ribosome data along with related programs and services. The offerings include phylogenetically ordered alignments of ribosomal RNA (rRNA) sequences, derived phylogenetic trees, rRNA secondary structure diagrams and various software packages for handling, analyzing and displaying alignments and trees. The data are available via ftp and electronic mail. Certain analytic services are also provided by the electronic mail server. PMID:8332524

  10. The Ribosomal Database Project.

    PubMed Central

    Maidak, B L; Larsen, N; McCaughey, M J; Overbeek, R; Olsen, G J; Fogel, K; Blandy, J; Woese, C R

    1994-01-01

    The Ribosomal Database Project (RDP) is a curated database that offers ribosome-related data, analysis services, and associated computer programs. The offerings include phylogenetically ordered alignments of ribosomal RNA (rRNA) sequences, derived phylogenetic trees, rRNA secondary structure diagrams, and various software for handling, analyzing and displaying alignments and trees. The data are available via anonymous ftp (rdp.life.uiuc.edu), electronic mail (server/rdp.life.uiuc.edu) and gopher (rdpgopher.life.uiuc.edu). The electronic mail server also provides ribosomal probe checking, approximate phylogenetic placement of user-submitted sequences, screening for chimeric nature of newly sequenced rRNAs, and automated alignment. PMID:7524021

  11. A system to simultaneously detect tick-borne pathogens based on the variability of the 16S ribosomal genes

    PubMed Central

    2013-01-01

    Background DNA microarrays can be used to quickly and sensitively identify several different pathogens in one step. Our previously developed DNA microarray, based on the detection of variable regions in the 16S rDNA gene (rrs), which are specific for each selected bacterial genus, allowed the concurrent detection of Borrelia spp., Anaplasma spp., Francisella spp., Rickettsia spp. and Coxiella spp. Methods In this study, we developed a comprehensive detection system consisting of a second generation DNA microarray and quantitative PCRs. New oligonucleotide capture probes specific for Borrelia burgdorferi s.l. genospecies and Candidatus Neoehrlichia mikurensis were included. This new DNA microarray system required substantial changes in solution composition, hybridization conditions and post-hybridization washes. Results This second generation chip displayed high specificity and sensitivity. The specificity of the capture probes was tested by hybridizing the DNA microarrays with Cy5-labeled, PCR-generated amplicons encoding the rrs genes of both target and non-target bacteria. The detection limit was determined to be 103 genome copies, which corresponds to 1–2 pg of DNA. A given sample was evaluated as positive if its mean fluorescence was at least 10% of the mean fluorescence of a positive control. Those samples with fluorescence close to the threshold were further analyzed using quantitative PCRs, developed to identify Francisella spp., Rickettsia spp. and Coxiella spp. Like the DNA microarray, the qPCRs were based on the genus specific variable regions of the rrs gene. No unspecific cross-reactions were detected. The detection limit for Francisella spp. was determined to be only 1 genome copy, for Coxiella spp. 10 copies, and for Rickettsia spp., 100 copies. Conclusions Our detection system offers a rapid method for the comprehensive identification of tick-borne bacteria, which is applicable to clinical samples. It can also be used to identify both pathogenic

  12. Biological activities of some Acacia spp. (Fabaceae) against new clinical isolates identified by ribosomal RNA gene-based phylogenetic analysis.

    PubMed

    Mahmoud, Mahmoud Fawzy; Alrumman, Sulaiman Abdullah; Hesham, Abd El-Latif

    2016-01-01

    Nowadays,most of the pathogenic bacteria become resistant to antibiotics. Therefore,the pharmaceutical properties of the natural plant extracts have become of interest to researchers as alternative antimicrobial agents. In this study,antibacterial activities of extract gained from Acacia etbaica, Acacia laeta, Acacia origena and Acacia pycnantha have been evaluated against isolated pathogenic bacteria (Strains MFM-01, MFM-10 and AH-09) using agar well diffusion methods.The bacterial strains were isolated from infected individuals,and their exact identification was detected on the basis of 16S rRNA gene amplification and sequence determination. Alignment results and the comparison of 16 SrRN A gene sequences of the isolates to 16 SrRN A gene sequences available in Gen Bank data base as well as the phylogenetic analysis confirmed the accurate position of the isolates as Klebsiella oxytoca strain MFM-01, Staphylococcus aureus strain MFM-10 and Klebsiella pneumoniae strain AH-09. Except for cold water, all tested solvents (Chloroform, petroleum ether, methanol, diethyl ether, and acetone) showed variation in their activity against studied bacteria. GC-MS analysis of ethanol extracts showed that four investigated Acacia species have different phyto components. Eight important pharmaceutical components were found in the legume of Acacia etbaica, seven in the legume of Acacia laeta, fifteen in the legume of Acacia origena and nine in the leaves of Acacia pycnantha. A dendrogram was constructed based on chemical composition, revealed that Acacia laeta is more closely related to Acacia etbaica forming on eclade, whereas Acacia origena less similar to other species. Our results demonstrated that, investigated plants and chemical compounds present could be used as promising antibacterial agents. PMID:26826814

  13. mtDNA ribosomal gene phylogeny of sea hares in the genus Aplysia (Gastropoda, Opisthobranchia, Anaspidea): implications for comparative neurobiology.

    PubMed

    Medina, M; Collins, T M; Walsh, P J

    2001-01-01

    Sea hares within the genus Aplysia are important neurobiological model organisms; as more studies based on different Aplysia species are appearing in the literature, a phylogenetic framework has become essential. We present a phylogenetic hypothesis for this genus, based on portions of two mitochondrial genes (12S and 16S). In addition, we reconstruct the evolution of several behavioral characters of interest to neurobiologists to illustrate the potential benefits of a phylogeny for the genus Aplysia. These benefits include determination of ancestral traits, direction and timing of evolution of characters, prediction of the distribution of traits, and identification of cases of independent acquisition of traits within lineages. This last benefit may prove especially useful in understanding the linkage between behaviors and their underlying neurological bases. PMID:12116938

  14. mtDNA ribosomal gene phylogeny of sea hares in the genus Aplysia (Gastropoda, Opisthobranchia, Anaspidea): Implications for comparative neurobiology

    SciTech Connect

    Medina, Monica; Collins, Timothy M.; Walsh, Patrick J.

    2000-08-10

    Sea hares within the genus Aplysia are important neurobiological model organisms, and as studies based on different Aplysia species appear in the literature, a phylogenetic framework has become essential. We present a phylogenetic hypothesis for this genus, based on portions of two mitochondrial genes (12S and 16S). In addition, we reconstruct the evolution of several behavioral characters of interest to neurobiologists in order to illustrate the potential benefits of a phylogeny for the genus Aplysia. These benefits include the determination of ancestral traits, the direction and timing of evolution of characters, prediction of the distribution of traits, and identification of cases of independent acquisition of traits within lineages. This last benefit may prove especially useful in understanding the linkage between behaviors and their underlying neurological basis.

  15. Molecular phylogeny of brachiopods and phoronids based on nuclear-encoded small subunit ribosomal RNA gene sequences

    PubMed Central

    L.Cohen, B.

    1998-01-01

    Brachiopod and phoronid phylogeny is inferred from SSU rDNA sequences of 28 articulate and nine inarticulate brachiopods, three phoronids, two ectoprocts and various outgroups, using gene trees reconstructed by weighted parsimony, distance and maximum likelihood methods. Of these sequences, 33 from brachiopods, two from phoronids and one each from an ectoproct and a priapulan are newly determined. The brachiopod sequences belong to 31 different genera and thus survey about 10% of extant genus-level diversity. Sequences determined in different laboratories and those from closely related taxa agree well, but evidence is presented suggesting that one published phoronid sequence (GenBank accession UO12648) is a brachiopod-phoronid chimaera, and this sequence is excluded from the analyses. The chiton, Acanthopleura, is identified as the phenetically proximal outgroup; other selected outgroups were chosen to allow comparison with recent, non-molecular analyses of brachiopod phylogeny. The different outgroups and methods of phylogenetic reconstruction lead to similar results, with differences mainly in the resolution of weakly supported ancient and recent nodes, including the divergence of inarticulate brachiopod sub-phyla, the position of the rhynchonellids in relation to long- and short-looped articulate brachiopod clades and the relationships of some articulate brachiopod genera and species. Attention is drawn to the problem presented by nodes that are strongly supported by non-molecular evidence but receive only low bootstrap resampling support. Overall, the gene trees agree with morphology-based brachiopod taxonomy, but novel relationships are tentatively suggested for thecideidine and megathyrid brachiopods. Articulate brachiopods are found to be monophyletic in all reconstructions, but monophyly of inarticulate brachiopods and the possible inclusion of phoronids in the inarticulate brachiopod clade are less strongly established. Phoronids are clearly excluded from

  16. The Ribosomal Database Project

    PubMed Central

    Olsen, Gary J.; Overbeek, Ross; Larsen, Niels; Marsh, Terry L.; McCaughey, Michael J.; Maciukenas, Michael A.; Kuan, Wen-Min; Macke, Thomas J.; Xing, Yuqing; Woese, Carl R.

    1992-01-01

    The Ribosomal Database Project (RDP) compiles ribosomal sequences and related data, and redistributes them in aligned and phylogenetically ordered form to its user community. It also offers various software packages for handling, analyzing and displaying sequences. In addition, the RDP offers (or will offer) certain analytic services. At present the project is in an intermediate stage of development. PMID:1598241

  17. Redescription of Rhizodomus tagatzi (Ciliophora: Spirotrichea: Tintinnida), based on morphology and small subunit ribosomal RNA gene sequence.

    PubMed

    Saccà, Alessandro; Strüder-Kypke, Michaela C; Lynn, Denis H

    2012-01-01

    Herein, we redescribe a tintinnid ciliate that is most commonly known as Tintinnopsis corniger Hada, 1964; but it has been described several times with different names, specifically Tintinnopsis nudicauda Paulmier, 1997 and Rhizodomus tagatzi Strelkow & Wirketis, 1950. Neotype material was collected from the water column of the coastal saline Lake Faro, a meromictic basin connected to the Straits of Messina, Central Mediterranean. The Lake Faro population is characterized by a hyaline or sparsely agglomerated lorica, which made it possible to observe in detail the basal layer structure, usually concealed by abundant incrusting particles. Along with an improved description of the lorica, we provide novel information, such as the general zooid morphology, the ciliary pattern, and the small subunit rRNA (SSU rRNA) gene sequence. Our phylogenetic analysis, based on the SSU rRNA, groups this species with Tintinnopsis radix, while the first taxonomic study designated it as R. tagatzi, introducing a new genus due to peculiarities in lorica morphology. We conclude that the species should be known as R. tagatzi, the senior synonym for the species. However, we do not transfer any other species to this genus, despite strong molecular similarities. Although it is obvious that the genus Tintinnopsis is in need of a thorough revision, current molecular and cytological information for this genus is too sparse, and the type species has not yet been redescribed with modern methods. PMID:22452414

  18. Karyotypic conservatism in samples of Characidium cf. zebra (Teleostei, Characiformes, Crenuchidae): Physical mapping of ribosomal genes and natural triploidy

    PubMed Central

    Pansonato-Alves, José Carlos; Oliveira, Claudio; Foresti, Fausto

    2011-01-01

    Basic and molecular cytogenetic analyses were performed in specimens of Characidium cf. zebra from five collection sites located throughout the Tietê, Paranapanema and Paraguay river basins. The diploid number in specimens from all samples was 2n = 50 with a karyotype composed of 32 metacentric and 18 submetacentric chromosomes in both males and females. Constitutive heterochromatin was present at the centromeric regions of all chromosomes and pair 23, had additional interstitial heterochromatic blocks on its long arms. The nucleolar organizer regions (NORs) were located on the long arms of pair 23, while the 5S rDNA sites were detected in different chromosomes among the studied samples. One specimen from the Alambari river was a natural triploid and had two extra chromosomes, resulting in 2n = 77. The remarkable karyotypic similarity among the specimens of C. cf. zebra suggests a close evolutionary relationship. On the other hand, the distinct patterns of 5S rDNA distribution may be the result of gene flow constraints during their evolutionary history. PMID:21734818

  19. A Sequence-Specific Interaction between the Saccharomyces cerevisiae rRNA Gene Repeats and a Locus Encoding an RNA Polymerase I Subunit Affects Ribosomal DNA Stability

    PubMed Central

    Cahyani, Inswasti; Cridge, Andrew G.; Engelke, David R.; Ganley, Austen R. D.

    2014-01-01

    The spatial organization of eukaryotic genomes is linked to their functions. However, how individual features of the global spatial structure contribute to nuclear function remains largely unknown. We previously identified a high-frequency interchromosomal interaction within the Saccharomyces cerevisiae genome that occurs between the intergenic spacer of the ribosomal DNA (rDNA) repeats and the intergenic sequence between the locus encoding the second largest RNA polymerase I subunit and a lysine tRNA gene [i.e., RPA135-tK(CUU)P]. Here, we used quantitative chromosome conformation capture in combination with replacement mapping to identify a 75-bp sequence within the RPA135-tK(CUU)P intergenic region that is involved in the interaction. We demonstrate that the RPA135-IGS1 interaction is dependent on the rDNA copy number and the Msn2 protein. Surprisingly, we found that the interaction does not govern RPA135 transcription. Instead, replacement of a 605-bp region within the RPA135-tK(CUU)P intergenic region results in a reduction in the RPA135-IGS1 interaction level and fluctuations in rDNA copy number. We conclude that the chromosomal interaction that occurs between the RPA135-tK(CUU)P and rDNA IGS1 loci stabilizes rDNA repeat number and contributes to the maintenance of nucleolar stability. Our results provide evidence that the DNA loci involved in chromosomal interactions are composite elements, sections of which function in stabilizing the interaction or mediating a functional outcome. PMID:25421713

  20. An Internal Ribosome Entry Site Directs Translation of the 3′-Gene from Pelargonium Flower Break Virus Genomic RNA: Implications for Infectivity

    PubMed Central

    Fernández-Miragall, Olga; Hernández, Carmen

    2011-01-01

    Pelargonium flower break virus (PFBV, genus Carmovirus) has a single-stranded positive-sense genomic RNA (gRNA) which contains five ORFs. The two 5′-proximal ORFs encode the replicases, two internal ORFs encode movement proteins, and the 3′-proximal ORF encodes a polypeptide (p37) which plays a dual role as capsid protein and as suppressor of RNA silencing. Like other members of family Tombusviridae, carmoviruses express ORFs that are not 5′-proximal from subgenomic RNAs. However, in one case, corresponding to Hisbiscus chlorotic ringspot virus, it has been reported that the 3′-proximal gene can be translated from the gRNA through an internal ribosome entry site (IRES). Here we show that PFBV also holds an IRES that mediates production of p37 from the gRNA, raising the question of whether this translation strategy may be conserved in the genus. The PFBV IRES was functional both in vitro and in vivo and either in the viral context or when inserted into synthetic bicistronic constructs. Through deletion and mutagenesis studies we have found that the IRES is contained within a 80 nt segment and have identified some structural traits that influence IRES function. Interestingly, mutations that diminish IRES activity strongly reduced the infectivity of the virus while the progress of the infection was favoured by mutations potentiating such activity. These results support the biological significance of the IRES-driven p37 translation and suggest that production of the silencing suppressor from the gRNA might allow the virus to early counteract the defence response of the host, thus facilitating pathogen multiplication and spread. PMID:21818349

  1. Stochastic and nonstochastic post-transcriptional silencing of chitinase and beta-1,3-glucanase genes involves increased RNA turnover-possible role for ribosome-independent RNA degradation.

    PubMed Central

    Holtorf, H; Schöb, H; Kunz, C; Waldvogel, R; Meins, F

    1999-01-01

    Stochastic and nonstochastic post-transcriptional gene silencing (PTGS) in Nicotiana sylvestris plants carrying tobacco class I chitinase (CHN) and beta-1,3-glucanase transgenes differs in incidence, stability, and pattern of expression. Measurements with inhibitors of RNA synthesis (cordycepin, actinomycin D, and alpha-amanitin) showed that both forms of PTGS are associated with increased sequence-specific degradation of transcripts, suggesting that increased RNA turnover may be a general feature of PTGS. The protein synthesis inhibitors cycloheximide and verrucarin A did not inhibit degradation of CHN RNA targeted for PTGS, confirming that PTGS-related RNA degradation does not depend on ongoing protein synthesis. Because verrucarin A, unlike cycloheximide, dissociates mRNA from ribosomes, our results also suggest that ribosome-associated RNA degradation pathways may not be involved in CHN PTGS. PMID:10072405

  2. A Comprehensive Selection of Reference Genes for RT-qPCR Analysis in a Predatory Lady Beetle, Hippodamia convergens (Coleoptera: Coccinellidae)

    PubMed Central

    Siegfried, Blair D.; Zhou, Xuguo

    2015-01-01

    Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a reliable, rapid, and reproducible technique for measuring and evaluating changes in gene expression. To facilitate gene expression studies and obtain more accurate RT-qPCR data, normalization relative to stable reference genes is required. In this study, expression profiles of seven candidate reference genes, including β-actin (Actin), elongation factor 1 α (EF1A), glyceralde hyde-3-phosphate dehydro-genase (GAPDH), cyclophilins A (CypA), vacuolar-type H+-ATPase (ATPase), 28S ribosomal RNA (28S), and 18S ribosomal RNA (18S) from Hippodamia convergens were investigated. H. convergens is an abundant predatory species in the New World, and has been widely used as a biological control agent against sap-sucking insect pests, primarily aphids. A total of four analytical methods, geNorm, Normfinder, BestKeeper, and the ΔCt method, were employed to evaluate the performance of these seven genes as endogenous controls under diverse experimental conditions. Additionally, RefFinder, a comprehensive evaluation platform integrating the four above mentioned algorithms, ranked the overall stability of these candidate genes. A suite of reference genes were specifically recommended for each experimental condition. Among them, 28S, EF1A, and CypA were the best reference genes across different development stages; GAPDH, 28S, and CypA were most stable in different tissues. GAPDH and CypA were most stable in female and male adults and photoperiod conditions, 28S and EF1A were most stable under a range of temperatures, Actin and CypA were most stable under dietary RNAi condition. This work establishes a standardized RT-qPCR analysis in H. convergens. Additionally, this study lays a foundation for functional genomics research in H. convergens and sheds light on the ecological risk assessment of RNAi-based biopesticides on this non-target biological control agent. PMID:25915640

  3. Precursor ribosomal ribonucleic acid and ribosome accumulation in vivo during the recovery of Salmonella typhimurium from thermal injury.

    PubMed

    Tomlins, R I; Ordal, Z J

    1971-07-01

    When cells of S. typhimurium were heated at 48 C for 30 min in phosphate buffer (pH 6.0), they became sensitive to Levine Eosin Methylene Blue Agar containing 2% NaCl (EMB-NaCl). The inoculation of injured cells into fresh growth medium supported the return of their normal tolerance to EMB-NaCl within 6 hr. The fractionation of ribosomal ribonucleic acid (rRNA) from unheated and heat-injured cells by polyacrylamide gel electrophoresis demonstrated that after injury the 16S RNA species was totally degraded and the 23S RNA was partially degraded. Sucrose gradient analysis demonstrated that after injury the 30S ribosomal subunit was totally destroyed and the sedimentation coefficient of the 50S particle was decreased to 47S. During the recovery of cells from thermal injury, four species of rRNA accumulated which were demonstrated to have the following sedimentation coefficients: 16, 17, 23, and 24S. Under identical recovery conditions, 22, 26, and 28S precursors of the 30S ribosomal subunit and 31 and 48S precursors of the 50S ribosomal subunit accumulated along with both the 30 and 50S mature particles. The addition of chloramphenicol to the recovery medium inhibited both the maturation of 17S RNA and the production of mature 30S ribosomal subunits, but permitted the accumulation of a single 22S precursor particle. Chloramphenicol did not affect either the maturation of 24S RNA or the mechanism of formation of 50S ribosomal subunits during recovery. Very little old ribosomal protein was associated with the new rRNA synthesized during recovery. New ribosomal proteins were synthesized during recovery and they were found associated with the new rRNA in ribosomal particles. The rate-limiting step in the recovery of S. typhimurium from thermal injury was in the maturation of the newly synthesized rRNA. PMID:4935315

  4. When ribosomes go bad: diseases of ribosome biogenesis

    PubMed Central

    Freed, Emily F.; Bleichert, Franziska; Dutca, Laura M.; Baserga, Susan J.

    2010-01-01

    Ribosomes are vital for cell growth and survival. Until recently, it was believed that mutations in ribosomes or ribosome biogenesis factors would be lethal, due to the essential nature of these complexes. However, in the last few decades, a number of diseases of ribosome biogenesis have been discovered. It remains a challenge in the field to elucidate the molecular mechanisms underlying them. PMID:20174677

  5. Increased Selectivity towards Cytoplasmic versus Mitochondrial Ribosome Confers Improved Efficiency of Synthetic Aminoglycosides in Fixing Damaged Genes: A Strategy for Treatment of Genetic Diseases Caused by Nonsense Mutations

    PubMed Central

    Kandasamy, Jeyakumar; Atia-Glikin, Dana; Shulman, Eli; Shapira, Katya; Shavit, Michal; Belakhov, Valery; Baasov, Timor

    2012-01-01

    Compelling evidence is now available that gentamicin and geneticin (G418) can induce mammalian ribosome to suppress disease-causing nonsense mutations and partially restore the expression of functional proteins. However, toxicity and relative lack of efficacy at subtoxic doses limit the use of gentamicin for suppression therapy. Although G418 exhibits strongest activity, it is very cytotoxic even at low doses. We describe here the first systematic development of the novel aminoglycoside (S)-11 exhibiting similar in vitro and ex vivo activity to that of G418, while its cell toxicity is significantly lower than those of gentamicin and G418. Using a series of biochemical assays, we provide proof of principle that antibacterial activity and toxicity of aminoglycosides can be dissected from their suppression activity. The data further indicate that the increased specificity towards cytoplasmic ribosome correlates with the increased activity, and that the decreased specificity towards mitochondrial ribosome confers to the lowered cytotoxicity. PMID:23148581

  6. Intra-Genomic Variation in the Ribosomal Repeats of Nematodes

    PubMed Central

    Bik, Holly M.; Fournier, David; Sung, Way; Bergeron, R. Daniel; Thomas, W. Kelley

    2013-01-01

    Ribosomal loci represent a major tool for investigating environmental diversity and community structure via high-throughput marker gene studies of eukaryotes (e.g. 18S rRNA). Since the estimation of species’ abundance is a major goal of environmental studies (by counting numbers of sequences), understanding the patterns of rRNA copy number across species will be critical for informing such high-throughput approaches. Such knowledge is critical, given that ribosomal RNA genes exist within multi-copy repeated arrays in a genome. Here we measured the repeat copy number for six nematode species by mapping the sequences from whole genome shotgun libraries against reference sequences for their rRNA repeat. This revealed a 6-fold variation in repeat copy number amongst taxa investigated, with levels of intragenomic variation ranging from 56 to 323 copies of the rRNA array. By applying the same approach to four C. elegans mutation accumulation lines propagated by repeated bottlenecking for an average of ~400 generations, we find on average a 2-fold increase in repeat copy number (rate of increase in rRNA estimated at 0.0285-0.3414 copies per generation), suggesting that rRNA repeat copy number is subject to selection. Within each Caenorhabditis species, the majority of intragenomic variation found across the rRNA repeat was observed within gene regions (18S, 28S, 5.8S), suggesting that such intragenomic variation is not a product of selection for rRNA coding function. We find that the dramatic variation in repeat copy number among these six nematode genomes would limit the use of rRNA in estimates of organismal abundance. In addition, the unique pattern of variation within a single genome was uncorrelated with patterns of divergence between species, reflecting a strong signature of natural selection for rRNA function. A better understanding of the factors that control or affect copy number in these arrays, as well as their rates and patterns of evolution, will be

  7. An overview of pre-ribosomal RNA processing in eukaryotes

    PubMed Central

    Henras, Anthony K; Plisson-Chastang, Célia; O'Donohue, Marie-Françoise; Chakraborty, Anirban; Gleizes, Pierre-Emmanuel

    2015-01-01

    Ribosomal RNAs are the most abundant and universal noncoding RNAs in living organisms. In eukaryotes, three of the four ribosomal RNAs forming the 40S and 60S subunits are borne by a long polycistronic pre-ribosomal RNA. A complex sequence of processing steps is required to gradually release the mature RNAs from this precursor, concomitant with the assembly of the 79 ribosomal proteins. A large set of trans-acting factors chaperone this process, including small nucleolar ribonucleoparticles. While yeast has been the gold standard for studying the molecular basis of this process, recent technical advances have allowed to further define the mechanisms of ribosome biogenesis in animals and plants. This renewed interest for a long-lasting question has been fueled by the association of several genetic diseases with mutations in genes encoding both ribosomal proteins and ribosome biogenesis factors, and by the perspective of new anticancer treatments targeting the mechanisms of ribosome synthesis. A consensus scheme of pre-ribosomal RNA maturation is emerging from studies in various kinds of eukaryotic organisms. However, major differences between mammalian and yeast pre-ribosomal RNA processing have recently come to light. WIREs RNA 2015, 6:225–242. doi: 10.1002/wrna.1269 PMID:25346433

  8. Characterization of three different clusters of 18S-26S ribosomal DNA genes in the sea urchin P. lividus: Genetic and epigenetic regulation synchronous to 5S rDNA.

    PubMed

    Bellavia, Daniele; Dimarco, Eufrosina; Caradonna, Fabio

    2016-04-15

    We previously reported the characterization 5S ribosomal DNA (rDNA) clusters in the common sea urchin Paracentrotus lividus and demonstrated the presence of DNA methylation-dependent silencing of embryo specific 5S rDNA cluster in adult tissue. In this work, we show genetic and epigenetic characterization of 18S-26S rDNA clusters in this specie. The results indicate the presence of three different 18S-26S rDNA clusters with different Non-Transcribed Spacer (NTS) regions that have different chromosomal localizations. Moreover, we show that the two largest clusters are hyper-methylated in the promoter-containing NTS regions in adult tissues, as in the 5S rDNA. These findings demonstrate an analogous epigenetic regulation in small and large rDNA clusters and support the logical synchronism in building ribosomes. In fact, all the ribosomal RNA genes must be synchronously and equally transcribed to perform their unique final product. PMID:26789074

  9. The ribosomal subunit assembly line

    PubMed Central

    Dlakić, Mensur

    2005-01-01

    Recent proteomic studies in Saccharomyces cerevisiae have identified nearly 200 proteins, other than the structural ribosomal proteins, that participate in the assembly of ribosomal subunits and their transport from the nucleus. In a separate line of research, proteomic studies of mature plant ribosomes have revealed considerable variability in the protein composition of individual ribosomes. PMID:16207363

  10. Alterations in the ribosomal machinery in cancer and hematologic disorders

    PubMed Central

    2012-01-01

    Ribosomes are essential components of the protein translation machinery and are composed of more than 80 unique large and small ribosomal proteins. Recent studies show that in addition to their roles in protein translation, ribosomal proteins are also involved in extra-ribosomal functions of DNA repair, apoptosis and cellular homeostasis. Consequently, alterations in the synthesis or functioning of ribosomal proteins can lead to various hematologic disorders. These include congenital anemias such as Diamond Blackfan anemia and Shwachman Diamond syndrome; both of which are associated with mutations in various ribosomal genes. Acquired uniallelic deletion of RPS14 gene has also been shown to lead to the 5q syndrome, a distinct subset of MDS associated with macrocytic anemia. Recent evidence shows that specific ribosomal proteins are overexpressed in liver, colon, prostate and other tumors. Ribosomal protein overexpression can promote tumorigenesis by interactions with the p53 tumor suppressor pathway and also by direct effects on various oncogenes. These data point to a broad role of ribosome protein alterations in hematologic and oncologic diseases. PMID:22709827

  11. The ribosome returned

    PubMed Central

    Moore, Peter B

    2009-01-01

    Since the mid-1990s, insights obtained from electron microscopy and X-ray crystallography have transformed our understanding of how the most important ribozyme in the cell, the ribosome, catalyzes protein synthesis. This review provides a brief account of how this structural revolution came to pass, and the impact it has had on our understanding of how the ribosome decodes messenger RNAs. PMID:19222865

  12. Mitochondrial ribosomal proteins (MRPs) of yeast.

    PubMed Central

    Graack, H R; Wittmann-Liebold, B

    1998-01-01

    Mitochondrial ribosomal proteins (MRPs) are the counterparts in that organelle of the cytoplasmic ribosomal proteins in the host. Although the MRPs fulfil similar functions in protein biosynthesis, they are distinct in number, features and primary structures from the latter. Most progress in the eludication of the properties of individual MRPs, and in the characterization of the corresponding genes, has been made in baker's yeast (Saccharomyces cerevisiae). To date, 50 different MRPs have been determined, although biochemical data and mutational analysis propose a total number which is substantially higher. Surprisingly, only a minority of the MRPs that have been characterized show significant sequence similarities to known ribosomal proteins from other sources, thus limiting the deduction of their functions by simple comparison of amino acid sequences. Further, individual MRPs have been characterized functionally by mutational studies, and the regulation of expression of MRP genes has been described. The interaction of the mitochondrial ribosomes with transcription factors specific for individual mitochondrial mRNAs, and the communication between mitochondria and the nucleus for the co-ordinated expression of ribosomal constituents, are other aspects of current MRP research. Although the mitochondrial translational system is still far from being described completely, the yeast MRP system serves as a model for other organisms, including that of humans. PMID:9445368

  13. Basic anatomy and tumor biology of the RPS6KA6 gene that encodes the p90 ribosomal S6 kinase-4

    PubMed Central

    Sun, Yuan; Cao, Shousong; Yang, Min; Wu, Sihong; Wang, Zhe; Lin, Xiukun; Song, Xiangrang; Liao, D.J.

    2012-01-01

    The RPS6KA6 gene encodes the p90 ribosomal S6 kinase-4 (RSK4) that is still largely uncharacterized. In this study we identified a new RSK4 transcription initiation site and several alternative splice sites with a 5’RACE approach. The resulting mRNA variants encompass four possible first start codons. The first 15 nucleotides (nt) of exon 22 in mouse and the penultimate exon in both human (exon 21) and mouse (exon 24) RSK4 underwent alternative splicing, although the penultimate exon deleted variant appeared mainly in cell clines, but not in most normal tissues. Demethylation agent 5-azacytidine inhibited the deletion of the penultimate exon whereas two indolocarbazole-derived inhibitors of cyclin dependent kinase 4 or 6 induced deletion of the first 39 nt from exon 21 of human RSK4. In all human cancer cell lines studied, the 90-kD wild type RSK4 was sparse but, surprisingly, several isoforms at or smaller than 72-kD were expressed as detected by seven different antibodies. On immunoblots, each of these smaller isoforms often appeared as a duplet or triplet and the levels of these isoforms varied greatly among different cell lines and culture conditions. Cyclin D1 inhibited RSK4 expression and serum starvation enhanced the inhibition, whereas c-Myc and RSK4 inhibited cyclin D1. The effects of RSK4 on cell growth, cell death and chemoresponse depended on the mRNA variant or the protein isoform expressed, on the specificity of the cell lines, as well as on the anchorage-dependent or -independent growth conditions and the in vivo situation. Moreover, we also observed that even a given cDNA might be expressed to multiple proteins; therefore, when using a cDNA, one needs to exclude this possibility before attribution of the biological results from the cDNA to the anticipated protein. Collectively, our results suggest that whether RSK4 is oncogenic or tumor suppressive depends on many factors. PMID:22614021

  14. PCR amplification of a multi-copy mitochondrial gene (cox3) improves detection of Cytauxzoon felis infection as compared to a ribosomal gene (18S).

    PubMed

    Schreeg, Megan E; Marr, Henry S; Griffith, Emily H; Tarigo, Jaime L; Bird, David M; Reichard, Mason V; Cohn, Leah A; Levy, Michael G; Birkenheuer, Adam J

    2016-07-30

    Cytauxzoon felis is a tick-transmitted protozoan parasite that infects felids. Clinical disease caused by acute C. felis infection rapidly progresses in domestic cats, leading to high morbidity and mortality. Accurately diagnosing cytauxzoonosis as soon as possible during acute infection would allow for earlier initiation of antiprotozoal therapy which could lead to higher survival rates. Molecular detection of parasite rRNA genes (18S) by PCR has previously been shown to be a sensitive method of diagnosing C. felis infections. Based on evidence from related apicomplexan species, we hypothesized that C. felis mitochondrial genes would exist at higher copy numbers than 18S and would be a more sensitive diagnostic target. In this study we have designed a PCR assay targeting the C. felis mitochondrial gene cytochrome c oxidase subunit III (cox3). Herein we demonstrate that (1) the cox3 PCR can detect as low as 1 copy of DNA target and can detect C. felis in samples with known mitochondrial sequence heterogeneity, (2) cox3 copy number is increased relative to 18S in blood and tissue samples from acutely infected cats, and (3) the cox3 PCR is more sensitive than 18S PCR for detection of C. felis during early infections. PMID:27369587

  15. Paradigms of ribosome synthesis: Lessons learned from ribosomal proteins

    PubMed Central

    Gamalinda, Michael; Woolford, John L

    2015-01-01

    The proteome in all cells is manufactured via the intricate process of translation by multimolecular factories called ribosomes. Nevertheless, these ribonucleoprotein particles, the largest of their kind, also have an elaborate assembly line of their own. Groundbreaking discoveries that bacterial ribosomal subunits can be self-assembled in vitro jumpstarted studies on how ribosomes are constructed. Until recently, ribosome assembly has been investigated almost entirely in vitro with bacterial small subunits under equilibrium conditions. In light of high-resolution ribosome structures and a more sophisticated toolkit, the past decade has been defined by a burst of kinetic studies in vitro and, importantly, also a shift to examining ribosome maturation in living cells, especially in eukaryotes. In this review, we summarize the principles governing ribosome assembly that emerged from studies focusing on ribosomal proteins and their interactions with rRNA. Understanding these paradigms has taken center stage, given the linkage between anomalous ribosome biogenesis and proliferative disorders. PMID:26779413

  16. Role of ribosomal protein mutations in tumor development (Review).

    PubMed

    Goudarzi, Kaveh M; Lindström, Mikael S

    2016-04-01

    Ribosomes are cellular machines essential for protein synthesis. The biogenesis of ribosomes is a highly complex and energy consuming process that initiates in the nucleolus. Recently, a series of studies applying whole-exome or whole-genome sequencing techniques have led to the discovery of ribosomal protein gene mutations in different cancer types. Mutations in ribosomal protein genes have for example been found in endometrial cancer (RPL22), T-cell acute lymphoblastic leukemia (RPL10, RPL5 and RPL11), chronic lymphocytic leukemia (RPS15), colorectal cancer (RPS20), and glioma (RPL5). Moreover, patients suffering from Diamond-Blackfan anemia, a bone marrow failure syndrome caused by mutant ribosomal proteins are also at higher risk for developing leukemia, or solid tumors. Different experimental models indicate potential mechanisms whereby ribosomal proteins may initiate cancer development. In particular, deregulation of the p53 tumor suppressor network and altered mRNA translation are mechanisms likely to be involved. We envisage that changes in expression and the occurrence of ribosomal protein gene mutations play important roles in cancer development. Ribosome biology constitutes a re-emerging vital area of basic and translational cancer research. PMID:26892688

  17. Role of ribosomal protein mutations in tumor development (Review)

    PubMed Central

    GOUDARZI, KAVEH M.; LINDSTRÖM, MIKAEL S.

    2016-01-01

    Ribosomes are cellular machines essential for protein synthesis. The biogenesis of ribosomes is a highly complex and energy consuming process that initiates in the nucleolus. Recently, a series of studies applying whole-exome or whole-genome sequencing techniques have led to the discovery of ribosomal protein gene mutations in different cancer types. Mutations in ribosomal protein genes have for example been found in endometrial cancer (RPL22), T-cell acute lymphoblastic leukemia (RPL10, RPL5 and RPL11), chronic lymphocytic leukemia (RPS15), colorectal cancer (RPS20), and glioma (RPL5). Moreover, patients suffering from Diamond-Blackfan anemia, a bone marrow failure syndrome caused by mutant ribosomal proteins are also at higher risk for developing leukemia, or solid tumors. Different experimental models indicate potential mechanisms whereby ribosomal proteins may initiate cancer development. In particular, deregulation of the p53 tumor suppressor network and altered mRNA translation are mechanisms likely to be involved. We envisage that changes in expression and the occurrence of ribosomal protein gene mutations play important roles in cancer development. Ribosome biology constitutes a re-emerging vital area of basic and translational cancer research. PMID:26892688

  18. [Ribosomal RNA Evolution

    NASA Technical Reports Server (NTRS)

    1997-01-01

    It is generally believed that an RNA World existed at an early stage in the history of life. During this early period, RNA molecules are seen to be potentially involved in both catalysis and the storage of genetic information. Translation presents several interrelated themes of inquiry for exobiology. First, it is essential, for understanding the very origin of life, how peptides and eventually proteins might have come to be made on the early Earth in a template directed manner. Second, it is necessary to understand how a machinery of similar complexity to that found in the ribosomes of modern organisms came to exist by the time of the last common ancestor (as detected by 16S rRNA sequence studies). Third, the ribosomal RNAs themselves likely had a very early origin and studies of their history may be very informative about the nature of the RNA World. Moreover, studies of these RNAs will contribute to a better understanding of the potential roles of RNA in early evolution.During the past year we have ave conducted a comparative study of four completely sequenced bacterial genoames. We have focused initially on conservation of gene order. The second component of the project continues to build on the model system for studying the validity of variant 5S rRNA sequences in the vicinity of the modern Vibrio proteolyticus 5S rRNA that we established earlier. This system has made it possible to conduct a detailed and extensive analysis of a local portion of the sequence space. These core methods have been used to construct numerous mutants during the last several years. Although it has been a secondary focus, this work has continued over the last year such that we now have in excess of 125 V. proteolyticus derived constructs which have been made and characterized. We have also continued high resolution NMR work on RNA oligomers originally initiated by G. Kenneth Smith who was funded by a NASA Graduate Student Researcher's Fellowship Award until May of 1996. Mr. Smith

  19. A tRNA methyltransferase paralog is important for ribosome stability and cell division in Trypanosoma brucei.

    PubMed

    Fleming, Ian M C; Paris, Zdeněk; Gaston, Kirk W; Balakrishnan, R; Fredrick, Kurt; Rubio, Mary Anne T; Alfonzo, Juan D

    2016-01-01

    Most eukaryotic ribosomes contain 26/28S, 5S, and 5.8S large subunit ribosomal RNAs (LSU rRNAs) in addition to the 18S rRNA of the small subunit (SSU rRNA). However, in kinetoplastids, a group of organisms that include medically important members of the genus Trypanosoma and Leishmania, the 26/28S large subunit ribosomal RNA is uniquely composed of 6 rRNA fragments. In addition, recent studies have shown the presence of expansion segments in the large ribosomal subunit (60S) of Trypanosoma brucei. Given these differences in structure, processing and assembly, T. brucei ribosomes may require biogenesis factors not found in other organisms. Here, we show that one of two putative 3-methylcytidine methyltransferases, TbMTase37 (a homolog of human methyltransferase-like 6, METTL6), is important for ribosome stability in T. brucei. TbMTase37 localizes to the nucleolus and depletion of the protein results in accumulation of ribosomal particles lacking srRNA 4 and reduced levels of polysome associated ribosomes. We also find that TbMTase37 plays a role in cytokinesis, as loss of the protein leads to multi-flagellated and multi-nucleated cells. PMID:26888608

  20. A tRNA methyltransferase paralog is important for ribosome stability and cell division in Trypanosoma brucei

    PubMed Central

    Fleming, Ian M. C.; Paris, Zdeněk; Gaston, Kirk W.; Balakrishnan, R.; Fredrick, Kurt; Rubio, Mary Anne T.; Alfonzo, Juan D.

    2016-01-01

    Most eukaryotic ribosomes contain 26/28S, 5S, and 5.8S large subunit ribosomal RNAs (LSU rRNAs) in addition to the 18S rRNA of the small subunit (SSU rRNA). However, in kinetoplastids, a group of organisms that include medically important members of the genus Trypanosoma and Leishmania, the 26/28S large subunit ribosomal RNA is uniquely composed of 6 rRNA fragments. In addition, recent studies have shown the presence of expansion segments in the large ribosomal subunit (60S) of Trypanosoma brucei. Given these differences in structure, processing and assembly, T. brucei ribosomes may require biogenesis factors not found in other organisms. Here, we show that one of two putative 3-methylcytidine methyltransferases, TbMTase37 (a homolog of human methyltransferase-like 6, METTL6), is important for ribosome stability in T. brucei. TbMTase37 localizes to the nucleolus and depletion of the protein results in accumulation of ribosomal particles lacking srRNA 4 and reduced levels of polysome associated ribosomes. We also find that TbMTase37 plays a role in cytokinesis, as loss of the protein leads to multi-flagellated and multi-nucleated cells. PMID:26888608

  1. Genome Mining for Ribosomally Synthesized Natural Products

    PubMed Central

    Velásquez, Juan E.; van der Donk, Wilfred

    2011-01-01

    In recent years, the number of known peptide natural products that are synthesized via the ribosomal pathway has rapidly grown. Taking advantage of sequence homology among genes encoding precursor peptides or biosynthetic proteins, in silico mining of genomes combined with molecular biology approaches has guided the discovery of a large number of new ribosomal natural products, including lantipeptides, cyanobactins, linear thiazole/oxazole-containing peptides, microviridins, lasso peptides, amatoxins, cyclotides, and conopeptides. In this review, we describe the strategies used for the identification of these ribosomally-synthesized and posttranslationally modified peptides (RiPPs) and the structures of newly identified compounds. The increasing number of chemical entities and their remarkable structural and functional diversity may lead to novel pharmaceutical applications. PMID:21095156

  2. Release of Nonstop Ribosomes Is Essential

    PubMed Central

    Feaga, Heather A.; Viollier, Patrick H.

    2014-01-01

    ABSTRACT Bacterial ribosomes frequently translate to the 3′ end of an mRNA without terminating at a stop codon. Almost all bacteria use the transfer-messenger RNA (tmRNA)-based trans-translation pathway to release these “nonstop” ribosomes and maintain protein synthesis capacity. trans-translation is essential in some species, but in others, such as Caulobacter crescentus, trans-translation can be inactivated. To determine why trans-translation is dispensable in C. crescentus, a Tn-seq screen was used to identify genes that specifically alter growth in cells lacking ssrA, the gene encoding tmRNA. One of these genes, CC1214, was essential in ΔssrA cells. Purified CC1214 protein could release nonstop ribosomes in vitro. CC1214 is a homolog of the Escherichia coli ArfB protein, and using the CC1214 sequence, ArfB homologs were identified in the majority of bacterial phyla. Most species in which ssrA has been deleted contain an ArfB homolog, suggesting that release of nonstop ribosomes may be essential in most or all bacteria. PMID:25389176

  3. Crystallography of ribosomal particles

    NASA Astrophysics Data System (ADS)

    Yonath, A.; Frolow, F.; Shoham, M.; Müssig, J.; Makowski, I.; Glotz, C.; Jahn, W.; Weinstein, S.; Wittmann, H. G.

    1988-07-01

    Several forms of three-dimensional crystals and two-dimensional sheets of intact ribosomes and their subunits have been obtained as a result of: (a) an extensive systematic investigation of the parameters involved in crystallization, (b) a development of an experimental procedure for controlling the volumes of the crystallization droplets, (c) a study of the nucleation process, and (d) introducing a delicate seeding procedure coupled with variations in the ratios of mono- and divalent ions in the crystallization medium. In all cases only biologically active particles could be crystallized, and the crystalline material retains its integrity and activity. Crystallographic data have been collected from crystals of 50S ribosomal subunits, using synchrotron radiation at temperatures between + 19 and - 180°C. Although at 4°C the higher resolution reflections decay within minutes in the synchrotron beam, at cryo-temperature there was hardly any radiation damage, and a complete set of data to about 6Åresolution could be collected from a single crystal. Heavy-atom clusters were used for soaking as well as for specific binding to the surface of the ribosomal subunits prior to crystallization. The 50S ribosomal subunits from a mutant of B. stearothermophilus which lacks the ribosomal protein BL11 crystallize isomorphously with in the native ones. Models, aimed to be used for low resolution phasing, have been reconstructed from two-dimensional sheets of 70S ribosomes and 50S subunits at 47 and 30Å, respectively. These models show the overall structure of these particles, the contact areas between the large and small subunits, the space where protein synthesis might take place and a tunnel which may provide the path for the nascent protein chain.

  4. The Ribosome Comes Alive

    PubMed Central

    2010-01-01

    This essay is a reflection on the ways the X-ray structures of the ribosome are helping in the interpretation of cryogenic electron microscopy (cryo-EM) density maps showing the translating ribosome in motion. Through advances in classification methods, cryo-EM and single-particle reconstruction methods have recently evolved to the point where they can yield an array of structures from a single sample (“story in a sample”), providing snapshots of an entire subprocess of translation, such as translocation or decoding. PMID:21072331

  5. The Ribosome Comes Alive.

    PubMed

    Frank, Joachim

    2010-06-18

    This essay is a reflection on the ways the X-ray structures of the ribosome are helping in the interpretation of cryogenic electron microscopy (cryo-EM) density maps showing the translating ribosome in motion. Through advances in classification methods, cryo-EM and single-particle reconstruction methods have recently evolved to the point where they can yield an array of structures from a single sample ("story in a sample"), providing snapshots of an entire subprocess of translation, such as translocation or decoding. PMID:21072331

  6. The site-specific ribosomal DNA insertion element R1Bm belongs to a class of non-long-terminal-repeat retrotransposons

    SciTech Connect

    Xiong, Y.; Eickbush, T.H.

    1988-01-01

    Two types of insertion elements, R1 and R2 (previously called type I and type II), are known to interrupt the 28S ribosomal genes of several insect species. In the silkmoth, Bombyx mori, each element occupies approximately 10% of the estimated 240 ribosomal DNA units, while at most only a few copies are located outside the ribosomal DNA units. The authors present here the complete nucleotide sequence of an R1 insertion from B. mori (R1Bm). This 5.1-kilobase element contains two overlapping open reading frames (ORFs) which together occupy 88% of its length. ORF1 is 461 amino acids in length and exhibits characteristics of retroviral gag genes. ORF2 is 1,051 amino acids in length and contains homology to reverse transcriptase-like enzymes. The analysis of 3' and 5' ends of independent isolates from the ribosomal locus supports the suggestion that R1 is still functioning as a transposable element. The precise location of the element within the genome implies that its transposition must occur with remarkable insertion sequence specificity. Comparison of the deduced amino acid sequences from six retrotransposons, R1 and R2 of B. mori, I factor and F element of Drosophila melanogaster, L1 of Mus domesticus, and Ingi of Trypanosoma brucei, reveals a relatively high level of sequence homology in the reverse transcriptase region. Like R1, these elements lack long terminal repeats. The authors therefore named this class of related elements the non-long-terminal-repeat (non-LTR) retrotransposons.

  7. A ribosome-inactivating protein in a Drosophila defensive symbiont.

    PubMed

    Hamilton, Phineas T; Peng, Fangni; Boulanger, Martin J; Perlman, Steve J

    2016-01-12

    Vertically transmitted symbionts that protect their hosts against parasites and pathogens are well known from insects, yet the underlying mechanisms of symbiont-mediated defense are largely unclear. A striking example of an ecologically important defensive symbiosis involves the woodland fly Drosophila neotestacea, which is protected by the bacterial endosymbiont Spiroplasma when parasitized by the nematode Howardula aoronymphium. The benefit of this defense strategy has led to the rapid spread of Spiroplasma throughout the range of D. neotestacea, although the molecular basis for this protection has been unresolved. Here, we show that Spiroplasma encodes a ribosome-inactivating protein (RIP) related to Shiga-like toxins from enterohemorrhagic Escherichia coli and that Howardula ribosomal RNA (rRNA) is depurinated during Spiroplasma-mediated protection of D. neotestacea. First, we show that recombinant Spiroplasma RIP catalyzes depurination of 28S rRNAs in a cell-free assay, as well as Howardula rRNA in vitro at the canonical RIP target site within the α-sarcin/ricin loop (SRL) of 28S rRNA. We then show that Howardula parasites in Spiroplasma-infected flies show a strong signal of rRNA depurination consistent with RIP-dependent modification and large decreases in the proportion of 28S rRNA intact at the α-sarcin/ricin loop. Notably, host 28S rRNA is largely unaffected, suggesting targeted specificity. Collectively, our study identifies a novel RIP in an insect defensive symbiont and suggests an underlying RIP-dependent mechanism in Spiroplasma-mediated defense. PMID:26712000

  8. A ribosome-inactivating protein in a Drosophila defensive symbiont

    PubMed Central

    Hamilton, Phineas T.; Peng, Fangni; Boulanger, Martin J.; Perlman, Steve J.

    2016-01-01

    Vertically transmitted symbionts that protect their hosts against parasites and pathogens are well known from insects, yet the underlying mechanisms of symbiont-mediated defense are largely unclear. A striking example of an ecologically important defensive symbiosis involves the woodland fly Drosophila neotestacea, which is protected by the bacterial endosymbiont Spiroplasma when parasitized by the nematode Howardula aoronymphium. The benefit of this defense strategy has led to the rapid spread of Spiroplasma throughout the range of D. neotestacea, although the molecular basis for this protection has been unresolved. Here, we show that Spiroplasma encodes a ribosome-inactivating protein (RIP) related to Shiga-like toxins from enterohemorrhagic Escherichia coli and that Howardula ribosomal RNA (rRNA) is depurinated during Spiroplasma-mediated protection of D. neotestacea. First, we show that recombinant Spiroplasma RIP catalyzes depurination of 28S rRNAs in a cell-free assay, as well as Howardula rRNA in vitro at the canonical RIP target site within the α-sarcin/ricin loop (SRL) of 28S rRNA. We then show that Howardula parasites in Spiroplasma-infected flies show a strong signal of rRNA depurination consistent with RIP-dependent modification and large decreases in the proportion of 28S rRNA intact at the α-sarcin/ricin loop. Notably, host 28S rRNA is largely unaffected, suggesting targeted specificity. Collectively, our study identifies a novel RIP in an insect defensive symbiont and suggests an underlying RIP-dependent mechanism in Spiroplasma-mediated defense. PMID:26712000

  9. Identification of stable reference genes for quantitative PCR in cells derived from chicken lymphoid organs.

    PubMed

    Borowska, D; Rothwell, L; Bailey, R A; Watson, K; Kaiser, P

    2016-02-01

    Quantitative polymerase chain reaction (qPCR) is a powerful technique for quantification of gene expression, especially genes involved in immune responses. Although qPCR is a very efficient and sensitive tool, variations in the enzymatic efficiency, quality of RNA and the presence of inhibitors can lead to errors. Therefore, qPCR needs to be normalised to obtain reliable results and allow comparison. The most common approach is to use reference genes as internal controls in qPCR analyses. In this study, expression of seven genes, including β-actin (ACTB), β-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-glucuronidase (GUSB), TATA box binding protein (TBP), α-tubulin (TUBAT) and 28S ribosomal RNA (r28S), was determined in cells isolated from chicken lymphoid tissues and stimulated with three different mitogens. The stability of the genes was measured using geNorm, NormFinder and BestKeeper software. The results from both geNorm and NormFinder were that the three most stably expressed genes in this panel were TBP, GAPDH and r28S. BestKeeper did not generate clear answers because of the highly heterogeneous sample set. Based on these data we will include TBP in future qPCR normalisation. The study shows the importance of appropriate reference gene normalisation in other tissues before qPCR analysis. PMID:26872627

  10. Mitomycin C Inhibits Ribosomal RNA

    PubMed Central

    Snodgrass, Ryan G.; Collier, Abby C.; Coon, Amy E.; Pritsos, Chris A.

    2010-01-01

    Mitomycin C (MMC) is a commonly used and extensively studied chemotherapeutic agent requiring biological reduction for activity. Damage to nuclear DNA is thought to be its primary mechanism of cell death. Due to a lack of evidence for significant MMC activation in the nucleus and for in vivo studies demonstrating the formation of MMC-DNA adducts, we chose to investigate alternative nucleic acid targets. Real-time reverse transcription-PCR was used to determine changes in mitochondrial gene expression induced by MMC treatment. Although no consistent effects on mitochondrial mRNA expression were observed, complementary results from reverse transcription-PCR experiments and gel-shift and binding assays demonstrated that MMC rapidly decreased the transcript levels of 18S ribosomal RNA in a concentration-dependent manner. Under hypoxic conditions, transcript levels of 18S rRNA decreased by 1.5-fold compared with untreated controls within 30 min. Recovery to base line required several hours, indicating that de novo synthesis of 18S was necessary. Addition of MMC to an in vitro translation reaction significantly decreased protein production in the cell-free system. Functional assays performed using a luciferase reporter construct in vivo determined that protein translation was inhibited, further confirming this mechanism of toxicity. The interaction of MMC with ribosomal RNA and subsequent inhibition of protein translation is consistent with mechanisms proposed for other natural compounds. PMID:20418373

  11. Bmi1 promotes erythroid development through regulating ribosome biogenesis

    PubMed Central

    Gao, Rui; Chen, Sisi; Kobayashi, Michihiro; Yu, Hao; Zhang, Yingchi; Wan, Yang; Young, Sara K.; Soltis, Anthony; Yu, Ming; Vemula, Sasidhar; Fraenkel, Ernest; Cantor, Alan; Antipin, Yevgeniy; Xu, Yang; Yoder, Mervin C.; Wek, Ronald C.; Ellis, Steven R.; Kapur, Reuben; Zhu, Xiaofan; Liu, Yan

    2015-01-01

    While Polycomb group protein Bmi1 is important for stem cell maintenance, its role in lineage commitment is largely unknown. We have identified Bmi1 as a novel regulator of erythroid development. Bmi1 is highly expressed in mouse erythroid progenitor cells and its deficiency impairs erythroid differentiation. BMI1 is also important for human erythroid development. Furthermore, we discovered that loss of Bmi1 in erythroid progenitor cells results in down-regulation of transcription of multiple ribosomal protein genes and impaired ribosome biogenesis. Bmi1 deficiency stabilizes p53 protein, leading to upregulation of p21 expression and subsequent G0/G1 cell cycle arrest. Genetic inhibition of p53 activity rescues the erythroid defects seen in the Bmi1 null mice, demonstrating that a p53-dependent mechanism underlies the pathophysiology of the anemia. Mechanistically, Bmi1 is associated with multiple ribosomal protein genes and may positively regulate their expression in erythroid progenitor cells. Thus, Bmi1 promotes erythroid development, at least in part through regulating ribosome biogenesis. Ribosomopathies are human disorders of ribosome dysfunction, including diamond blackfan anemia (DBA) and 5q- syndrome, in which genetic abnormalities cause impaired ribosome biogenesis, resulting in specific clinical phenotypes. We observed that BMI1 expression in human hematopoietic stem and progenitor cells (HSPCs) from patients with DBA is correlated with the expression of some ribosomal protein genes, suggesting that BMI1 deficiency may play a pathological role in DBA and other ribosomopathies. PMID:25385494

  12. Bmi1 promotes erythroid development through regulating ribosome biogenesis.

    PubMed

    Gao, Rui; Chen, Sisi; Kobayashi, Michihiro; Yu, Hao; Zhang, Yingchi; Wan, Yang; Young, Sara K; Soltis, Anthony; Yu, Ming; Vemula, Sasidhar; Fraenkel, Ernest; Cantor, Alan; Antipin, Yevgeniy; Xu, Yang; Yoder, Mervin C; Wek, Ronald C; Ellis, Steven R; Kapur, Reuben; Zhu, Xiaofan; Liu, Yan

    2015-03-01

    While Polycomb group protein Bmi1 is important for stem cell maintenance, its role in lineage commitment is largely unknown. We have identified Bmi1 as a novel regulator of erythroid development. Bmi1 is highly expressed in mouse erythroid progenitor cells and its deficiency impairs erythroid differentiation. BMI1 is also important for human erythroid development. Furthermore, we discovered that loss of Bmi1 in erythroid progenitor cells results in decreased transcription of multiple ribosomal protein genes and impaired ribosome biogenesis. Bmi1 deficiency stabilizes p53 protein, leading to upregulation of p21 expression and subsequent G0/G1 cell cycle arrest. Genetic inhibition of p53 activity rescues the erythroid defects seen in the Bmi1 null mice, demonstrating that a p53-dependent mechanism underlies the pathophysiology of the anemia. Mechanistically, Bmi1 is associated with multiple ribosomal protein genes and may positively regulate their expression in erythroid progenitor cells. Thus, Bmi1 promotes erythroid development, at least in part through regulating ribosome biogenesis. Ribosomopathies are human disorders of ribosome dysfunction, including Diamond-Blackfan anemia (DBA) and 5q- syndrome, in which genetic abnormalities cause impaired ribosome biogenesis, resulting in specific clinical phenotypes. We observed that BMI1 expression in human hematopoietic stem and progenitor cells from patients with DBA is correlated with the expression of some ribosomal protein genes, suggesting that BMI1 deficiency may play a pathological role in DBA and other ribosomopathies. PMID:25385494

  13. Ribosome-dependent activation of stringent control.

    PubMed

    Brown, Alan; Fernández, Israel S; Gordiyenko, Yuliya; Ramakrishnan, V

    2016-06-01

    In order to survive, bacteria continually sense, and respond to, environmental fluctuations. Stringent control represents a key bacterial stress response to nutrient starvation that leads to rapid and comprehensive reprogramming of metabolic and transcriptional patterns. In general, transcription of genes for growth and proliferation is downregulated, while those important for survival and virulence are upregulated. Amino acid starvation is sensed by depletion of the aminoacylated tRNA pools, and this results in accumulation of ribosomes stalled with non-aminoacylated (uncharged) tRNA in the ribosomal A site. RelA is recruited to stalled ribosomes and activated to synthesize a hyperphosphorylated guanosine analogue, (p)ppGpp, which acts as a pleiotropic secondary messenger. However, structural information about how RelA recognizes stalled ribosomes and discriminates against aminoacylated tRNAs is missing. Here we present the cryo-electron microscopy structure of RelA bound to the bacterial ribosome stalled with uncharged tRNA. The structure reveals that RelA utilizes a distinct binding site compared to the translational factors, with a multi-domain architecture that wraps around a highly distorted A-site tRNA. The TGS (ThrRS, GTPase and SpoT) domain of RelA binds the CCA tail to orient the free 3' hydroxyl group of the terminal adenosine towards a β-strand, such that an aminoacylated tRNA at this position would be sterically precluded. The structure supports a model in which association of RelA with the ribosome suppresses auto-inhibition to activate synthesis of (p)ppGpp and initiate the stringent response. Since stringent control is responsible for the survival of pathogenic bacteria under stress conditions, and contributes to chronic infections and antibiotic tolerance, RelA represents a good target for the development of novel antibacterial therapeutics. PMID:27279228

  14. Motion of individual ribosomes along mRNA

    NASA Astrophysics Data System (ADS)

    Visscher, Koen

    2004-11-01

    Ribosomes move along messenger RNA to translate a sequence of ribonucleotides into a corresponding sequence of amino acids that make up a protein. Efficient motion of ribosomes along the mRNA requires hydrolysis of GTP, converting chemical energy into mechanical work, like better known molecular motors such as kinesin. However, motion is just one of the many tasks of the ribosome, whereas for kinesin, motion itself is the main goal. In keeping with these functional differences, the ribosome is also much larger consisting of more than 50 proteins and with half of its mass made up of ribosomal RNA. Such structural complexity enables indirect ways of coupling GTP hydrolysis to directed motion. In order to elucidate the mechanochemical coupling in ribosomes we have developed a single-molecule assay based on using optical tweezers to record the motion of individual ribosomes along mRNA. Translation rates of 2-4 codons/s have been observed. However, when increasing the force opposing motion, we observe backward slippage of ribosomes along homopolymeric poly(U) messages. Currently, it is not clear if the motor operates in reverse or if backward motion has become completely uncoupled from GTP hydrolysis. Interestingly, force-induced backward motion is of biological relevance because of its possible role in -1 frameshifting, a mechanism used by viruses to regulate gene expression at the level of translation.

  15. Ribosome Assembly as Antimicrobial Target

    PubMed Central

    Nikolay, Rainer; Schmidt, Sabine; Schlömer, Renate; Deuerling, Elke; Nierhaus, Knud H.

    2016-01-01

    Many antibiotics target the ribosome and interfere with its translation cycle. Since translation is the source of all cellular proteins including ribosomal proteins, protein synthesis and ribosome assembly are interdependent. As a consequence, the activity of translation inhibitors might indirectly cause defective ribosome assembly. Due to the difficulty in distinguishing between direct and indirect effects, and because assembly is probably a target in its own right, concepts are needed to identify small molecules that directly inhibit ribosome assembly. Here, we summarize the basic facts of ribosome targeting antibiotics. Furthermore, we present an in vivo screening strategy that focuses on ribosome assembly by a direct fluorescence based read-out that aims to identify and characterize small molecules acting as primary assembly inhibitors. PMID:27240412

  16. Ribosome Assembly as Antimicrobial Target.

    PubMed

    Nikolay, Rainer; Schmidt, Sabine; Schlömer, Renate; Deuerling, Elke; Nierhaus, Knud H

    2016-01-01

    Many antibiotics target the ribosome and interfere with its translation cycle. Since translation is the source of all cellular proteins including ribosomal proteins, protein synthesis and ribosome assembly are interdependent. As a consequence, the activity of translation inhibitors might indirectly cause defective ribosome assembly. Due to the difficulty in distinguishing between direct and indirect effects, and because assembly is probably a target in its own right, concepts are needed to identify small molecules that directly inhibit ribosome assembly. Here, we summarize the basic facts of ribosome targeting antibiotics. Furthermore, we present an in vivo screening strategy that focuses on ribosome assembly by a direct fluorescence based read-out that aims to identify and characterize small molecules acting as primary assembly inhibitors. PMID:27240412

  17. Structural insights into ribosome translocation.

    PubMed

    Ling, Clarence; Ermolenko, Dmitri N

    2016-09-01

    During protein synthesis, tRNA and mRNA are translocated from the A to P to E sites of the ribosome thus enabling the ribosome to translate one codon of mRNA after the other. Ribosome translocation along mRNA is induced by the universally conserved ribosome GTPase, elongation factor G (EF-G) in bacteria and elongation factor 2 (EF-2) in eukaryotes. Recent structural and single-molecule studies revealed that tRNA and mRNA translocation within the ribosome is accompanied by cyclic forward and reverse rotations between the large and small ribosomal subunits parallel to the plane of the intersubunit interface. In addition, during ribosome translocation, the 'head' domain of small ribosomal subunit undergoes forward- and back-swiveling motions relative to the rest of the small ribosomal subunit around the axis that is orthogonal to the axis of intersubunit rotation. tRNA/mRNA translocation is also coupled to the docking of domain IV of EF-G into the A site of the small ribosomal subunit that converts the thermally driven motions of the ribosome and tRNA into the forward translocation of tRNA/mRNA inside the ribosome. Despite recent and enormous progress made in the understanding of the molecular mechanism of ribosome translocation, the sequence of structural rearrangements of the ribosome, EF-G and tRNA during translocation is still not fully established and awaits further investigation. WIREs RNA 2016, 7:620-636. doi: 10.1002/wrna.1354 For further resources related to this article, please visit the WIREs website. PMID:27117863

  18. Ribosomal DNA organization patterns within the dinoflagellate genus Alexandrium as revealed by FISH: life cycle and evolutionary implications.

    PubMed

    Figueroa, Rosa Isabel; Cuadrado, Angeles; Stüken, Anke; Rodríguez, Francisco; Fraga, Santiago

    2014-05-01

    Dinoflagellates are a group of protists whose genome differs from that of other eukaryotes in terms of size (contains up to 250pg per haploid cell), base composition, chromosomal organization, and gene expression. But rDNA gene mapping of the active nucleolus in this unusual eukaryotic genome has not been carried out thus far. Here we used FISH in dinoflagellate species belonging to the genus Alexandrium (genome sizes ranging from 21 to 170 pg of DNA per haploid genome) to localize the sequences encoding the 18S, 5.8S, and 28S rRNA genes. The results can be summarized as follows: 1) Each dinoflagellate cell contains only one active nucleolus, with no hybridization signals outside it. However, the rDNA organization varies among species, from repetitive clusters forming discrete nuclear organizer regions (NORs) in some to specialized "ribosomal chromosomes" in other species. The latter chromosomes, never reported before in other eukaryotes, are mainly formed by rDNA genes and appeared in the species with the highest DNA content. 2) Dinoflagellate chromosomes are first characterized by several eukaryotic features, such as structural differentiation (centromere-like constrictions), size differences (dot chromosomes), and SAT (satellite) chromosomes. 3) NOR patterns prove to be useful in discriminating between cryptic species and life cycle stages in protists. PMID:24846057

  19. Metabolic Labeling in the Study of Mammalian Ribosomal RNA Synthesis.

    PubMed

    Stefanovsky, Victor Y; Moss, Tom

    2016-01-01

    RNA metabolic labeling is a method of choice in the study of dynamic changes in the rate of gene transcription and RNA processing. It is particularly applicable to transcription of the ribosomal RNA genes and their processing products due to the very high levels of ribosomal RNA synthesis. Metabolic labeling can detect changes in ribosomal RNA transcription that occur within a few minutes as opposed to the still widely used RT-PCR or Northern blot procedures that measure RNA pool sizes and at best are able to detect changes occurring over several hours or several days. Here, we describe a metabolic labeling technique applicable to the measurement of ribosomal RNA synthesis and processing rates, as well as to the determination of RNA Polymerase I transcription elongation rates. PMID:27576716

  20. Hindered proton collectivity in the proton-rich nucleus 28S: Possible magic number Z = 16

    NASA Astrophysics Data System (ADS)

    Togano, Y.; Yamada, Y.; Iwasa, N.; Yamada, K.; Motobayashi, T.; Aoi, N.; Baba, H.; Bishop, S.; Cai, X.; Doornenbal, P.; Fang, D.; Furukawa, T.; Ieki, K.; Kawabata, T.; Kanno, S.; Kobayashi, N.; Kondo, Y.; Kuboki, T.; Kume, N.; Kurita, K.; Kurokawa, M.; Ma, Y. G.; Matsuo, Y.; Murakami, H.; Matsushita, M.; Nakamura, T.; Okada, K.; Ota, S.; Satou, Y.; Shimoura, S.; Shioda, R.; Tanaka, K. N.; Takeuchi, S.; Tian, W.; Wang, H.; Wang, J.; Yoneda, K.

    2012-10-01

    The reduced transition probability B(E2;0gs+→21+) for the proton-rich nucleus 28S was determined experimentally using intermediate-energy Coulomb excitation. The resultant B(E2) value 181(31) e2fm4 is smaller than those of neighboring N = 12 isotones and Z = 16 isotopes. The double ratio |Mn/Mp|/(N/Z) of the 0gs+→21+ transition in 28S was obtained to be 1.9(2) by evaluating the Mn value from the known B(E2) value of the mirror nucleus 28Mg, showing the hindrance of proton collectivity relative to that of neutrons. These results indicate the emergence of the magic number Z = 16 in 28S.

  1. Ribosome recycling induces optimal translation rate at low ribosomal availability.

    PubMed

    Marshall, E; Stansfield, I; Romano, M C

    2014-09-01

    During eukaryotic cellular protein synthesis, ribosomal translation is made more efficient through interaction between the two ends of the messenger RNA (mRNA). Ribosomes reaching the 3' end of the mRNA can thus recycle and begin translation again on the same mRNA, the so-called 'closed-loop' model. Using a driven diffusion lattice model of translation, we study the effects of ribosome recycling on the dynamics of ribosome flow and density on the mRNA. We show that ribosome recycling induces a substantial increase in ribosome current. Furthermore, for sufficiently large values of the recycling rate, the lattice does not transition directly from low to high ribosome density, as seen in lattice models without recycling. Instead, a maximal current phase becomes accessible for much lower values of the initiation rate, and multiple phase transitions occur over a wide region of the phase plane. Crucially, we show that in the presence of ribosome recycling, mRNAs can exhibit a peak in protein production at low values of the initiation rate, beyond which translation rate decreases. This has important implications for translation of certain mRNAs, suggesting that there is an optimal concentration of ribosomes at which protein synthesis is maximal, and beyond which translational efficiency is impaired. PMID:25008084

  2. Ribosome recycling induces optimal translation rate at low ribosomal availability

    PubMed Central

    Marshall, E.; Stansfield, I.; Romano, M. C.

    2014-01-01

    During eukaryotic cellular protein synthesis, ribosomal translation is made more efficient through interaction between the two ends of the messenger RNA (mRNA). Ribosomes reaching the 3′ end of the mRNA can thus recycle and begin translation again on the same mRNA, the so-called ‘closed-loop’ model. Using a driven diffusion lattice model of translation, we study the effects of ribosome recycling on the dynamics of ribosome flow and density on the mRNA. We show that ribosome recycling induces a substantial increase in ribosome current. Furthermore, for sufficiently large values of the recycling rate, the lattice does not transition directly from low to high ribosome density, as seen in lattice models without recycling. Instead, a maximal current phase becomes accessible for much lower values of the initiation rate, and multiple phase transitions occur over a wide region of the phase plane. Crucially, we show that in the presence of ribosome recycling, mRNAs can exhibit a peak in protein production at low values of the initiation rate, beyond which translation rate decreases. This has important implications for translation of certain mRNAs, suggesting that there is an optimal concentration of ribosomes at which protein synthesis is maximal, and beyond which translational efficiency is impaired. PMID:25008084

  3. Yeast Ribosomal Protein L40 Assembles Late into Precursor 60 S Ribosomes and Is Required for Their Cytoplasmic Maturation*

    PubMed Central

    Fernández-Pevida, Antonio; Rodríguez-Galán, Olga; Díaz-Quintana, Antonio; Kressler, Dieter; de la Cruz, Jesús

    2012-01-01

    Most ribosomal proteins play important roles in ribosome biogenesis and function. Here, we have examined the contribution of the essential ribosomal protein L40 in these processes in the yeast Saccharomyces cerevisiae. Deletion of either the RPL40A or RPL40B gene and in vivo depletion of L40 impair 60 S ribosomal subunit biogenesis. Polysome profile analyses reveal the accumulation of half-mers and a moderate reduction in free 60 S ribosomal subunits. Pulse-chase, Northern blotting, and primer extension analyses in the L40-depleted strain clearly indicate that L40 is not strictly required for the precursor rRNA (pre-rRNA) processing reactions but contributes to optimal 27 SB pre-rRNA maturation. Moreover, depletion of L40 hinders the nucleo-cytoplasmic export of pre-60 S ribosomal particles. Importantly, all these defects most likely appear as the direct consequence of impaired Nmd3 and Rlp24 release from cytoplasmic pre-60 S ribosomal subunits and their inefficient recycling back into the nucle(ol)us. In agreement, we show that hemagglutinin epitope-tagged L40A assembles in the cytoplasm into almost mature pre-60 S ribosomal particles. Finally, we have identified that the hemagglutinin epitope-tagged L40A confers resistance to sordarin, a translation inhibitor that impairs the function of eukaryotic elongation factor 2, whereas the rpl40a and rpl40b null mutants are hypersensitive to this antibiotic. We conclude that L40 is assembled at a very late stage into pre-60 S ribosomal subunits and that its incorporation into 60 S ribosomal subunits is a prerequisite for subunit joining and may ensure proper functioning of the translocation process. PMID:22995916

  4. The nuclear elongation factor-1α gene: a promising marker for phylogenetic studies of Triatominae (Hemiptera: Reduviidae).

    PubMed

    Díaz, Sebastián; Triana-Chávez, Omar; Gómez-Palacio, Andrés

    2016-09-01

    Molecular systematics is a remarkable approach for understanding the taxonomic traits and allows the exploration of the inter-population dynamics of several species in the Triatominae subfamily that are involved in Trypanosoma cruzi transmission. Compared to other relevant species that transmit vector-borne diseases, such as some species of the Diptera, there are relatively few nuclear genetic markers available for systematic studies in the Triatominae subfamily. Molecular systematic studies performed on Triatominae are based on mitochondrial gene fragments and, less frequently, on nuclear ribosomal genes or spacers. Due to the fact that these markers can occasionally present problems such as nuclear mitochondrial genes (NUMTs) or intra-genomic variation for high gene copy numbers, it is necessary to use additional nuclear markers to more reliably address the molecular evolution of Triatominae. In this study, we performed phylogenetic analysis using the nuclear elongation factor-1 alpha (EF-1α) gene in individuals from 12 species belonging to the Triatomini and Rhodniini tribes. Genetic diversities and phylogenetic topologies were compared with those obtained for the mitochondrial 16S rRNA and Cytochrome b (cyt b) genes, as well as for the D2 variable region of the ribosomal 28S rRNA gene. These results indicate that the EF-1α marker exhibits an intermediate level of diversity compared to mitochondrial and nuclear ribosomal genes, and that phylogenetic analysis based on EF-1α is highly informative for resolving deep phylogenetic relationships in Triatominae, such as tribe or genera. PMID:27268149

  5. Development of a Newcastle disease virus vector expressing a foreign gene through an internal ribosomal entry site provides direct proof for a sequential transcription mechanism

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Newcastle disease virus (NDV) has been developed as a vector to express foreign genes for vaccine and gene therapy purposes. The foreign genes are usually inserted into a non-coding region of the NDV genome as an independent transcription unit (ITU). Based on the well-accepted “stop-start” transcr...

  6. Expression analyses of Brachiaria brizantha genes encoding ribosomal proteins BbrizRPS8, BbrizRPS15a, and BbrizRPL41 during development of ovaries and anthers.

    PubMed

    Lacerda, Ana Luiza Machado; Dusi, Diva Maria de Alencar; Alves, Elizangela Ribeiro; Rodrigues, Júlio Carlyle Macedo; Gomes, Ana Cristina Menezes Mendes; Carneiro, Vera Tavares de Campos

    2013-04-01

    Brachiaria brizantha is a forage grass of the Poaceae family. Introduced from Africa, it is largely used for beef cattle production in Brazil. Brachiaria reproduces sexually or asexually by apomixis, and development of biotechnological tools for gene transfer is being researched to support the breeding programs. The molecular bases of reproduction have not yet been fully elucidated; it is known that gametophyte formation and main reproductive events occur inside the anthers and ovaries. There is therefore much interest in identifying genes expressed in these organs and their corresponding upstream regulatory sequences. In this work we characterized three cDNA from ovaries of B. brizantha plants (CL 09, CL10, and CL21) which show similarity in databases with genes encoding ribosomal proteins S8, S15a, and L41 and were named BbrizRPS8, BbrizRPS15a, and BbrizRPL41, respectively. These clones show higher expression in ovaries, anthers and roots, mitotically active tissues, when compared to leaves of B. brizantha. Localization of transcripts of BbrizRPS8, BbrizRPS15a, and BbrizRPL41 was investigated in the reproductive organs, ovaries, and anthers, from the beginning of development up to maturity. Their activity was higher in early stages of anther development, while expression was detected in all developmental stages in the ovaries, except for BbrizS15a, which was detected only in synergids of apomictic plants. PMID:22833119

  7. Identification of airborne bacterial and fungal species in the clinical microbiology laboratory of a university teaching hospital employing ribosomal DNA (rDNA) PCR and gene sequencing techniques.

    PubMed

    Nagano, Yuriko; Walker, Jim; Loughrey, Anne; Millar, Cherie; Goldsmith, Colin; Rooney, Paul; Elborn, Stuart; Moore, John

    2009-06-01

    Universal or "broad-range" PCR-based ribosomal DNA (rDNA) was performed on a collection of 58 isolates (n = 30 bacteria + 28 fungi), originating from environmental air from several locations within a busy clinical microbiology laboratory, supporting a university teaching hospital. A total of 10 bacterial genera were identified including both Gram-positive and Gram-negative genera. Gram-positive organisms accounted for 27/30 (90%) of total bacterial species, consisting of seven genera and included (in descending order of frequency) Staphylococcus, Micrococcus, Corynebacterium, Paenibacillus, Arthrobacter, Janibacter and Rothia. Gram-negative organisms were less frequently isolated 3/30 (10%) and comprised three genera, including Moraxella, Psychrobacter and Haloanella. Eight fungal genera were identified among the 28 fungal organisms isolated, including (in descending order of frequency) Cladosporium, Penicillium, Aspergillus, Thanatephorus, Absidia, Eurotium, Paraphaeosphaeria and Tritirachium, with Cladosporium accounting for 10/28 (35.7%) of the total fungal isolates. In conclusion, this study identified the presence of 10 bacterial and eight fungal genera in the air within the laboratory sampled. Although this reflected diversity of the microorganisms present, none of these organisms have been described previously as having an inhalational route of laboratory-acquired infection. Therefore, we believe that the species of organisms identified and the concentration levels of these airborne contaminants determined, do not pose a significant health and safety threat for immunocompotent laboratory personnel and visitors. PMID:20183192

  8. Ribosomes in a Stacked Array

    PubMed Central

    Yamashita, Yui; Kadokura, Yoshitomo; Sotta, Naoyuki; Fujiwara, Toru; Takigawa, Ichigaku; Satake, Akiko; Onouchi, Hitoshi; Naito, Satoshi

    2014-01-01

    Expression of CGS1, which codes for an enzyme of methionine biosynthesis, is feedback-regulated by mRNA degradation in response to S-adenosyl-l-methionine (AdoMet). In vitro studies revealed that AdoMet induces translation arrest at Ser-94, upon which several ribosomes stack behind the arrested one, and mRNA degradation occurs at multiple sites that presumably correspond to individual ribosomes in a stacked array. Despite the significant contribution of stacked ribosomes to inducing mRNA degradation, little is known about the ribosomes in the stacked array. Here, we assigned the peptidyl-tRNA species of the stacked second and third ribosomes to their respective codons and showed that they are arranged at nine-codon intervals behind the Ser-94 codon, indicating tight stacking. Puromycin reacts with peptidyl-tRNA in the P-site, releasing the nascent peptide as peptidyl-puromycin. This reaction is used to monitor the activity of the peptidyltransferase center (PTC) in arrested ribosomes. Puromycin reaction of peptidyl-tRNA on the AdoMet-arrested ribosome, which is stalled at the pre-translocation step, was slow. This limited reactivity can be attributed to the peptidyl-tRNA occupying the A-site at this step rather than to suppression of PTC activity. In contrast, puromycin reactions of peptidyl-tRNA with the stacked second and third ribosomes were slow but were not as slow as pre-translocation step ribosomes. We propose that the anticodon end of peptidyl-tRNA resides in the A-site of the stacked ribosomes and that the stacked ribosomes are stalled at an early step of translocation, possibly at the P/E hybrid state. PMID:24652291

  9. Ribosomal Mutations in Streptococcus pneumoniae Clinical Isolates

    PubMed Central

    Pihlajamäki, Marja; Kataja, Janne; Seppälä, Helena; Elliot, John; Leinonen, Maija; Huovinen, Pentti; Jalava, Jari

    2002-01-01

    Eleven clinical isolates of Streptococcus pneumoniae, isolated in Finland during 1996 to 2000, had an unusual macrolide resistance phenotype. They were resistant to macrolides and streptogramin B but susceptible, intermediate, or low-level resistant to lincosamides. No acquired macrolide resistance genes were detected from the strains. The isolates were found to have mutations in domain V of the 23S rRNA or ribosomal protein L4. Seven isolates had an A2059C mutation in two to four out of the four alleles encoding the 23S rRNA, two isolates had an A2059G mutation in two alleles, one isolate had a C2611G mutation in all four alleles, and one isolate had a 69GTG71-to-69TPS71 substitution in ribosomal protein L4. PMID:11850244

  10. Direct ribosomal binding by a cellular inhibitor of translation

    PubMed Central

    Colón-Ramos, Daniel A; Shenvi, Christina L; Weitzel, Douglas H; Gan, Eugene C; Matts, Robert; Cate, Jamie; Kornbluth, Sally

    2009-01-01

    During apoptosis and under conditions of cellular stress, several signaling pathways promote inhibition of cap-dependent translation while allowing continued translation of specific messenger RNAs encoding regulatory and stress-response proteins. We report here that the apoptotic regulator Reaper inhibits protein synthesis by binding directly to the 40S ribosomal subunit. This interaction does not affect either ribosomal association of initiation factors or formation of 43S or 48S complexes. Rather, it interferes with late initiation events upstream of 60S subunit joining, apparently modulating start-codon recognition during scanning. CrPV IRES–driven translation, involving direct ribosomal recruitment to the start site, is relatively insensitive to Reaper. Thus, Reaper is the first known cellular ribosomal binding factor with the potential to allow selective translation of mRNAs initiating at alternative start codons or from certain IRES elements. This function of Reaper may modulate gene expression programs to affect cell fate. PMID:16429152

  11. The impact of transcriptional tuning on in vitro integrated rRNA transcription and ribosome construction

    PubMed Central

    Fritz, Brian R.; Jewett, Michael C.

    2014-01-01

    In vitro ribosome construction could enable studies of ribosome assembly and function, provide a route toward constructing minimal cells for synthetic biology, and permit the construction of ribosome variants with new functions. Toward these long-term goals, we recently reported on an integrated, one-pot ribosomal RNA synthesis (rRNA), ribosome assembly, and translation technology (termed iSAT) for the construction of Escherichia coli ribosomes in crude ribosome-free S150 extracts. Here, we aimed to improve the activity of iSAT through transcriptional tuning. Specifically, we increased transcriptional efficiency through 3′ modifications to the rRNA gene sequences, optimized plasmid and polymerase concentrations, and demonstrated the use of a T7-promoted rRNA operon for stoichiometrically balanced rRNA synthesis and native rRNA processing. Our modifications produced a 45-fold improvement in iSAT protein synthesis activity, enabling synthesis of 429 ± 15 nmol/l green fluorescent protein in 6 h batch reactions. Further, we show that the translational activity of ribosomes purified from iSAT reactions is about 20% the activity of native ribosomes purified directly from E. coli cells. Looking forward, we believe iSAT will enable unique studies to unravel the systems biology of ribosome biogenesis and open the way to new methods for making and studying ribosomal variants. PMID:24792158

  12. Rational Extension of the Ribosome Biogenesis Pathway Using Network-Guided Genetics

    PubMed Central

    Li, Zhihua; Lee, Insuk; Moradi, Emily; Hung, Nai-Jung; Johnson, Arlen W.; Marcotte, Edward M.

    2009-01-01

    Biogenesis of ribosomes is an essential cellular process conserved across all eukaryotes and is known to require >170 genes for the assembly, modification, and trafficking of ribosome components through multiple cellular compartments. Despite intensive study, this pathway likely involves many additional genes. Here, we employ network-guided genetics—an approach for associating candidate genes with biological processes that capitalizes on recent advances in functional genomic and proteomic studies—to computationally identify additional ribosomal biogenesis genes. We experimentally evaluated >100 candidate yeast genes in a battery of assays, confirming involvement of at least 15 new genes, including previously uncharacterized genes (YDL063C, YIL091C, YOR287C, YOR006C/TSR3, YOL022C/TSR4). We associate the new genes with specific aspects of ribosomal subunit maturation, ribosomal particle association, and ribosomal subunit nuclear export, and we identify genes specifically required for the processing of 5S, 7S, 20S, 27S, and 35S rRNAs. These results reveal new connections between ribosome biogenesis and mRNA splicing and add >10% new genes—most with human orthologs—to the biogenesis pathway, significantly extending our understanding of a universally conserved eukaryotic process. PMID:19806183

  13. Assessing the translational landscape of myogenic differentiation by ribosome profiling

    PubMed Central

    de Klerk, Eleonora; Fokkema, Ivo F.A.C.; Thiadens, Klaske A.M.H.; Goeman, Jelle J.; Palmblad, Magnus; den Dunnen, Johan T.; von Lindern, Marieke; ‘t Hoen, Peter A.C.

    2015-01-01

    The formation of skeletal muscles is associated with drastic changes in protein requirements known to be safeguarded by tight control of gene transcription and mRNA processing. The contribution of regulation of mRNA translation during myogenesis has not been studied so far. We monitored translation during myogenic differentiation of C2C12 myoblasts, using a simplified protocol for ribosome footprint profiling. Comparison of ribosome footprints to total RNA showed that gene expression is mostly regulated at the transcriptional level. However, a subset of transcripts, enriched for mRNAs encoding for ribosomal proteins, was regulated at the level of translation. Enrichment was also found for specific pathways known to regulate muscle biology. We developed a dedicated pipeline to identify translation initiation sites (TISs) and discovered 5333 unannotated TISs, providing a catalog of upstream and alternative open reading frames used during myogenesis. We identified 298 transcripts with a significant switch in TIS usage during myogenesis, which was not explained by alternative promoter usage, as profiled by DeepCAGE. Also these transcripts were enriched for ribosomal protein genes. This study demonstrates that differential mRNA translation controls protein expression of specific subsets of genes during myogenesis. Experimental protocols, analytical workflows, tools and data are available through public repositories (http://lumc.github.io/ribosome-profiling-analysis-framework/). PMID:25873627

  14. Streptomycin affinity depends on 13 amino acids forming a loop in homology modelled ribosomal S12 protein (rpsL gene) of Lysinibacillus sphaericus DSLS5 associated with marine sponge (Tedania anhelans).

    PubMed

    Suriyanarayanan, Balasubramanian; Lakshmi, Praveena Pothuraju; Santhosh, Ramachandran Sarojini; Dhevendaran, Kandasamy; Priya, Balakrishnan; Krishna, Shivaani

    2016-06-01

    Streptomycin, an antibiotic used against microbial infections, inhibits the protein synthesis by binding to ribosomal protein S12, encoded by rpsL12 gene, and associated mutations cause streptomycin resistance. A streptomycin resistant, Lysinibacillus sphaericus DSLS5 (MIC >300 µg/mL for streptomycin), was isolated from a marine sponge (Tedania anhelans). The characterisation of rpsL12 gene showed a region having similarity to long terminal repeat sequences of murine lukemia virus which added 13 amino acids for loop formation in RpsL12; in addition, a K56R mutation which corresponds to K43R mutation present in streptomycin-resistant Escherichia coli is also present. The RpsL12 protein was modelled and compared with that of Lysinibacillus boronitolerans, Escherichia coli and Mycobacterium tuberculosis. The modelled proteins docked with streptomycin indicate compound had less affinity. The effect of loop on streptomycin resistance was analysed by constructing three different models of RpsL12 by, (i) removing both loop and mutation, (ii) removing the loop alone while retaining the mutation and (iii) without mutation having loop. The results showed that the presence of loop causes streptomycin resistance (decreases the affinity), and it further enhanced in the presence of mutation at 56th codon. Further study will help in understanding the evolution of streptomycin resistance in organisms. PMID:26198082

  15. Conditional expression of RPA190, the gene encoding the largest subunit of yeast RNA polymerase I: effects of decreased rRNA synthesis on ribosomal protein synthesis.

    PubMed Central

    Wittekind, M; Kolb, J M; Dodd, J; Yamagishi, M; Mémet, S; Buhler, J M; Nomura, M

    1990-01-01

    The synthesis of ribosomal proteins (r proteins) under the conditions of greatly reduced RNA synthesis were studied by using a strain of the yeast Saccharomyces cerevisiae in which the production of the largest subunit (RPA190) of RNA polymerase I was controlled by the galactose promoter. Although growth on galactose medium was normal, the strain was unable to sustain growth when shifted to glucose medium. This growth defect was shown to be due to a preferential decrease in RNA synthesis caused by deprivation of RNA polymerase I. Under these conditions, the accumulation of r proteins decreased to match the rRNA synthesis rate. When proteins were pulse-labeled for short periods, no or only a weak decrease was observed in the differential synthesis rate of several r proteins (L5, L39, L29 and/or L28, L27 and/or S21) relative to those of control cells synthesizing RPA190 from the normal promoter. Degradation of these r proteins synthesized in excess was observed during subsequent chase periods. Analysis of the amounts of mRNAs for L3 and L29 and their locations in polysomes also suggested that the synthesis of these proteins relative to other cellular proteins were comparable to those observed in control cells. However, Northern analysis of several r-protein mRNAs revealed that the unspliced precursor mRNA for r-protein L32 accumulated when rRNA synthesis rates were decreased. This result supports the feedback regulation model in which excess L32 protein inhibits the splicing of its own precursor mRNA, as proposed by previous workers (M. D. Dabeva, M. A. Post-Beittenmiller, and J. R. Warner, Proc. Natl. Acad. Sci. USA 83:5854-5857, 1986). Images PMID:2183018

  16. Requirement for SAPK-JNK signaling in the induction of apoptosis by ribosomal stress in REH lymphoid leukemia cells.

    PubMed

    Johnson, C R; Jiffar, T; Fischer, U M; Ruvolo, P P; Jarvis, W D

    2003-11-01

    The present studies examined performance of SAPK cascades and apoptotic commitment following ribosomal trauma in REH lymphoid leukemia cells. Ribostatic insults included disruption of ribosomal activity by mechanistically dissimilar agents such as blasticidin-S (BCS) (which binds 28S-rRNA to block peptidyl bond formation), kasugamycin (KSM) (which binds 18S-rRNA to prevent translational initiation), and cycloheximide (CHX) (which blocks A-site to P-site translocation of peptidyl-tRNA). Exposure of REH cells to BCS elicited DNA degradation and apoptotic cytolysis. BCS stimulated JNK1/JNK2 and p38, and their shared targets c-Jun and ATF2. Inhibition of JNK1/JNK2 (but not of p38) antagonized blasticidin-induced apoptosis, whereas targeting alternative ribosomal sites with KSM or CHX limited translation, but failed to activate the SAPK cascade or initiate apoptosis. Our findings indicate that interference with 28S-rRNA by BCS initiates apoptosis in REH cells through recruitment of SAPK-JNK signaling. Disparities between the lethal actions of BCS, KSM, and CHX appear to reflect established differences in the subribosomal targets of these agents. We propose that the SAPK cascade comprises an essential mechanism for the transduction of specific lethal stress signals emanating from active ribosomes, and that interference with the 28S-rRNA, rather than the peptidyl transfer center of the large subunit, is critical to apoptotic commitment. PMID:12970763

  17. The complete structure of the large subunit of the mammalian mitochondrial ribosome.

    PubMed

    Greber, Basil J; Boehringer, Daniel; Leibundgut, Marc; Bieri, Philipp; Leitner, Alexander; Schmitz, Nikolaus; Aebersold, Ruedi; Ban, Nenad

    2014-11-13

    Mitochondrial ribosomes (mitoribosomes) are extensively modified ribosomes of bacterial descent specialized for the synthesis and insertion of membrane proteins that are critical for energy conversion and ATP production inside mitochondria. Mammalian mitoribosomes, which comprise 39S and 28S subunits, have diverged markedly from the bacterial ribosomes from which they are derived, rendering them unique compared to bacterial, eukaryotic cytosolic and fungal mitochondrial ribosomes. We have previously determined at 4.9 Å resolution the architecture of the porcine (Sus scrofa) 39S subunit, which is highly homologous to the human mitoribosomal large subunit. Here we present the complete atomic structure of the porcine 39S large mitoribosomal subunit determined in the context of a stalled translating mitoribosome at 3.4 Å resolution by cryo-electron microscopy and chemical crosslinking/mass spectrometry. The structure reveals the locations and the detailed folds of 50 mitoribosomal proteins, shows the highly conserved mitoribosomal peptidyl transferase active site in complex with its substrate transfer RNAs, and defines the path of the nascent chain in mammalian mitoribosomes along their idiosyncratic exit tunnel. Furthermore, we present evidence that a mitochondrial tRNA has become an integral component of the central protuberance of the 39S subunit where it architecturally substitutes for the absence of the 5S ribosomal RNA, a ubiquitous component of all cytoplasmic ribosomes. PMID:25271403

  18. A Unique Box in 28S rRNA Is Shared by the Enigmatic Insect Order Zoraptera and Dictyoptera

    PubMed Central

    Dang, Kai; Wu, Haoyang; Wang, Ying; Xie, Qiang; Bu, Wenjun

    2013-01-01

    The position of the Zoraptera remains one of the most challenging and uncertain concerns in ordinal-level phylogenies of the insects. Zoraptera have been viewed as having a close relationship with five different groups of Polyneoptera, or as being allied to the Paraneoptera or even Holometabola. Although rDNAs have been widely used in phylogenetic studies of insects, the application of the complete 28S rDNA are still scattered in only a few orders. In this study, a secondary structure model of the complete 28S rRNAs of insects was reconstructed based on all orders of Insecta. It was found that one length-variable region, D3-4, is particularly distinctive. The length and/or sequence of D3-4 is conservative within each order of Polyneoptera, but it can be divided into two types between the different orders of the supercohort, of which the enigmatic order Zoraptera and Dictyoptera share one type, while the remaining orders of Polyneoptera share the other. Additionally, independent evidence from phylogenetic results support the clade (Zoraptera+Dictyoptera) as well. Thus, the similarity of D3-4 between Zoraptera and Dictyoptera can serve as potentially valuable autapomorphy or synapomorphy in phylogeny reconstruction. The clades of (Plecoptera+Dermaptera) and ((Grylloblattodea+Mantophasmatodea)+(Embiodea+Phasmatodea)) were also recovered in the phylogenetic study. In addition, considering the other studies based on rDNAs, this study reached the highest congruence with previous phylogenetic studies of Holometabola based on nuclear protein coding genes or morphology characters. Future comparative studies of secondary structures across deep divergences and additional taxa are likely to reveal conserved patterns, structures and motifs that can provide support for major phylogenetic lineages. PMID:23301099

  19. Congruence between nuclear and mitochondrial genes in Demospongiae: a new hypothesis for relationships within the G4 clade (Porifera: Demospongiae).

    PubMed

    Morrow, C C; Picton, B E; Erpenbeck, D; Boury-Esnault, N; Maggs, C A; Allcock, A L

    2012-01-01

    The current morphological classification of the Demospongiae G4 clade was tested using large subunit ribosomal RNA (LSU rRNA) sequences from 119 taxa. Fifty-three mitochondrial cytochrome oxidase 1 (CO1) barcoding sequences were also analysed to test whether the 28S phylogeny could be recovered using an independent gene. This is the largest and most comprehensive study of the Demospongiae G4 clade. The 28S and CO1 genetrees result in congruent clades but conflict with the current morphological classification. The results confirm the polyphyly of Halichondrida, Hadromerida, Dictyonellidae, Axinellidae and Poecilosclerida and show that several of the characters used in morphological classifications are homoplasious. Robust clades are clearly shown and a new hypothesis for relationships of taxa allocated to G4 is proposed. PMID:22001855

  20. Synthesis of Amplified DNA That Codes for Ribosomal RNA

    PubMed Central

    Crippa, Marco; Tocchini-Valentini, Glauco P.

    1971-01-01

    During the amplification stage in ovaries, the complete repetitive unit of the DNA that codes for ribosomal RNA in Xenopus appears to be transcribed. This large RNA transcript is found in a complex with DNA. Substitution experiments with 5-bromodeoxyuridine do not show any evidence that a complete amplified cistron is used as a template for further amplification. A derivative of rifampicin, 2′,5′-dimethyl-N(4′)benzyl-N(4′)[desmethyl] rifampicin, preferentially inhibits the DNA synthesis responsible for ribosomal gene amplification. These results are consistent with the hypothesis that RNA-dependent DNA synthesis is involved in gene amplification. PMID:5288254

  1. Amplification of 16S ribosomal RNA genes of autotrophic ammonia-oxidizing bacteria demonstrates the ubiquity of nitrosospiras in the environment.

    PubMed

    Hiorns, W D; Hastings, R C; Head, I M; McCarthy, A J; Saunders, J R; Pickup, R W; Hall, G H

    1995-11-01

    Oligonucleotide sequences selected from the 16S rRNA genes of various species of ammonia-oxidizing bacteria were evaluated as specific PCR amplification primers and probes. The specificities of primer pairs for eubacterial, Nitrosospira and Nitrosomonas rRNA genes were established with sequence databases, and the primer pairs were used to amplify DNA from laboratory cultures and environmental samples. Eubacterial rRNA genes amplified from samples of soil and activated sludge hybridized with an oligonucleotide probe specific for Nitrosospira spp., but not with a Nitrosomonas-specific probe. Lakewater and sediment samples were analysed using a nested PCR technique in which eubacterial rRNA genes were subjected to a secondary amplification with Nitrosomonas or Nitrosospira specific primers. Again, the presence of Nitrosospira DNA, but not Nitrosomonas DNA, was detected and this was confirmed by hybridization of the amplified DNA with an internal oligonucleotide probe. Enrichments of lakewater and sediment samples, incubated for two weeks in the presence of ammonium, produced nitrite and were found to contain DNA from both Nitrosospira and Nitrosomonas as determined by nested PCR amplification and probing of 16S rRNA genes. This demonstrates that Nitrosospira spp. are widespread in the environment. The implications of the detection of Nitrosomonas DNA only after enrichment culture are discussed. PMID:8535507

  2. Comparison of Virulence Gene Identification, Ribosomal Spacer PCR, and Pulsed-Field Gel Electrophoresis for Typing of Staphylococcus aureus Strains Isolated from Cases of Subclinical Bovine Mastitis in the United States.

    PubMed

    Adkins, Pamela R F; Middleton, John R; Fox, Lawrence K

    2016-07-01

    Staphylococcus aureus is one of the most important pathogens causing contagious mastitis in dairy cattle worldwide. The objectives of this study were to determine if recently described S. aureus genotype B was present among previously characterized isolates from cases of bovine intramammary infection in the United States and to compare pulsed-field gel electrophoresis (PFGE) to the combination of ribosomal spacer PCR (RS-PCR) and virulence gene identification for typing of S. aureus strains. The hypothesis was that isolates that were previously characterized as contagious would be identified as genotype B and that the results of the two strain-typing methods would be comparable. Isolates were selected from a collection of S. aureus isolates from eight dairy farms. Mammary quarter milk somatic cell count (SCC) and N-acetyl-β-d-gluconaminidase (NAGase) activity data were known and used to evaluate strain pathogenicity. RS-PCR was performed with conventional gel electrophoresis, and PCR was used for toxin gene identification. RS-PCR patterns were associated with a specific virulence gene pattern, as previously reported. Five RS-PCR banding patterns were identified. None of the isolates were characterized as genotype B. No association between RS-PCR types and milk SCC was found; however, NAGase activity was significantly higher in milk from mammary glands infected with RS-PCR banding type 1 (RSP type 1) than in milk from those infected with RSP type 2. The discriminatory power values were 1.0 and 0.46 for PFGE and RS-PCR, respectively. These data suggest that genotype B may have a limited geographic distribution and that PFGE is more discriminatory than RS-PCR performed with conventional gel electrophoresis for typing of S. aureus isolates of bovine origin. PMID:27194685

  3. A comparison of two real-time polymerase chain reaction assays using hybridization probes targeting either 16S ribosomal RNA or a subsurface lipoprotein gene for detecting leptospires in canine urine.

    PubMed

    Gentilini, Fabio; Zanoni, Renato Giulio; Zambon, Elisa; Turba, Maria Elena

    2015-11-01

    Leptospires are excreted in the urine of infected animals, and the prompt detection of leptospiral DNA using polymerase chain reaction (PCR) is increasingly being used. However, contradictory data has emerged concerning the diagnostic accuracy of the most popular PCR assays that target either the 16S ribosomal RNA (rrs) or the subsurface lipoprotein (LipL32) genes. In order to clarify the effect of the gene target, a novel hydrolysis probe-based, quantitative real-time PCR (qPCR) assay targeting the LipL32 gene was developed, validated, and then compared directly to the previously described rrs hydrolysis probe-based qPCR using a convenience collection of canine urine samples. The novel LipL32 qPCR assay was linear from 5.9 × 10(6) to 59 genome equivalents per reaction. Both the LipL32 and the rrs qPCR assays showed a limit of detection of 10 target copies per reaction indicating an approximately equivalent analytical sensitivity. Both assays amplified all 20 pathogenic leptospiral strains tested but did not amplify a representative collection of bacteria commonly found in voided canine urine. When the field samples were assayed, 1 and 5 out of 184 samples yielded an amplification signal in the LipL32 and rrs assays, respectively. Nevertheless, when the limit of detection was considered as the cutoff for interpreting findings, the 4 discordant cases were judged as negative. In conclusion, our study confirmed that both LipL32 and rrs are suitable targets for qPCR for the detection of leptospiral DNA in canine urine. However, the rrs target requires the mandatory use of a cutoff value in order to correctly interpret spurious amplifications. PMID:26450835

  4. Targeted cancer therapy with ribosome biogenesis inhibitors: a real possibility?

    PubMed Central

    Brighenti, Elisa; Treré, Davide; Derenzini, Massimo

    2015-01-01

    The effects of many chemotherapeutic drugs on ribosome biogenesis have been underestimated for a long time. Indeed, many drugs currently used for cancer treatment – and which are known to either damage DNA or hinder DNA synthesis – have been shown to exert their toxic action mainly by inhibiting rRNA synthesis or maturation. Moreover, there are new drugs that have been proposed recently for cancer chemotherapy, which only hinder ribosome biogenesis without any genotoxic activity. Even though ribosome biogenesis occurs in both normal and cancer cells, whether resting or proliferating, there is evidence that the selective inhibition of ribosome biogenesis may, in some instances, result in a selective damage to neoplastic cells. The higher sensitivity of cancer cells to inhibitors of rRNA synthesis appears to be the consequence of either the loss of the mechanisms controlling the cell cycle progression or the acquisition of activating oncogene and inactivating tumor suppressor gene mutations that up-regulate the ribosome biogenesis rate. This article reviews those cancer cell characteristics on which the selective cancer cell cytotoxicity induced by the inhibitors of ribosome biogenesis is based. PMID:26415219

  5. The small ribosomal protein S12P gene rpsL as an efficient positive selection marker in allelic exchange mutation systems for Corynebacterium glutamicum.

    PubMed

    Kim, Il Kwon; Jeong, Weol Kyu; Lim, Seong Han; Hwang, In Kwan; Kim, Young Ho

    2011-01-01

    We report that the mutant rpsL K43R in streptomycin-resistant and lysine-producing Corynebacterium glutamicum is responsible for streptomycin resistance. In addition, we describe its effective application in gene modification in C. glutamicum. PMID:20951172

  6. Maize reas1 Mutant Stimulates Ribosome Use Efficiency and Triggers Distinct Transcriptional and Translational Responses.

    PubMed

    Qi, Weiwei; Zhu, Jie; Wu, Qiao; Wang, Qun; Li, Xia; Yao, Dongsheng; Jin, Ying; Wang, Gang; Wang, Guifeng; Song, Rentao

    2016-02-01

    Ribosome biogenesis is a fundamental cellular process in all cells. Impaired ribosome biogenesis causes developmental defects; however, its molecular and cellular bases are not fully understood. We cloned a gene responsible for a maize (Zea mays) small seed mutant, dek* (for defective kernel), and found that it encodes Ribosome export associated1 (ZmReas1). Reas1 is an AAA-ATPase that controls 60S ribosome export from the nucleus to the cytoplasm after ribosome maturation. dek* is a weak mutant allele with decreased Reas1 function. In dek* cells, mature 60S ribosome subunits are reduced in the nucleus and cytoplasm, but the proportion of actively translating polyribosomes in cytosol is significantly increased. Reduced phosphorylation of eukaryotic initiation factor 2α and the increased elongation factor 1α level indicate an enhancement of general translational efficiency in dek* cells. The mutation also triggers dramatic changes in differentially transcribed genes and differentially translated RNAs. Discrepancy was observed between differentially transcribed genes and differentially translated RNAs, indicating distinct cellular responses at transcription and translation levels to the stress of defective ribosome processing. DNA replication and nucleosome assembly-related gene expression are selectively suppressed at the translational level, resulting in inhibited cell growth and proliferation in dek* cells. This study provides insight into cellular responses due to impaired ribosome biogenesis. PMID:26645456

  7. Phylogenetic analysis based on full-length large subunit ribosomal RNA gene sequence comparison reveals that Neospora caninum is more closely related to Hammondia heydorni than to Toxoplasma gondii.

    PubMed

    Mugridge, N B; Morrison, D A; Heckeroth, A R; Johnson, A M; Tenter, A M

    1999-10-01

    Since its first description in the late 1980s, Neospora caninum has been recognised as a prominent tissue cyst-forming parasite due to its ability to induce congenital disease and abortion in animals, especially cattle. It is found worldwide and is a cause of significant economic losses for the livestock industry. However, its place within the family Sarcocystidae, like that of several other taxa, remains unresolved. Neospora caninum shares several morphological and life cycle characters with Hammondia heydorni, although it is most commonly thought of as being a close relative of Toxoplasma gondii. This study presents information regarding the phylogenetic relationship of N. caninum to species currently classified into the genus Hammondia, as well as to two strains (RH and ME49) of T. gondii based on the full-length large subunit ribosomal RNA gene. Phylogenetic analyses using two alignment strategies and three different tree-building methods showed that the two species in the genus Hammondia are paraphyletic. Neospora caninum was shown to form a monophyletic clade with H. heydorni instead of T. gondii, which in turn was shown to be most closely related to H. hammondi. The finding that N. caninum and H. heydorni are closely related phylogenetically may aid the elucidation of currently unknown aspects of their biology and epidemiology, and suggests that H. heydorni should be considered in the differential diagnosis of N. caninum from other apicomplexan parasites. PMID:10608441

  8. [Analysis of the sequences of internal transcribed spacers ITS1, ITS2 and the 5.8S ribosomal gene of species of the Amaranthus genus].

    PubMed

    Slugina, M A; Torres Minho, K; Filiushin, M A

    2014-01-01

    Analysis of the sequence ITS1-5.8S-ITS2 in 11 samples of the amaranth species (Amaranthus caudatus, A. cruentus, A. hybridus, A. tricolor, A. paniculatus, A. hypohondriacus) was performed. It has been shown that the variability of the sequences of the intergenic spacers ITS1, ITS2 and 5.8S rRNA gene of the amaranth species analyzed is extremely low. A possible secondary structure of the 5.8S rRNA molecule was determined for the first time; three conservative motifs were identified. A single nucleotide substitution found in A. hybridus did not change the loop topology. In the sample of Celosia cristata taken as an external group, a four-nucleotide insertion in the 5'-end of the gene and a one-nucleotide deletion in the fourth hairpin not affecting the general topology of the 5.8S rRNA molecule were found. PMID:25739312

  9. A continental-wide perspective: the genepool of nuclear encoded ribosomal DNA and single-copy gene sequences in North American Boechera (Brassicaceae).

    PubMed

    Kiefer, Christiane; Koch, Marcus A

    2012-01-01

    74 of the currently accepted 111 taxa of the North American genus Boechera (Brassicaceae) were subject to pyhlogenetic reconstruction and network analysis. The dataset comprised 911 accessions for which ITS sequences were analyzed. Phylogenetic analyses yielded largely unresolved trees. Together with the network analysis confirming this result this can be interpreted as an indication for multiple, independent, and rapid diversification events. Network analyses were superimposed with datasets describing i) geographical distribution, ii) taxonomy, iii) reproductive mode, and iv) distribution history based on phylogeographic evidence. Our results provide first direct evidence for enormous reticulate evolution in the entire genus and give further insights into the evolutionary history of this complex genus on a continental scale. In addition two novel single-copy gene markers, orthologues of the Arabidopsis thaliana genes At2g25920 and At3g18900, were analyzed for subsets of taxa and confirmed the findings obtained through the ITS data. PMID:22606266

  10. The mechanics of ribosomal translocation.

    PubMed

    Achenbach, John; Nierhaus, Knud H

    2015-07-01

    The ribosome translates the sequence of codons of an mRNA into the corresponding sequence of amino acids as it moves along the mRNA with a codon-step width of about 10 Å. The movement of the million-dalton complex ribosome is triggered by the universal elongation factor G (EF2 in archaea and eukaryotes) and is termed translocation. Unraveling the molecular details of translocation is one of the most challenging tasks of current ribosome research. In the last two years, enormous progress has been obtained by highly-resolved X-ray and cryo-electron microscopic structures as well as by sophisticated biochemical approaches concerning the trigger and control of the movement of the tRNA2·mRNA complex inside the ribosome during translocation. This review inspects and surveys these achievements. PMID:25514765

  11. The Ribosomal Database Project (RDP).

    PubMed Central

    Maidak, B L; Olsen, G J; Larsen, N; Overbeek, R; McCaughey, M J; Woese, C R

    1996-01-01

    The Ribosomal Database Project (RDP) is a curated database that offers ribosome-related data, analysis services and associated computer programs. The offerings include phylogenetically ordered alignments of ribosomal RNA (rRNA) sequences, derived phylogenetic trees, rRNA secondary structure diagrams and various software for handling, analyzing and displaying alignments and trees. The data are available via anonymous ftp (rdp.life.uiuc.edu), electronic mail (server@rdp.life.uiuc.edu), gopher (rdpgopher.life.uiuc.edu) and World Wide Web (WWW)(http://rdpwww.life.uiuc.edu/). The electronic mail and WWW servers provide ribosomal probe checking, screening for possible chimeric rRNA sequences, automated alignment and approximate phylogenetic placement of user-submitted sequences on an existing phylogenetic tree. PMID:8594608

  12. Chicken rRNA Gene Cluster Structure

    PubMed Central

    Dyomin, Alexander G.; Koshel, Elena I.; Kiselev, Artem M.; Saifitdinova, Alsu F.; Galkina, Svetlana A.; Fukagawa, Tatsuo; Kostareva, Anna A.

    2016-01-01

    Ribosomal RNA (rRNA) genes, whose activity results in nucleolus formation, constitute an extremely important part of genome. Despite the extensive exploration into avian genomes, no complete description of avian rRNA gene primary structure has been offered so far. We publish a complete chicken rRNA gene cluster sequence here, including 5’ETS (1836 bp), 18S rRNA gene (1823 bp), ITS1 (2530 bp), 5.8S rRNA gene (157 bp), ITS2 (733 bp), 28S rRNA gene (4441 bp) and 3’ETS (343 bp). The rRNA gene cluster sequence of 11863 bp was assembled from raw reads and deposited to GenBank under KT445934 accession number. The assembly was validated through in situ fluorescent hybridization analysis on chicken metaphase chromosomes using computed and synthesized specific probes, as well as through the reference assembly against de novo assembled rRNA gene cluster sequence using sequenced fragments of BAC-clone containing chicken NOR (nucleolus organizer region). The results have confirmed the chicken rRNA gene cluster validity. PMID:27299357

  13. Genome-wide polysomal analysis of a yeast strain with mutated ribosomal protein S9

    PubMed Central

    Pnueli, Lilach; Arava, Yoav

    2007-01-01

    Background The yeast ribosomal protein S9 (S9) is located at the entrance tunnel of the mRNA into the ribosome. It is known to play a role in accurate decoding and its bacterial homolog (S4) has recently been shown to be involved in opening RNA duplexes. Here we examined the effects of changing the C terminus of S9, which is rich in acidic amino acids and extends out of the ribosome surface. Results We performed a genome-wide analysis to reveal effects at the transcription and translation levels of all yeast genes. While negligible relative changes were observed in steady-state mRNA levels, a significant number of mRNAs appeared to have altered ribosomal density. Notably, 40% of the genes having reliable signals changed their ribosomal association by more than one ribosome. Yet, no general correlations with physical or functional features of the mRNA were observed. Ribosome Density Mapping (RDM) along four of the mRNAs with increased association revealed an increase in ribosomal density towards the end of the coding region for at least two of them. Read-through analysis did not reveal any increase in read-through of a premature stop codon by the mutant strain. Conclusion The ribosomal protein rpS9 appears to be involved in the translation of many mRNAs, since altering its C terminus led to a significant change in ribosomal association of many mRNAs. We did not find strong correlations between these changes and several physical features of the mRNA, yet future studies with advanced tools may allow such correlations to be determined. Importantly, our results indicate an accumulation of ribosomes towards the end of the coding regions of some mRNAs. This suggests an involvement of S9 in ribosomal dissociation during translation termination. PMID:17711575

  14. Phylogenetic relationships among Japanese species of the family Sciuridae (Mammalia, Rodentia), inferred from nucleotide sequences of mitochondrial 12S ribosomal RNA genes.

    PubMed

    Oshida, T; Masuda, R; Yoshida, M C

    1996-08-01

    In order to investigate phylogenetic relationships of the family Sciuridae living in Japan, we sequenced partial regions (379 bases) of mitochondrial 12S rRNA genes in six species of Japanese and other Asian squirrels. Phylogenetic trees constructed by sequence data indicated that two genera of flying squirrels (Petaurista and Pteromys) were clustered in a group distinct from non-flying squirrels, suggesting a possible monophyletic relationships of these flying squirrels. The evolutionary distance between the Japanese squirrel (Sciurus lis) from Honshu island and the Eurasian red squirrel (Sciurus vulgaris) from Hokkaido island was comparable to intraspecific distances of the remaining species examined. PMID:8940915

  15. Ribosome dynamics and the evolutionary history of ribosomes

    NASA Astrophysics Data System (ADS)

    Fox, George E.; Paci, Maxim; Tran, Quyen; Petrov, Anton S.; Williams, Loren D.

    2015-09-01

    The ribosome is a dynamic nanomachine responsible for coded protein synthesis. Its major subsystems were essentially in place at the time of the last universal common ancestor (LUCA). Ribosome evolutionary history thus potentially provides a window into the pre- LUCA world. This history begins with the origins of the peptidyl transferase center where the actual peptide is synthesized and then continues over an extended timeframe as additional functional centers including the GTPase center are added. The large ribosomal RNAs (rRNAs) have grown over time by an accretion process and a model exists that proposes a relative age of each accreted element. We have compared atomic resolution ribosome structures before and after EF-G bound GTP hydrolysis and thereby identified the location of 23 pivot points in the large rRNAs that facilitate ribosome dynamics. Pivots in small subunit helices h28 and h44 appear to be especially central to the process and according to the accretion model significantly older than the other helices containing pivots. Overall, the results suggest that ribosomal dynamics occurred in two phases. In the first phase, an inherently mobile h28/h44 combination provided the flexibility needed to create a dynamic ribosome that was essentially a Brownian machine. This addition likely made coded peptide synthesis possible by facilitating movement of a primitive mRNA. During the second phase, addition of pivoting elements and the creation of a factor binding site allowed the regulation of the inherent motion created by h28/h44. All of these events likely occurred before LUCA.

  16. Characterization of silk gland ribosomes from a bivoltine caddisfly, Stenopsyche marmorata: translational suppression of a silk protein in cold conditions.

    PubMed

    Nomura, Takaomi; Ito, Miho; Kanamori, Mai; Shigeno, Yuta; Uchiumi, Toshio; Arai, Ryoichi; Tsukada, Masuhiro; Hirabayashi, Kimio; Ohkawa, Kousaku

    2016-01-01

    Larval Stenopsyche marmorata constructs food capture nets and fixed retreats underwater using self-produced proteinaceous silk fibers. In the Chikuma River (Nagano Prefecture, Japan) S. marmorata has a bivoltine life cycle; overwintering larvae grow slowly with reduced net spinning activity in winter. We recently reported constant transcript abundance of S. marmorata silk protein 1 (Smsp-1), a core S. marmorata silk fiber component, in all seasons, implying translational suppression in the silk gland during winter. Herein, we prepared and characterized silk gland ribosomes from seasonally collected S. marmorata larvae. Ribosomes from silk glands immediately frozen in liquid nitrogen (LN2) after dissection exhibited comparable translation elongation activity in spring, summer, and autumn. Conversely, silk glands obtained in winter did not contain active ribosomes and Smsp-1. Ribosomes from silk glands immersed in ice-cold physiological saline solution for approximately 4 h were translationally inactive, despite summer collection and Smsp-1 expression. The ribosomal inactivation occurs because of defects in the formation of 80S ribosomes, presumably due to splitting of 60S subunits containing 28S rRNA with central hidden break, in response to cold stress. These results suggest a novel-type ribosome-regulated translation control mechanism. PMID:26646291

  17. Replication of ribosomal DNA in Xenopus laevis.

    PubMed

    Bozzoni, I; Baldari, C T; Amaldi, F; Buongiorno-Nardelli, M

    1981-09-01

    The study of the localization of the replication origins of rDNA in Xenopus laevis has been approached by two different methods. 1. The DNA of X. laevis larvae was fractionated by CsCl gradient centrifugation in bulk and ribosomal DNA and examined in the electron microscope. In bulk DNA, clusters of microbubbles, which are related with the origins of replication, appear to be spaced along the DNA molecules at intervals comparable with the size of the 'average' replicon of X. laevis. In ribosomal DNA, the distance between adjacent clusters is much shorter and corresponds to the size of the rDNA repeating unit. When ribosomal DNA was submitted to digestion with restriction enzymes (Eco RI and HindIII) the microbubbles are observed in the non-transcribed spacer-containing fragment. 2. Cultured cells of X. laevis were synchronized by mitotic selection and incubated with 5-fluoro-2-deoxyuridine for a time longer than the G1 phase. This treatment synchronizes the replicons and allows them to start replicating very slowly. It was thus possible to obtain a preferential labelling of the regions containing the origins. The analysis by gel electrophoresis of the Eco Ri-digested rDNA showed that the radioactivity was preferentially incorporated in the fragments which contain the non-transcribed spacer. The results of these two approaches indicate that the rRNA gene cluster consists of multiple units of replication, possibly one per gene unit. Furthermore they show that the origins of replication are localized into the non-transcribed spacer. PMID:7297565

  18. Neuron-Like Networks Between Ribosomal Proteins Within the Ribosome.

    PubMed

    Poirot, Olivier; Timsit, Youri

    2016-01-01

    From brain to the World Wide Web, information-processing networks share common scale invariant properties. Here, we reveal the existence of neural-like networks at a molecular scale within the ribosome. We show that with their extensions, ribosomal proteins form complex assortative interaction networks through which they communicate through tiny interfaces. The analysis of the crystal structures of 50S eubacterial particles reveals that most of these interfaces involve key phylogenetically conserved residues. The systematic observation of interactions between basic and aromatic amino acids at the interfaces and along the extension provides new structural insights that may contribute to decipher the molecular mechanisms of signal transmission within or between the ribosomal proteins. Similar to neurons interacting through "molecular synapses", ribosomal proteins form a network that suggest an analogy with a simple molecular brain in which the "sensory-proteins" innervate the functional ribosomal sites, while the "inter-proteins" interconnect them into circuits suitable to process the information flow that circulates during protein synthesis. It is likely that these circuits have evolved to coordinate both the complex macromolecular motions and the binding of the multiple factors during translation. This opens new perspectives on nanoscale information transfer and processing. PMID:27225526

  19. Neuron-Like Networks Between Ribosomal Proteins Within the Ribosome

    PubMed Central

    Poirot, Olivier; Timsit, Youri

    2016-01-01

    From brain to the World Wide Web, information-processing networks share common scale invariant properties. Here, we reveal the existence of neural-like networks at a molecular scale within the ribosome. We show that with their extensions, ribosomal proteins form complex assortative interaction networks through which they communicate through tiny interfaces. The analysis of the crystal structures of 50S eubacterial particles reveals that most of these interfaces involve key phylogenetically conserved residues. The systematic observation of interactions between basic and aromatic amino acids at the interfaces and along the extension provides new structural insights that may contribute to decipher the molecular mechanisms of signal transmission within or between the ribosomal proteins. Similar to neurons interacting through “molecular synapses”, ribosomal proteins form a network that suggest an analogy with a simple molecular brain in which the “sensory-proteins” innervate the functional ribosomal sites, while the “inter-proteins” interconnect them into circuits suitable to process the information flow that circulates during protein synthesis. It is likely that these circuits have evolved to coordinate both the complex macromolecular motions and the binding of the multiple factors during translation. This opens new perspectives on nanoscale information transfer and processing. PMID:27225526

  20. Dissecting the transcriptional phenotype of ribosomal protein deficiency: implications for Diamond-Blackfan Anemia.

    PubMed

    Aspesi, Anna; Pavesi, Elisa; Robotti, Elisa; Crescitelli, Rossella; Boria, Ilenia; Avondo, Federica; Moniz, Hélène; Da Costa, Lydie; Mohandas, Narla; Roncaglia, Paola; Ramenghi, Ugo; Ronchi, Antonella; Gustincich, Stefano; Merlin, Simone; Marengo, Emilio; Ellis, Steven R; Follenzi, Antonia; Santoro, Claudio; Dianzani, Irma

    2014-07-25

    Defects in genes encoding ribosomal proteins cause Diamond Blackfan Anemia (DBA), a red cell aplasia often associated with physical abnormalities. Other bone marrow failure syndromes have been attributed to defects in ribosomal components but the link between erythropoiesis and the ribosome remains to be fully defined. Several lines of evidence suggest that defects in ribosome synthesis lead to "ribosomal stress" with p53 activation and either cell cycle arrest or induction of apoptosis. Pathways independent of p53 have also been proposed to play a role in DBA pathogenesis. We took an unbiased approach to identify p53-independent pathways activated by defects in ribosome synthesis by analyzing global gene expression in various cellular models of DBA. Ranking-Principal Component Analysis (Ranking-PCA) was applied to the identified datasets to determine whether there are common sets of genes whose expression is altered in these different cellular models. We observed consistent changes in the expression of genes involved in cellular amino acid metabolic process, negative regulation of cell proliferation and cell redox homeostasis. These data indicate that cells respond to defects in ribosome synthesis by changing the level of expression of a limited subset of genes involved in critical cellular processes. Moreover, our data support a role for p53-independent pathways in the pathophysiology of DBA. PMID:24835311

  1. Dissecting the transcriptional phenotype of ribosomal protein deficiency: implications for Diamond-Blackfan Anemia

    PubMed Central

    Aspesi, Anna; Pavesi, Elisa; Robotti, Elisa; Crescitelli, Rossella; Boria, Ilenia; Avondo, Federica; Moniz, Hélène; Da Costa, Lydie; Mohandas, Narla; Roncaglia, Paola; Ramenghi, Ugo; Ronchi, Antonella; Gustincich, Stefano; Merlin, Simone; Marengo, Emilio; Ellis, Steven R.; Follenzi, Antonia; Santoro, Claudio; Dianzani, Irma

    2014-01-01

    Defects in genes encoding ribosomal proteins cause Diamond Blackfan Anemia (DBA), a red cell aplasia often associated with physical abnormalities. Other bone marrow failure syndromes have been attributed to defects in ribosomal components but the link between erythropoiesis and the ribosome remains to be fully defined. Several lines of evidence suggest that defects in ribosome synthesis lead to “ribosomal stress” with p53 activation and either cell cycle arrest or induction of apoptosis. Pathways independent of p53 have also been proposed to play a role in DBA pathogenesis. We took an unbiased approach to identify p53-independent pathways activated by defects in ribosome synthesis by analyzing global gene expression in various cellular models of DBA. Ranking-Principal Component Analysis (Ranking-PCA) was applied to the identified datasets to determine whether there are common sets of genes whose expression is altered in these different cellular models. We observed consistent changes in the expression of genes involved in cellular amino acid metabolic process, negative regulation of cell proliferation and cell redox homeostasis. These data indicate that cells respond to defects in ribosome synthesis by changing the level of expression of a limited subset of genes involved in critical cellular processes. Moreover, our data support a role for p53-independent pathways in the pathophysiology of DBA. PMID:24835311

  2. Paralogues of nuclear ribosomal genes conceal phylogenetic signals within the invasive Asian fish tapeworm lineage: evidence from next generation sequencing data.

    PubMed

    Brabec, Jan; Kuchta, Roman; Scholz, Tomáš; Littlewood, D Timothy J

    2016-08-01

    Complete mitochondrial genomes and nuclear rRNA operons of eight geographically distinct isolates of the Asian fish tapeworm Schyzocotyle acheilognathi (syn. Bothriocephalus acheilognathi), representing the parasite's global diversity spanning four continents, were fully characterised using an Illumina sequencing platform. This cestode species represents an extreme example of a highly invasive, globally distributed pathogen of veterinary importance with exceptionally low host specificity unseen elsewhere within the parasitic flatworms. In addition to eight specimens of S. acheilognathi, we fully characterised its closest known relative and the only congeneric species, Schyzocotyle nayarensis, from cyprinids in the Indian subcontinent. Since previous nucleotide sequence data on the Asian fish tapeworm were restricted to a single molecular locus of questionable phylogenetic utility-the nuclear rRNA genes-separating internal transcribed spacers-the mitogenomic data presented here offer a unique opportunity to gain the first detailed insights into both the intraspecific phylogenetic relationships and population genetic structure of the parasite, providing key baseline information for future research in the field. Additionally, we identify a previously unnoticed source of error and demonstrate the limited utility of the nuclear rRNA sequences, including the internal transcribed spacers that has likely misled most of the previous molecular phylogenetic and population genetic estimates on the Asian fish tapeworm. PMID:27155330

  3. Initiation factor 2 stabilizes the ribosome in a semirotated conformation.

    PubMed

    Ling, Clarence; Ermolenko, Dmitri N

    2015-12-29

    Intersubunit rotation and movement of the L1 stalk, a mobile domain of the large ribosomal subunit, have been shown to accompany the elongation cycle of translation. The initiation phase of protein synthesis is crucial for translational control of gene expression; however, in contrast to elongation, little is known about the conformational rearrangements of the ribosome during initiation. Bacterial initiation factors (IFs) 1, 2, and 3 mediate the binding of initiator tRNA and mRNA to the small ribosomal subunit to form the initiation complex, which subsequently associates with the large subunit by a poorly understood mechanism. Here, we use single-molecule FRET to monitor intersubunit rotation and the inward/outward movement of the L1 stalk of the large ribosomal subunit during the subunit-joining step of translation initiation. We show that, on subunit association, the ribosome adopts a distinct conformation in which the ribosomal subunits are in a semirotated orientation and the L1 stalk is positioned in a half-closed state. The formation of the semirotated intermediate requires the presence of an aminoacylated initiator, fMet-tRNA(fMet), and IF2 in the GTP-bound state. GTP hydrolysis by IF2 induces opening of the L1 stalk and the transition to the nonrotated conformation of the ribosome. Our results suggest that positioning subunits in a semirotated orientation facilitates subunit association and support a model in which L1 stalk movement is coupled to intersubunit rotation and/or IF2 binding. PMID:26668356

  4. The N-terminal extension of Escherichia coli ribosomal protein L20 is important for ribosome assembly, but dispensable for translational feedback control

    PubMed Central

    GUILLIER, MAUDE; ALLEMAND, FRÉDÉRIC; GRAFFE, MONIQUE; RAIBAUD, SOPHIE; DARDEL, FRÉDÉRIC; SPRINGER, MATHIAS; CHIARUTTINI, CLAUDE

    2005-01-01

    The Escherichia coli autoregulatory ribosomal protein L20 consists of two structurally distinct domains. The C-terminal domain is globular and sits on the surface of the large ribosomal subunit whereas the N-terminal domain has an extended shape and penetrates deep into the RNA-rich core of the subunit. Many other ribosomal proteins have analogous internal or terminal extensions. However, the biological functions of these extended domains remain obscure. Here we show that the N-terminal tail of L20 is important for ribosome assembly in vivo. Indeed, a truncated version of L20 without its N-terminal tail is unable to complement the deletion of rplT, the gene encoding L20. In addition, this L20 truncation confers a lethal-dominant phenotype, suggesting that the N-terminal domain is essential for cell growth because it could be required for ribosome assembly. Supporting this hypothesis, partial deletions of the N-terminal tail of the protein are shown to cause a slow-growth phenotype due to altered ribosome assembly in vivo as large amounts of intermediate 40S ribosomal particles accumulate. In addition to being a ribosomal protein, L20 also acts as an autogenous repressor. Using L20 truncations, we also show that the N-terminal tail of L20 is dispensable for autogenous control. PMID:15840820

  5. The extended loops of ribosomal proteins uL4 and uL22 of Escherichia coli contribute to ribosome assembly and protein translation.

    PubMed

    Lawrence, Marlon G; Shamsuzzaman, Md; Kondopaka, Maithri; Pascual, Clarence; Zengel, Janice M; Lindahl, Lasse

    2016-07-01

    Nearly half of ribosomal proteins are composed of a domain on the ribosome surface and a loop or extension that penetrates into the organelle's RNA core. Our previous work showed that ribosomes lacking the loops of ribosomal proteins uL4 or uL22 are still capable of entering polysomes. However, in those experiments we could not address the formation of mutant ribosomes, because we used strains that also expressed wild-type uL4 and uL22. Here, we have focused on ribosome assembly and function in strains in which loop deletion mutant genes are the ONLY: sources of uL4 or uL22 protein. The uL4 and uL22 loop deletions have different effects, but both mutations result in accumulation of immature particles that do not accumulate in detectable amounts in wild-type strains. Thus, our results suggest that deleting the loops creates kinetic barriers in the normal assembly pathway, possibly resulting in assembly via alternate pathway(s). Furthermore, deletion of the uL4 loop results in cold-sensitive ribosome assembly and function. Finally, ribosomes carrying either of the loop-deleted proteins responded normally to the secM translation pausing peptide, but the uL4 mutant responded very inefficiently to the cmlA(crb) pause peptide. PMID:27257065

  6. The extended loops of ribosomal proteins uL4 and uL22 of Escherichia coli contribute to ribosome assembly and protein translation

    PubMed Central

    Lawrence, Marlon G.; Shamsuzzaman, Md; Kondopaka, Maithri; Pascual, Clarence; Zengel, Janice M.; Lindahl, Lasse

    2016-01-01

    Nearly half of ribosomal proteins are composed of a domain on the ribosome surface and a loop or extension that penetrates into the organelle's RNA core. Our previous work showed that ribosomes lacking the loops of ribosomal proteins uL4 or uL22 are still capable of entering polysomes. However, in those experiments we could not address the formation of mutant ribosomes, because we used strains that also expressed wild-type uL4 and uL22. Here, we have focused on ribosome assembly and function in strains in which loop deletion mutant genes are the only sources of uL4 or uL22 protein. The uL4 and uL22 loop deletions have different effects, but both mutations result in accumulation of immature particles that do not accumulate in detectable amounts in wild-type strains. Thus, our results suggest that deleting the loops creates kinetic barriers in the normal assembly pathway, possibly resulting in assembly via alternate pathway(s). Furthermore, deletion of the uL4 loop results in cold-sensitive ribosome assembly and function. Finally, ribosomes carrying either of the loop-deleted proteins responded normally to the secM translation pausing peptide, but the uL4 mutant responded very inefficiently to the cmlAcrb pause peptide. PMID:27257065

  7. Does hybridization increase evolutionary rate? Data from the 28S-rDNA D8 domain in echinoderms.

    PubMed

    Chenuil, Anne; Egea, Emilie; Rocher, Caroline; Touzet, Hélène; Féral, Jean-Pierre

    2008-11-01

    The divergent domain D8 of the large ribosomal RNA is very variable and extended in vertebrates compared to other eukaryotes. We provide data from 31 species of echinoderms and present the first comparative analysis of the D8 in nonvertebrate deuterostomes. In addition, we obtained 16S mitochondrial DNA sequences for the sea urchin taxa and analyzed single-strand conformation polymorphism (SSCP) of D8 in several populations within the species complex Echinocardium cordatum. A common secondary structure supported by compensatory substitutions and indels is inferred for echinoderms. Variation mostly arises at the tip of the longest stem (D8a), and the most variable taxa also display the longest and most stable D8. The most stable variants are the only ones displaying bulges in the terminal part of the stem, suggesting that selection, rather than maximizing stability of the D8 secondary structure, maintains it in a given range. Striking variation in D8 evolutionary rates was evidenced among sea urchins, by comparison with both 16S mitochondrial DNA and paleontological data. In Echinocardium cordatum and Strongylocentrotus pallidus and S. droebachiensis, belonging to very distant genera, the increase in D8 evolutionary rate is extreme. Their highly stable D8 secondary structures rule out the possibility of pseudogenes. These taxa are the only ones in which interspecific hybridization was reported. We discuss how evolutionary rates may be affected in nuclear relative to mitochondrial genes after hybridization, by selective or mutational processes such as gene silencing and concerted evolution. PMID:18949506

  8. Functional Role of Ribosomal Signatures

    PubMed Central

    Chen, Ke; Eargle, John; Sarkar, Krishnarjun; Gruebele, Martin; Luthey-Schulten, Zaida

    2010-01-01

    Although structure and sequence signatures in ribosomal RNA and proteins are defining characteristics of the three domains of life and instrumental in constructing the modern phylogeny, little is known about their functional roles in the ribosome. In this work, the largest coevolving RNA/protein signatures in the bacterial 30S ribosome are investigated both experimentally and computationally through all-atom molecular-dynamics simulations. The complex includes the N-terminal fragment of the ribosomal protein S4, which is a primary binding protein that initiates 30S small subunit assembly from the 5′ domain, and helix 16 (h16), which is part of the five-way junction in 16S rRNA. Our results show that the S4 N-terminus signature is intrinsically disordered in solution, whereas h16 is relatively stable by itself. The dynamic disordered property of the protein is exploited to couple the folding and binding process to the five-way junction, and the results provide insight into the mechanism for the early and fast binding of S4 in the assembly of the ribosomal small subunit. PMID:21156135

  9. Ribosome biogenesis: emerging evidence for a central role in the regulation of skeletal muscle mass†

    PubMed Central

    Chaillou, Thomas; Kirby, Tyler J.; McCarthy, John J.

    2016-01-01

    The ribosome is a supramolecular ribonucleoprotein complex that functions at the heart of the translation machinery to convert mRNA into protein. Ribosome biogenesis is the primary determinant of translational capacity of the cell and accordingly has an essential role in the control of cell growth in eukaryotes. Cumulative evidence supports the hypothesis that ribosome biogenesis has an important role in the regulation of skeletal muscle mass. The purpose of this review is to, first, summarize the main mechanisms known to regulate ribosome biogenesis and, second, put forth the hypothesis that ribosome biogenesis is a central mechanism used by skeletal muscle to regulate protein synthesis and control skeletal muscle mass in response to anabolic and catabolic stimuli. The mTORC1 and Wnt/β-catenin/c-myc signaling pathways are discussed as the major pathways that work in concert with each of the three RNA polymerases (RNA Pol I, II and III) in regulating ribosome biogenesis. Consistent with our hypothesis, activation of these two pathways has been shown to be associated with ribosome biogenesis during skeletal muscle hypertrophy. Although further study is required, the finding that ribosome biogenesis is altered under catabolic states, in particular during disuse atrophy, suggests that its activation represents a novel therapeutic target to reduce or prevent muscle atrophy. Lastly, the emerging field of ribosome specialization is discussed and its potential role in the regulation of gene expression during periods of skeletal muscle plasticity. PMID:24604615

  10. Natural populations of lactic acid bacteria associated with silage fermentation as determined by phenotype, 16S ribosomal RNA and recA gene analysis.

    PubMed

    Pang, Huili; Qin, Guangyong; Tan, Zhongfang; Li, Zongwei; Wang, Yanping; Cai, Yimin

    2011-05-01

    One hundred and fifty-six strains isolated from corn (Zea mays L.), forage paddy rice (Oryza sativa L.), sorghum (Sorghum bicolor L.) and alfalfa (Medicago sativa L.) silages prepared on dairy farms were screened, of which 110 isolates were considered to be lactic acid bacteria (LAB) according to their Gram-positive and catalase-negative characteristics and, mainly, the lactic acid metabolic products. These isolates were divided into eight groups (A-H) based on the following properties: morphological and biochemical characteristics, γ-aminobutyric acid production capacity, and 16S rRNA gene sequences. They were identified as Weissella cibaria (36.4%), Weissella confusa (9.1%), Leuconostoc citreum (5.3%), Leuconostoc lactis (4.9%), Leuconostoc pseudomesenteroides (8.0%), Lactococcus lactis subsp. lactis (4.5%), Lactobacillus paraplantarum (4.5%) and Lactobacillus plantarum (27.3%). W. cibaria and W. confusa were mainly present in corn silages, and L. plantarum was dominant on sorghum and forage paddy rice silages, while L. pseudomesenteroides, L. plantarum and L. paraplantarum were the dominant species in alfalfa silage. The corn, sorghum and forage paddy rice silages were well preserved with lower pH values and ammonia-N concentrations, but had higher lactic acid content, while the alfalfa silage had relatively poor quality with higher pH values and ammonia-N concentrations, and lower lactic acid content. The present study confirmed the diversity of LAB species inhabiting silages. It showed that the differing natural populations of LAB on these silages might influence fermentation quality. These results will enable future research on the relationship between LAB species and silage fermentation quality, and will enhance the screening of appropriate inoculants aimed at improving such quality. PMID:21282025

  11. Epidemiology of sporadic (non-epidemic) cases of Trichophyton tonsurans infection in Japan based on PCR-RFLP analysis of non-transcribed spacer region of ribosomal RNA gene.

    PubMed

    Mochizuki, Takashi; Kawasaki, Masako; Anzawa, Kazushi; Fujita, Jun; Ushigami, Tsuyoshi; Takeda, Kiminobu; Sano, Ayako; Takahashi, Yoko; Kamei, Katsuhiko

    2008-05-01

    A number of cases of Trichophyton tonsurans infection have been reported among sportsmen and women participating in wrestling, judo, and sumo wrestling in Japan, but there have also been sporadic reports of cases with no history of contact with these sports. A molecular method using restriction enzyme analysis of PCR-amplified fragments targeting the non-transcribed spacer region (NTS) of ribosomal RNA gene in fungal nuclei was applied to T. tonsurans strains isolated from sporadic cases in Japan. Five of 6 molecular types recorded in Japan, i.e., NTS types I, II, IV, V, and VI, and two new types, designated NTS VII and NTS VIII, were observed among 10 strains isolated from sporadic cases. The NTS IV strains, considered not to be related to the present epidemic, were found to be the most prevalent molecular type accounting for 4 of the 10 strains isolated. NTS I was the most prevalent type in the current epidemic in Japan, but it was cultured from only one patient who was later noted to be the daughter of a retired judo practitioner. Four subjects had histories of living abroad and were considered to have been infected outside Japan. The strains in these cases were NTS II, V, VI, and VII. The results of this study suggested that the NTS IV strains were originally present in Japan at a low incidence, but that there has been a recent influx of NTS I, II, V, VI, and VII from abroad, which has been accompanied by the secondary spread of strains from wrestlers and practitioners of martial arts to the general community. PMID:18503175

  12. Structure and stability of variants of the sarcin-ricin loop of 28S rRNA: NMR studies of the prokaryotic SRL and a functional mutant.

    PubMed Central

    Seggerson, K; Moore, P B

    1998-01-01

    NMR has been used to examine the conformational properties of two variants of the sarcin-ricin loop (SRL) from eukaryotic 28S rRNA, which is essential for elongation factor interactions with the ribosome: (1) its bacterial homologue, which lacks two of the bases that flank the conserved 12-nt sequence in the middle of the SRL, but which is functionally equivalent, and (2) a functionally active variant of the eukaryotic SRL in which the bulged G within the conserved sequence is replaced by an A. The data indicate that, although the bacterial SRL is less stable than the eukaryotic SRL, its conformation is closely similar. Furthermore, even though replacement of the bulged G in the SRL with an A seriously destabilizes the center of the loop, its effect on the overall conformation of the SRL appears to be modest. In the course of this work, it was serendipitously discovered that at neutral pH, the C8 proton of the bulged G, in both PRO-SRL and E73, exchanges about 10 times faster than it does in GMP. PMID:9769095

  13. RNA structures regulating ribosomal protein biosynthesis in bacilli.

    PubMed

    Deiorio-Haggar, Kaila; Anthony, Jon; Meyer, Michelle M

    2013-07-01

    In Bacilli, there are three experimentally validated ribosomal-protein autogenous regulatory RNAs that are not shared with E. coli. Each of these RNAs forms a unique secondary structure that interacts with a ribosomal protein encoded by a downstream gene, namely S4, S15, and L20. Only one of these RNAs that interacts with L20 is currently found in the RNA Families Database. We created, or modified, existing structural alignments for these three RNAs and used them to perform homology searches. We have determined that each structure exhibits a narrow phylogenetic distribution, mostly relegated to the Firmicute class Bacilli. This work, in conjunction with other similar work, demonstrates that there are most likely many non-homologous RNA regulatory elements regulating ribosomal protein biosynthesis that still await discovery and characterization in other bacterial species. PMID:23611891

  14. RNA structures regulating ribosomal protein biosynthesis in bacilli

    PubMed Central

    Deiorio-Haggar, Kaila; Anthony, Jon; Meyer, Michelle M.

    2013-01-01

    In Bacilli, there are three experimentally validated ribosomal-protein autogenous regulatory RNAs that are not shared with E. coli. Each of these RNAs forms a unique secondary structure that interacts with a ribosomal protein encoded by a downstream gene, namely S4, S15, and L20. Only one of these RNAs that interacts with L20 is currently found in the RNA Families Database. We created, or modified, existing structural alignments for these three RNAs and used them to perform homology searches. We have determined that each structure exhibits a narrow phylogenetic distribution, mostly relegated to the Firmicute class Bacilli. This work, in conjunction with other similar work, demonstrates that there are most likely many non-homologous RNA regulatory elements regulating ribosomal protein biosynthesis that still await discovery and characterization in other bacterial species. PMID:23611891

  15. Silencing of RNA helicase II/Gualpha inhibits mammalian ribosomal RNA production.

    PubMed

    Henning, Dale; So, Rolando B; Jin, Runyan; Lau, Lester F; Valdez, Benigno C

    2003-12-26

    The intricate production of ribosomal RNA is well defined in yeast, but its complexity in higher organisms is barely understood. We recently showed that down-regulation of nucleolar protein RNA helicase II/Gualpha (RH-II/Gualpha or DDX21) in Xenopus oocytes inhibited processing of 20 S rRNA to 18 S and contributed to degradation of 28 S rRNA (Yang, H., Zhou, J., Ochs, R. L., Henning, D., Jin, R., and Valdez, B. C. (2003) J. Biol. Chem. 278, 38847-38859). Since no nucleolar RNA helicase has been functionally characterized in mammalian cells, we used short interfering RNA to search for functions for RH-II/Gualpha and its paralogue RH-II/Gubeta in rRNA production. Silencing of RH-II/Gualpha by more than 80% in HeLa cells resulted in an almost 80% inhibition of 18 and 28 S rRNA production. This inhibition could be reversed by exogenous expression of wild type RH-II/Gualpha. A helicase-deficient mutant form having ATPase activity was able to rescue the production of 28 S but not 18 S rRNA. A phenotype exhibiting inhibition of 18 S and 28 S rRNA production was also observed when the paralogue RH-II/Gubeta was overexpressed. Both down-regulation of RH-II/Gualpha and overexpression of RH-II/Gubeta slowed cell proliferation. The opposite effects of the two paralogues suggest antagonistic functions. PMID:14559904

  16. Comprehensive Molecular Structure of the Eukaryotic Ribosome

    PubMed Central

    Taylor, Derek J.; Devkota, Batsal; Huang, Andrew D.; Topf, Maya; Narayanan, Eswar; Sali, Andrej; Harvey, Stephen C.; Frank, Joachim

    2009-01-01

    Despite the emergence of a large number of X-ray crystallographic models of the bacterial 70S ribosome over the past decade, an accurate atomic model of the eukaryotic 80S ribosome is still not available. Eukaryotic ribosomes possess more ribosomal proteins and ribosomal RNA than bacterial ribosomes, which are implicated in extra-ribosomal functions in the eukaryotic cells. By combining cryo-EM with RNA and protein homology modeling, we obtained an atomic model of the yeast 80S ribosome complete with all ribosomal RNA expansion segments and all ribosomal proteins for which a structural homolog can be identified. Mutation or deletion of 80S ribosomal proteins can abrogate maturation of the ribosome, leading to several human diseases. We have localized one such protein unique to eukaryotes, rpS19e, whose mutations are associated with Diamond-Blackfan anemia in humans. Additionally, we characterize crucial and novel interactions between the dynamic stalk base of the ribosome with eukaryotic elongation factor 2. PMID:20004163

  17. New ribosomes for new memories?

    PubMed Central

    Hernández, A Iván; Alarcon, Juan M; Allen, Kim D

    2015-01-01

    Widely thought to be a housekeeping process, the regulation and synthesis of rRNA emerges as a potentially central mechanism for the maintenance of synaptic plasticity and memory. We have recently shown that an essential component of late-phase synaptic plasticity is rRNA biosynthesis — the rate-limiting step in the production of new ribosomes. We hypothesize that a particular population of ribosomes is generated upon learning-associated neural activity to alter the rate of synthesis of plasticity factors at tagged synapses that will support the maintenance of synaptic plasticity and memory. PMID:26479998

  18. Riproximin is a recently discovered type II ribosome inactivating protein with potential for treating cancer.

    PubMed

    Adwan, Hassan; Bayer, Helene; Pervaiz, Asim; Sagini, Micah; Berger, Martin R

    2014-11-01

    The development of new anticancer drugs is a salient problem and the traditional use of plants is a potentially rich source of information for detecting new molecules with antineoplastic activity. Riproximin is a recently detected cytotoxic type II ribosome inactivating protein with high selectivity for certain tumor cell lines. Its activity was recognized as the main component in a plant powder used by African healers for treating cancer. By ribulose bisphosphate carboxylase gene sequencing analysis, the powder was identified to be derived from the plant Ximenia americana. The cDNA sequence of riproximin was identified, the protein was modeled to contain one A- and a B-chain, respectively, and a reliable purification procedure from kernels of X. americana was established. Riproximin displays high but differential antiproliferative activity in a panel of human and rodent cancer cell lines, with concentrations inhibiting cell proliferation by 50% (IC50 values) that diverge by a factor of 100. Consistent antineoplastic activity was detected in colorectal and pancreatic cancer liver metastasis models in rats. The cytotoxic mechanism of action was determined to be based on cellular uptake of riproximin followed by its A-chain prompted depurination of the 28S ribosomal RNA and induction of unfolded protein response. Riproximin's specificity depended on its B-chain connected binding to cell surface glycans, the presence of which is crucial for subsequent internalization into cells and cytotoxicity. These N- and O-glycans include bi- and tri-antennary NA structures (NA2/NA3) as well as Tn3 structures (clustered Tn antigen). Riproximin was found to crosslink proteins with N- and O-glycan structure, thus indicating both types of binding sites on its B chain. Due to this crosslinking ability, riproximin is expected to show prominent cytotoxicity towards cells expressing both, NA2/NA3 and clustered Tn structures. Apart from the properties of riproximin, the plant X. americana

  19. Chromatographic Purification of Highly Active Yeast Ribosomes

    PubMed Central

    Meskauskas, Arturas; Leshin, Jonathan A.; Dinman, Jonathan D.

    2011-01-01

    Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes. PMID:22042245

  20. A putative precursor for the small ribosomal RNA from mitochondria of Saccharomyces cerevisiae.

    PubMed Central

    Osinga, K A; Evers, R F; Van der Laan, J C; Tabak, H F

    1981-01-01

    We have characterized a putative precursor RNA (15.5S) for the 15S ribosomal RNA in mitochondria of Saccharomyces cerevisiae. Hybrids were formed with mitochondrial RNA and mtDNA fragments terminally labelled at restriction sites located within the gene coding for 15S ribosomal RNA and treated with S1 nuclease (Berk, A.J. and Sharp, J.A. (1977) 12, 721-732). Sites of resistant hybrids were measured by agarose gel electrophoresis and end points of RNAs determined. The 15.5S RNA is approximately 80 nucleotides longer than the 15S ribosomal RNA, with the extra sequences being located at the 5'-end. Both 15S ribosomal RNA and 15.5S RNA are fully localised within a 2000 base pair HapII fragment. This putative precursor and the mature 15S ribosomal RNA are also found in petite mutants which retain the 15S ribosomal RNA gene. The petite mutant with the smallest genetic complexity has its end point of deletion (junction) just outside the HapII site located in the 5' flank of the 15S ribosomal RNA genes as determined by S1 nuclease analysis. This leaves a DNA stretch approximately 300 base pairs long where an initiation signal for mitochondrial transcription may be present. Images PMID:6262728

  1. Glycogen synthase kinase-3 is involved in regulation of ribosome biogenesis in yeast.

    PubMed

    Yabuki, Yukari; Kodama, Yushi; Katayama, Masako; Sakamoto, Akiko; Kanemaru, Hirofumi; Wan, Kun; Mizuta, Keiko

    2014-01-01

    Secretory defects cause transcriptional repression of both ribosomal proteins and ribosomal RNA genes in Saccharomyces cerevisiae. Rrs1, a trans-acting factor that participates in ribosome biogenesis, is involved in the signaling pathway induced by secretory defects. Here, we found that Rrs1 interacts with two homologs of the glycogen synthase kinase-3 (GSK-3), Rim11, and Mrk1. Rrs1 possesses a repetitive consensus amino acid sequence for phosphorylation by GSK-3, and mutation of this sequence abolished the interaction of Rrs1 with Rim11 and Mrk1. Although this mutation did not affect vegetative cell growth or secretory response, disruption of all four genes encoding GSK-3 homologs, especially Mck1, diminished the transcriptional repression of ribosomal protein genes in response to secretory defects. Among the four GSK-3 kinases, Mck1 appears to be the primary mediator of this response, while the other GSK-3 kinases contribute redundantly. PMID:25035982

  2. AMPLIFICATION OF RIBOSOMAL RNA SEQUENCES

    EPA Science Inventory

    This book chapter offers an overview of the use of ribosomal RNA sequences. A history of the technology traces the evolution of techniques to measure bacterial phylogenetic relationships and recent advances in obtaining rRNA sequence information. The manual also describes procedu...

  3. Studies on Pea Ribosomal Proteins

    PubMed Central

    Lin, Chu-Yung; Chia, Subrina Li-Li; Travis, Robert L.; Key, Joe L.

    1975-01-01

    Ribosomal subunits prepared by NH4Cl dissociation (0.5 m) of the monomeric ribosomes were much less active in in vitro protein synthesis than those prepared by KCl dissociation. The decrease in activity correlated with a detachment of some proteins (L2 and L9 as shown by gel electrophoresis) within the 60S ribosomal subunits. Subunits prepared with 0.3 m NH4Cl retained L2 and L9, but the activity remained low. Incubation of these 60S subunits in TKM buffer (50 mm tris [pH 7.5], 20 mm KCl, and 5 mm MgCl2) for 20 min at 37 C restored the activity almost to the level of those obtained by KCl dissociation. Treatment of the 0.3 m NH4Cl-derived 60S subunits with a protein reagent, Procion brilliant blue, prior to extraction of the ribosomal proteins resulted in the loss of L2 and L9, showing that these proteins were made accessible for dye binding. These observations suggest that a considerable degree of unfolding of the 60S subunit occurs at 0.3 m NH4Cl (this apparently leads to a preferential detachment of L2 and L9 at 0.5 m NH4Cl) and that the activity of the purified subunits depends not only on the presence of L2 and L9 but also on the organization of these proteins within the 60S subunits. Images PMID:16659254

  4. High Precision Analysis of Translational Pausing by Ribosome Profiling in Bacteria Lacking EFP

    PubMed Central

    Woolstenhulme, Christopher J.; Guydosh, Nicholas R.; Green, Rachel; Buskirk, Allen R.

    2015-01-01

    Summary Ribosome profiling is a powerful method for globally assessing the activity of ribosomes in a cell. Despite its application in many organisms, ribosome profiling studies in bacteria have struggled to obtain the resolution necessary to precisely define translational pauses. Here we report improvements that yield much higher resolution in E. coli profiling data, enabling us to more accurately assess ribosome pausing and refine earlier studies of the impact of polyproline motifs on elongation. We comprehensively characterize pausing at proline-rich motifs in the absence of elongation factor EFP. We find that only a small fraction of genes with strong pausing motifs have reduced ribosome density downstream and identify features that explain this phenomenon. These features allow us to predict which proteins likely have reduced output in the efp knockout strain. PMID:25843707

  5. PCR Primers for Metazoan Mitochondrial 12S Ribosomal DNA Sequences

    PubMed Central

    Machida, Ryuji J.; Kweskin, Matthew; Knowlton, Nancy

    2012-01-01

    Background Assessment of the biodiversity of communities of small organisms is most readily done using PCR-based analysis of environmental samples consisting of mixtures of individuals. Known as metagenetics, this approach has transformed understanding of microbial communities and is beginning to be applied to metazoans as well. Unlike microbial studies, where analysis of the 16S ribosomal DNA sequence is standard, the best gene for metazoan metagenetics is less clear. In this study we designed a set of PCR primers for the mitochondrial 12S ribosomal DNA sequence based on 64 complete mitochondrial genomes and then tested their efficacy. Methodology/Principal Findings A total of the 64 complete mitochondrial genome sequences representing all metazoan classes available in GenBank were downloaded using the NCBI Taxonomy Browser. Alignment of sequences was performed for the excised mitochondrial 12S ribosomal DNA sequences, and conserved regions were identified for all 64 mitochondrial genomes. These regions were used to design a primer pair that flanks a more variable region in the gene. Then all of the complete metazoan mitochondrial genomes available in NCBI's Organelle Genome Resources database were used to determine the percentage of taxa that would likely be amplified using these primers. Results suggest that these primers will amplify target sequences for many metazoans. Conclusions/Significance Newly designed 12S ribosomal DNA primers have considerable potential for metazoan metagenetic analysis because of their ability to amplify sequences from many metazoans. PMID:22536450

  6. Ribosome dimerization is essential for the efficient regrowth of Bacillus subtilis.

    PubMed

    Akanuma, Genki; Kazo, Yuka; Tagami, Kazumi; Hiraoka, Hirona; Yano, Koichi; Suzuki, Shota; Hanai, Ryo; Nanamiya, Hideaki; Kato-Yamada, Yasuyuki; Kawamura, Fujio

    2016-03-01

    Ribosome dimers are a translationally inactive form of ribosomes found in Escherichia coli and many other bacterial cells. In this study, we found that the 70S ribosomes of Bacillus subtilis dimerized during the early stationary phase and these dimers remained in the cytoplasm until regrowth was initiated. Ribosome dimerization during the stationary phase required the hpf gene, which encodes a homologue of the E. coli hibernation-promoting factor (Hpf). The expression of hpf was induced at an early stationary phase and its expression was observed throughout the rest of the experimental period, including the entire 6 h of the stationary phase. Ribosome dimerization followed the induction of hpf in WT cells, but the dimerization was impaired in cells harbouring a deletion in the hpf gene. Although the absence of ribosome dimerization in these Hpf-deficient cells did not affect their viability in the stationary phase, their ability to regrow from the stationary phase decreased. Thus, following the transfer of stationary-phase cells to fresh LB medium, Δhpf mutant cells grew slower than WT cells. This observed lag in growth of Δhpf cells was probably due to a delay in restoring their translational activity. During regrowth, the abundance of ribosome dimers in WT cells decreased with a concomitant increase in the abundance of 70S ribosomes and growth rate. These results suggest that the ribosome dimers, by providing 70S ribosomes to the cells, play an important role in facilitating rapid and efficient regrowth of cells under nutrient-rich conditions. PMID:26743942

  7. The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments

    PubMed Central

    Ingolia, Nicholas T.; Brar, Gloria A.; Rouskin, Silvia; McGeachy, Anna M.; Weissman, Jonathan S.

    2012-01-01

    Recent studies highlight the importance of translational control in determining protein abundance, underscoring the value of measuring gene expression at the level of translation. We present a protocol for genome-wide, quantitative analysis of in vivo translation by deep sequencing. This ribosome profiling approach maps the exact positions of ribosomes on transcripts by nuclease footprinting. The nuclease-protected mRNA fragments are converted into a DNA library suitable for deep sequencing using a strategy that minimizes bias. The abundance of different footprint fragments in deep sequencing data reports on the amount of translation of a gene. Additionally, footprints reveal the exact regions of the transcriptome that are translated. To better define translated reading frames, we describe an adaptation that reveals the sites of translation initiation by pre-treating cells with harringtonine to immobilize initiating ribosomes. The protocol we describe requires 5–7 days to generate a completed ribosome profiling sequencing library. Sequencing and data analysis requires a further 4 – 5 days. PMID:22836135

  8. Bypass of the pre-60S ribosomal quality control as a pathway to oncogenesis.

    PubMed

    Sulima, Sergey O; Patchett, Stephanie; Advani, Vivek M; De Keersmaecker, Kim; Johnson, Arlen W; Dinman, Jonathan D

    2014-04-15

    Ribosomopathies are a class of diseases caused by mutations that affect the biosynthesis and/or functionality of the ribosome. Although they initially present as hypoproliferative disorders, such as anemia, patients have elevated risk of hyperproliferative disease (cancer) by midlife. Here, this paradox is explored using the rpL10-R98S (uL16-R98S) mutant yeast model of the most commonly identified ribosomal mutation in acute lymphoblastic T-cell leukemia. This mutation causes a late-stage 60S subunit maturation failure that targets mutant ribosomes for degradation. The resulting deficit in ribosomes causes the hypoproliferative phenotype. This 60S subunit shortage, in turn, exerts pressure on cells to select for suppressors of the ribosome biogenesis defect, allowing them to reestablish normal levels of ribosome production and cell proliferation. However, suppression at this step releases structurally and functionally defective ribosomes into the translationally active pool, and the translational fidelity defects of these mutants culminate in destabilization of selected mRNAs and shortened telomeres. We suggest that in exchange for resolving their short-term ribosome deficits through compensatory trans-acting suppressors, cells are penalized in the long term by changes in gene expression that ultimately undermine cellular homeostasis. PMID:24706786

  9. Control of ribosome traffic by position-dependent choice of synonymous codons

    NASA Astrophysics Data System (ADS)

    Mitarai, Namiko; Pedersen, Steen

    2013-10-01

    Messenger RNA (mRNA) encodes a sequence of amino acids by using codons. For most amino acids, there are multiple synonymous codons that can encode the amino acid. The translation speed can vary from one codon to another, thus there is room for changing the ribosome speed while keeping the amino acid sequence and hence the resulting protein. Recently, it has been noticed that the choice of the synonymous codon, via the resulting distribution of slow- and fast-translated codons, affects not only on the average speed of one ribosome translating the mRNA but also might have an effect on nearby ribosomes by affecting the appearance of ‘traffic jams’ where multiple ribosomes collide and form queues. To test this ‘context effect’ further, we here investigate the effect of the sequence of synonymous codons on the ribosome traffic by using a ribosome traffic model with codon-dependent rates, estimated from experiments. We compare the ribosome traffic on wild-type (WT) sequences and sequences where the synonymous codons were swapped randomly. By simulating translation of 87 genes, we demonstrate that the WT sequences, especially those with a high bias in codon usage, tend to have the ability to reduce ribosome collisions, hence optimizing the cellular investment in the translation apparatus. The magnitude of such reduction of the translation time might have a significant impact on the cellular growth rate and thereby have importance for the survival of the species.

  10. A protein residing at the subunit interface of the bacterial ribosome.

    PubMed

    Agafonov, D E; Kolb, V A; Nazimov, I V; Spirin, A S

    1999-10-26

    Surface labeling of Escherichia coli ribosomes with the use of the tritium bombardment technique has revealed a minor unidentified ribosome-bound protein (spot Y) that is hidden in the 70S ribosome and becomes highly labeled on dissociation of the 70S ribosome into subunits. In the present work, the N-terminal sequence of the protein Y was determined and its gene was identified as yfia, an ORF located upstream the phe operon of E. coli. This 12.7-kDa protein was isolated and characterized. An affinity of the purified protein Y for the 30S subunit, but not for the 50S ribosomal subunit, was shown. The protein proved to be exposed on the surface of the 30S subunit. The attachment of the 50S subunit resulted in hiding the protein Y, thus suggesting the protein location at the subunit interface in the 70S ribosome. The protein was shown to stabilize ribosomes against dissociation. The possible role of the protein Y as ribosome association factor in translation is discussed. PMID:10535924

  11. Hypoxic stress-induced changes in ribosomes of maize seedling roots. [Zea mays L

    SciTech Connect

    Bailey-Serres, J.; Freeling, M. )

    1990-11-01

    The hypoxic stress response of Zea mays L. seedling roots involves regulation of gene expression at transcriptional and posttranscriptional levels. We investigated the effect of hypoxia on the translational machinery of seedling roots. The levels of monoribosomes and ribosomal subunits increased dramatically within 1 hour of stress. Prolonged hypoxia resulted in continued accumulation of nontranslating ribosomes, as well as increased levels of small polyribosomes. The return of seedlings to normal aerobic conditions resulted in recovery of normal polyribosome levels. Comparison of ribosomal proteins from control and hypoxic roots revealed differences in quantity and electrophoretic mobility. In vivo labeling of roots with ({sup 35}S)methionine revealed variations in newly synthesized ribosomal proteins. In vivo labeling of roots with ({sup 32}P)orthophosphate revealed a major reduction in the phosphorylation of a 31 kilodalton ribosomal protein in hypoxic stressed roots. In vitro phosphorylation of ribosomal proteins by endogenous kinases was used to probe for differences in ribosome structure and composition. The patterns of in vitro kinased phosphoproteins of ribosomes from control and hypoxic roots were not identical. Variation in phosphoproteins of polyribosomes from control and hypoxic roots, as well as among polyribosomes from hypoxic roots were observed. These results indicate that modification of the translational machinery occurs in response to hypoxic stress.

  12. Sequence variation within the rRNA gene loci of 12 Drosophila species

    PubMed Central

    Stage, Deborah E.; Eickbush, Thomas H.

    2007-01-01

    Concerted evolution maintains at near identity the hundreds of tandemly arrayed ribosomal RNA (rRNA) genes and their spacers present in any eukaryote. Few comprehensive attempts have been made to directly measure the identity between the rDNA units. We used the original sequencing reads (trace archives) available through the whole-genome shotgun sequencing projects of 12 Drosophila species to locate the sequence variants within the 7.8–8.2 kb transcribed portions of the rDNA units. Three to 18 variants were identified in >3% of the total rDNA units from 11 species. Species where the rDNA units are present on multiple chromosomes exhibited only minor increases in sequence variation. Variants were 10–20 times more abundant in the noncoding compared with the coding regions of the rDNA unit. Within the coding regions, variants were three to eight times more abundant in the expansion compared with the conserved core regions. The distribution of variants was largely consistent with models of concerted evolution in which there is uniform recombination across the transcribed portion of the unit with the frequency of standing variants dependent upon the selection pressure to preserve that sequence. However, the 28S gene was found to contain fewer variants than the 18S gene despite evolving 2.5-fold faster. We postulate that the fewer variants in the 28S gene is due to localized gene conversion or DNA repair triggered by the activity of retrotransposable elements that are specialized for insertion into the 28S genes of these species. PMID:17989256

  13. Modifying the maker: Oxygenases target ribosome biology.

    PubMed

    Zhuang, Qinqin; Feng, Tianshu; Coleman, Mathew L

    2015-01-01

    The complexity of the eukaryotic protein synthesis machinery is partly driven by extensive and diverse modifications to associated proteins and RNAs. These modifications can have important roles in regulating translation factor activity and ribosome biogenesis and function. Further investigation of 'translational modifications' is warranted considering the growing evidence implicating protein synthesis as a critical point of gene expression control that is commonly deregulated in disease. New evidence suggests that translation is a major new target for oxidative modifications, specifically hydroxylations and demethylations, which generally are catalyzed by a family of emerging oxygenase enzymes that act at the interface of nutrient availability and metabolism. This review summarizes what is currently known about the role or these enzymes in targeting rRNA synthesis, protein translation and associated cellular processes. PMID:26779412

  14. Modifying the maker: Oxygenases target ribosome biology

    PubMed Central

    Zhuang, Qinqin; Feng, Tianshu; Coleman, Mathew L

    2015-01-01

    The complexity of the eukaryotic protein synthesis machinery is partly driven by extensive and diverse modifications to associated proteins and RNAs. These modifications can have important roles in regulating translation factor activity and ribosome biogenesis and function. Further investigation of ‘translational modifications’ is warranted considering the growing evidence implicating protein synthesis as a critical point of gene expression control that is commonly deregulated in disease. New evidence suggests that translation is a major new target for oxidative modifications, specifically hydroxylations and demethylations, which generally are catalyzed by a family of emerging oxygenase enzymes that act at the interface of nutrient availability and metabolism. This review summarizes what is currently known about the role or these enzymes in targeting rRNA synthesis, protein translation and associated cellular processes. PMID:26779412

  15. Positive modulation of RNA polymerase III transcription by ribosomal proteins

    SciTech Connect

    Dieci, Giorgio; Carpentieri, Andrea; Amoresano, Angela; Ottonello, Simone

    2009-02-06

    A yeast nuclear fraction of unknown composition, named TFIIIE, was reported previously to enhance transcription of tRNA and 5S rRNA genes in vitro. We show that TFIIIE activity co-purifies with a specific subset of ribosomal proteins (RPs) which, as revealed by chromatin immunoprecipitation analysis, generally interact with tRNA and 5S rRNA genes, but not with a Pol II-specific promoter. Only Rpl6Ap and Rpl6Bp, among the tested RPs, were found associated to a TATA-containing tRNA{sup Ile}(TAT) gene. The RPL6A gene also emerged as a strong multicopy suppressor of a conditional mutation in the basal transcription factor TFIIIC, while RPL26A and RPL14A behaved as weak suppressors. The data delineate a novel extra-ribosomal role for one or a few RPs which, by influencing 5S rRNA and tRNA synthesis, could play a key role in the coordinate regulation of the different sub-pathways required for ribosome biogenesis and functionality.

  16. Alcoholic Liver Disease and the Mitochondrial Ribosome

    PubMed Central

    Cahill, Alan; Sykora, Peter

    2009-01-01

    Summary Chronic alcohol consumption has been shown to severely compromise mitochondrial protein synthesis. Hepatic mitochondria isolated from alcoholic animals contain decreased levels of respiratory complexes and display depressed respiration rates when compared to pair-fed controls. One underlying mechanism for this involves ethanol-elicited alterations in the structural and functional integrity of the mitochondrial ribosome. Ethanol feeding results in ribosomal changes that include decreased sedimentation rates, larger hydrodynamic volumes, increased levels of unassociated subunits and changes in the levels of specific ribosomal proteins. The methods presented in this chapter detail how to isolate mitochondrial ribosomes, determine ribosomal activity, separate ribosomes into nucleic acid and protein, and perform two-dimensional nonequilibrium pH gradient electrophoretic polyacrylamide gel electrophoresis to separate and subsequently identify mitochondrial ribosomal proteins. PMID:18369931

  17. [About the ribosomal biogenesis in human].

    PubMed

    Tafforeau, Lionel

    2015-01-01

    Ribosomes are cellular ribonucleoprotein particles required for a fundamental mechanism, translation of the genetic information into proteins. Ribosome biogenesis is a highly complex pathway involving many maturation steps: ribosomal RNA (rRNA) synthesis, rRNA processing, pre-rRNA modifications, its assembly with ribosomal proteins in the nuceolus, export of the subunit precursors to the nucleoplasm and the cytoplasm. Ribosome biogenesis has mainly being investigated in yeast during these last 25 years. However, recent works have shown that, despite many similarities between yeast and human ribosome structure and biogenesis, human pre-rRNA processing is far more complex than in yeast. In order to better understand diseases related to a malfunction in ribosome synthesis, the ribosomopathies, research should be conducted directly in human cells and animal models. PMID:26152166

  18. Intersubunit movement is required for ribosomal translocation

    PubMed Central

    Horan, Lucas H.; Noller, Harry F.

    2007-01-01

    Translocation of tRNA and mRNA during protein synthesis is believed to be coupled to structural changes in the ribosome. The “ratchet model,” based on cryo-EM reconstructions of ribosome complexes, invokes relative movement of the 30S and 50S ribosomal subunits in this process; however, evidence that directly demonstrates a requirement for intersubunit movement during translocation is lacking. To address this problem, we created an intersubunit disulfide cross-link to restrict potential movement. The cross-linked ribosomes were unable to carry out polypeptide synthesis; this inhibition was completely reversed upon reduction of the disulfide bridge. In vitro assays showed that the cross-linked ribosomes were specifically blocked in elongation factor G-dependent translocation. These findings show that intersubunit movement is required for ribosomal translocation, accounting for the universal two-subunit architecture of ribosomes. PMID:17360328

  19. The ribosomal RNA transcription unit of Entamoeba invadens: accumulation of unprocessed pre-rRNA and a long non coding RNA during encystation.

    PubMed

    Ojha, Sandeep; Singh, Nishant; Bhattacharya, Alok; Bhattacharya, Sudha

    2013-01-01

    The ribosomal RNA genes in Entamoeba spp. are located on extrachromosomal circular molecules. Unlike model organisms where rRNA transcription stops during growth stress, Entamoeba histolytica continues transcription; but unprocessed pre-rRNA accumulates during stress, along with a novel class of circular transcripts from the 5'-external transcribed spacer (ETS). To determine the fate of rRNA transcription during stage conversion between trophozoite to cyst we analyzed Entamoeba invadens, a model system for differentiation studies in Entamoeba. We characterized the complete rDNA transcription unit by mapping the ends of pre-rRNA and mature rRNAs. The 3' end of mature 28S rRNA was located 321 nt downstream of the end predicted by sequence homology with E. histolytica. The major processing sites were mapped in external and internal transcribed spacers. The promoter located within 146 nt upstream of 5' ETS was used to transcribe the pre-rRNA. On the other hand, a second promoter located at the 3' end of 28S rDNA was used to transcribe almost the entire intergenic spacer into a long non coding (nc) RNA (>10 kb). Interestingly we found that the levels of pre-rRNA and long ncRNA, measured by northern hybridization, decreased initially in cells shifted to encystation medium, after which they began to increase and reached high levels by 72 h when mature cysts were formed. Unlike E. histolytica, no circular transcripts were found in E. invadens. E. histolytica and E. invadens express fundamentally different ncRNAs from the rDNA locus, which may reflect their adaptation to different hosts (human and reptiles, respectively). This is the first description of rDNA organization and transcription in E. invadens, and provides the framework for further studies on regulation of rRNA synthesis during cyst formation. PMID:24200639

  20. The importance of ribosome production, and the 5S RNP–MDM2 pathway, in health and disease

    PubMed Central

    Pelava, Andria; Schneider, Claudia; Watkins, Nicholas J.

    2016-01-01

    Ribosomes are abundant, large RNA–protein complexes that are the source of all protein synthesis in the cell. The production of ribosomes is an extremely energetically expensive cellular process that has long been linked to human health and disease. More recently, it has been shown that ribosome biogenesis is intimately linked to multiple cellular signalling pathways and that defects in ribosome production can lead to a wide variety of human diseases. Furthermore, changes in ribosome production in response to nutrient levels in the diet lead to metabolic re-programming of the liver. Reduced or abnormal ribosome production in response to cellular stress or mutations in genes encoding factors critical for ribosome biogenesis causes the activation of the tumour suppressor p53, which leads to re-programming of cellular transcription. The ribosomal assembly intermediate 5S RNP (ribonucleoprotein particle), containing RPL5, RPL11 and the 5S rRNA, accumulates when ribosome biogenesis is blocked. The excess 5S RNP binds to murine double minute 2 (MDM2), the main p53-suppressor in the cell, inhibiting its function and leading to p53 activation. Here, we discuss the involvement of ribosome biogenesis in the homoeostasis of p53 in the cell and in human health and disease. PMID:27528756

  1. The importance of ribosome production, and the 5S RNP-MDM2 pathway, in health and disease.

    PubMed

    Pelava, Andria; Schneider, Claudia; Watkins, Nicholas J

    2016-08-15

    Ribosomes are abundant, large RNA-protein complexes that are the source of all protein synthesis in the cell. The production of ribosomes is an extremely energetically expensive cellular process that has long been linked to human health and disease. More recently, it has been shown that ribosome biogenesis is intimately linked to multiple cellular signalling pathways and that defects in ribosome production can lead to a wide variety of human diseases. Furthermore, changes in ribosome production in response to nutrient levels in the diet lead to metabolic re-programming of the liver. Reduced or abnormal ribosome production in response to cellular stress or mutations in genes encoding factors critical for ribosome biogenesis causes the activation of the tumour suppressor p53, which leads to re-programming of cellular transcription. The ribosomal assembly intermediate 5S RNP (ribonucleoprotein particle), containing RPL5, RPL11 and the 5S rRNA, accumulates when ribosome biogenesis is blocked. The excess 5S RNP binds to murine double minute 2 (MDM2), the main p53-suppressor in the cell, inhibiting its function and leading to p53 activation. Here, we discuss the involvement of ribosome biogenesis in the homoeostasis of p53 in the cell and in human health and disease. PMID:27528756

  2. Maize reas1 Mutant Stimulates Ribosome Use Efficiency and Triggers Distinct Transcriptional and Translational Responses1[OPEN

    PubMed Central

    Qi, Weiwei; Zhu, Jie; Wu, Qiao; Wang, Qun; Li, Xia; Yao, Dongsheng; Jin, Ying; Wang, Gang; Wang, Guifeng

    2016-01-01

    Ribosome biogenesis is a fundamental cellular process in all cells. Impaired ribosome biogenesis causes developmental defects; however, its molecular and cellular bases are not fully understood. We cloned a gene responsible for a maize (Zea mays) small seed mutant, dek* (for defective kernel), and found that it encodes Ribosome export associated1 (ZmReas1). Reas1 is an AAA-ATPase that controls 60S ribosome export from the nucleus to the cytoplasm after ribosome maturation. dek* is a weak mutant allele with decreased Reas1 function. In dek* cells, mature 60S ribosome subunits are reduced in the nucleus and cytoplasm, but the proportion of actively translating polyribosomes in cytosol is significantly increased. Reduced phosphorylation of eukaryotic initiation factor 2α and the increased elongation factor 1α level indicate an enhancement of general translational efficiency in dek* cells. The mutation also triggers dramatic changes in differentially transcribed genes and differentially translated RNAs. Discrepancy was observed between differentially transcribed genes and differentially translated RNAs, indicating distinct cellular responses at transcription and translation levels to the stress of defective ribosome processing. DNA replication and nucleosome assembly-related gene expression are selectively suppressed at the translational level, resulting in inhibited cell growth and proliferation in dek* cells. This study provides insight into cellular responses due to impaired ribosome biogenesis. PMID:26645456

  3. Karyotypic Variability in Ribosomal DNA Subchromosome Size among Colpodid Ciliates, a Possible Tool To Differentiate Colpodid Species

    PubMed Central

    Martin, A.; Palacios, G.; Olmo, A.; Martin-Gonzalez, A.; Ruiz-Perez, L. M.; Gutierrez, J. C.

    1997-01-01

    Pulsed-field gel electrophoresis has been applied to analyze the karyotypic variability among colpodid ciliates. The 18S ribosomal gene was found at different locations in the electrophoretic pattern, and these size variations in the ribosomal DNA subchromosomal molecule seem to be species specific. This could potentially be a useful new tool with which to differentiate colpodid ciliates. PMID:16535582

  4. Ribosomal crystallography: from crystal growth to initial phasing

    NASA Astrophysics Data System (ADS)

    Thygesen, J.; Krumbholz, S.; Levin, I.; Zaytzev-Bashan, A.; Harms, J.; Bartels, H.; Schlünzen, F.; Hansen, H. A. S.; Bennett, W. S.; Volkmann, N.; Agmon, I.; Eisenstein, M.; Dribin, A.; Maltz, E.; Sagi, I.; Morlang, S.; Fua, M.; Franceschi, F.; Weinstein, S.; Böddeker, N.; Sharon, R.; Anagnostopoulos, K.; Peretz, M.; Geva, M.; Berkovitch-Yellin, Z.; Yonath, A.

    1996-10-01

    Preliminary phases were determined by the application of the isomorphous replacement method at low and intermediate resolution for structure factor amplitudes collected from crystals of large and small ribosomal subunits from halophilic and thermophilic bacteria. Derivatization was performed with dense heavy atom clusters, either by soaking or by specific covalent binding prior to the crystallization. The resulting initial electron density maps contain features comparable in size to those expected for the corresponding particles. The packing arrangements of these maps have been compared with motifs observed by electron microscopy in positively stained thin sections of embedded three-dimensional crystals, as well as with phase sets obtained by ab-initio computations. Aimed at higher resolution phasing, procedures are being developed for multi-site binding of relatively small dense metal clusters at selected locations. Potential sites are being inserted either by mutagenesis or by chemical modifications to facilitate cluster binding to the large halophilic and the small thermophilic ribosomal subunits which yield crystals diffracting to the highest resolution obtained so far for ribosomes, 2.9 and 7.3 Å, respectively. For this purpose the surfaces of these ribosomal particles have been characterized and conditions for quantitative reversible detachment of selected ribosomal proteins have been found. The corresponding genes are being cloned, sequenced, mutated to introduce the reactive side-groups (mainly cysteines) and overexpressed. To assist the interpretation of the anticipated electron density maps, sub-ribosomal stable complexes were isolated from H50S. One of these complexes is composed of two proteins and the other is made of a stretch of the rRNA and a protein. For exploiting the exposed parts of the surface of these complexes for heavy atom binding and for attempting the determination of their three-dimensional structure, their components are being produced

  5. Origins of the plant chloroplasts and mitochondria based on comparisons of 5S ribosomal RNAs

    NASA Technical Reports Server (NTRS)

    Delihas, N.; Fox, G. E.

    1987-01-01

    In this paper, we provide macromolecular comparisons utilizing the 5S ribosomal RNA structure to suggest extant bacteria that are the likely descendants of chloroplast and mitochondria endosymbionts. The genetic stability and near universality of the 5S ribosomal gene allows for a useful means to study ancient evolutionary changes by macromolecular comparisons. The value in current and future ribosomal RNA comparisons is in fine tuning the assignment of ancestors to the organelles and in establishing extant species likely to be descendants of bacteria involved in presumed multiple endosymbiotic events.

  6. Ribosomal Protein Rps26 Influences 80S Ribosome Assembly in Saccharomyces cerevisiae

    PubMed Central

    Belyy, Alexander; Levanova, Nadezhda; Tabakova, Irina; Rospert, Sabine

    2016-01-01

    ABSTRACT The eukaryotic ribosome consists of a small (40S) and a large (60S) subunit. Rps26 is one of the essential ribosomal proteins of the 40S subunit and is encoded by two almost identical genes, RPS26a and RPS26b. Previous studies demonstrated that Rps26 interacts with the 5′ untranslated region of mRNA via the eukaryote-specific 62-YXXPKXYXK-70 (Y62–K70) motif. Those observations suggested that this peptide within Rps26 might play an important and specific role during translation initiation. By using alanine-scanning mutagenesis and engineered strains of the yeast Saccharomyces cerevisiae, we found that single amino acid substitutions within the Y62–K70 motif of Rps26 did not affect the in vivo function of the protein. In contrast, complete deletion of the Y62–K70 segment was lethal. The simultaneous replacement of five conserved residues within the Y62–K70 segment by alanines resulted in growth defects under stress conditions and produced distinct changes in polysome profiles that were indicative of the accumulation of free 60S subunits. Human Rps26 (Rps26-Hs), which displays significant homology with yeast Rps26, supported the growth of an S. cerevisiae Δrps26a Δrps26b strain. However, the Δrps26a Δrps26b double deletion strain expressing Rps26-Hs displayed substantial growth defects and an altered ratio of 40S/60S ribosomal subunits. The combined data strongly suggest that the eukaryote-specific motif within Rps26 does not play a specific role in translation initiation. Rather, the data indicate that Rps26 as a whole is necessary for proper assembly of the 40S subunit and the 80S ribosome in yeast. IMPORTANCE Rps26 is an essential protein of the eukaryotic small ribosomal subunit. Previous experiments demonstrated an interaction between the eukaryote-specific Y62–K70 segment of Rps26 and the 5′ untranslated region of mRNA. The data suggested a specific role of the Y62–K70 motif during translation initiation. Here, we report that single

  7. Neutron scattering in the ribosome structure

    NASA Astrophysics Data System (ADS)

    Serdyuk, Igor N.

    1997-02-01

    Thermal neutron scattering has become a powerful instrument for studying the ribosome and its components. The application of neutron scattering allowed to establish some principal features of the ribosome structure: non-homogeneous distribution of the RNA and protein within ribosomal particles, the RNA role as a framework in the arrangement and maintenance of the structure of ribosomal particles, and the globular character of ribosomal proteins. The use of selective deuteration of separate ribosomal proteins in combination with the triangulation method revealed mutual spatial arrangement (the 3D-map) of all the ribosomal proteins within the small particle and in the most part of the large ribosomal particle. An essential impact has been made in the structural studies of ribosomes with the development of novel experimental approaches: triple isotopic substitution and spin contrast variation. These approaches with direct interpretation of spherical harmonics provide new possibilities for constructing models of ribosomal particles, opening principally new perspectives for joint use of X-ray synchrotron diffraction in crystals and small-angle neutron scattering in solution.

  8. Alveolate phylogeny inferred using concatenated ribosomal proteins.

    PubMed

    Bachvaroff, Tsvetan R; Handy, Sara M; Place, Allen R; Delwiche, Charles F

    2011-01-01

    Dinoflagellates and apicomplexans are a strongly supported monophyletic group in rDNA phylogenies, although this phylogeny is not without controversy, particularly between the two groups. Here we use concatenated protein-coding genes from expressed sequence tags or genomic data to construct phylogenies including "typical" dinophycean dinoflagellates, a parasitic syndinian dinoflagellate, Amoebophrya sp., and two related species, Oxyrrhis marina, and Perkinsus marinus. Seventeen genes encoding proteins associated with the ribosome were selected for phylogenetic analysis. The dataset was limited for the most part by data availability from the dinoflagellates. Forty-five taxa from four major lineages were used: the heterokont outgroup, ciliates, dinoflagellates, and apicomplexans. Amoebophrya sp. was included in this phylogeny as a sole representative of the enigmatic marine alveolate or syndinian lineage. The atypical dinoflagellate O. marina, usually excluded from rDNA analyses due to long branches, was also included. The resulting phylogenies were well supported in concatenated analyses with only a few unstable or weakly supported branches; most features were consistent when different lineages were pruned from the tree or different genes were concatenated. The least stable branches involved the placement of Cryptosporidium spp. within the Apicomplexa and the relationships between P. marinus, Amoebophrya sp., and O. marina. Both bootstrap and approximately unbiased test results confirmed that P. marinus, Amoebophrya sp., O. marina, and the remaining dinoflagellates form a monophyletic lineage to the exclusion of Apicomplexa. PMID:21518081

  9. Dynamic evolution of mitochondrial ribosomal proteins in Holozoa.

    PubMed

    Scheel, Bettina M; Hausdorf, Bernhard

    2014-07-01

    We studied the highly dynamic evolution of mitochondrial ribosomal proteins (MRPs) in Holozoa. Most major clades within Holozoa are characterized by gains and/or losses of MRPs. The usefulness of gains of MRPs as rare genomic changes in phylogenetics is undermined by the high frequency of secondary losses. However, phylogenetic analyses of the MRP sequences provide evidence for the Acrosomata hypothesis, a sister group relationship between Ctenophora and Bilateria. An extensive restructuring of the mitochondrial genome and, as a consequence, of the mitochondrial ribosomes occurred in the ancestor of metazoans. The last MRP genes encoded in the mitochondrial genome were either moved to the nuclear genome or were lost. The strong decrease in size of the mitochondrial genome was probably caused by selection for rapid replication of mitochondrial DNA during oogenesis in the metazoan ancestor. A phylogenetic analysis of MRPL56 sequences provided evidence for a horizontal gene transfer of the corresponding MRP gene between metazoans and Dictyostelidae (Amoebozoa). The hypothesis that the requisition of additional MRPs compensated for a loss of rRNA segments in the mitochondrial ribosomes is corroborated by a significant negative correlation between the number of MRPs and length of the rRNA. Newly acquired MRPs evolved faster than bacterial MRPs and positions in eukaryote-specific MRPs were more strongly affected by coevolution than positions in prokaryotic MRPs in accordance with the necessity to fit these proteins into the pre-existing structure of the mitoribosome. PMID:24631858

  10. Detecting actively translated open reading frames in ribosome profiling data.

    PubMed

    Calviello, Lorenzo; Mukherjee, Neelanjan; Wyler, Emanuel; Zauber, Henrik; Hirsekorn, Antje; Selbach, Matthias; Landthaler, Markus; Obermayer, Benedikt; Ohler, Uwe

    2016-02-01

    RNA-sequencing protocols can quantify gene expression regulation from transcription to protein synthesis. Ribosome profiling (Ribo-seq) maps the positions of translating ribosomes over the entire transcriptome. We have developed RiboTaper (available at https://ohlerlab.mdc-berlin.de/software/), a rigorous statistical approach that identifies translated regions on the basis of the characteristic three-nucleotide periodicity of Ribo-seq data. We used RiboTaper with deep Ribo-seq data from HEK293 cells to derive an extensive map of translation that covered open reading frame (ORF) annotations for more than 11,000 protein-coding genes. We also found distinct ribosomal signatures for several hundred upstream ORFs and ORFs in annotated noncoding genes (ncORFs). Mass spectrometry data confirmed that RiboTaper achieved excellent coverage of the cellular proteome. Although dozens of novel peptide products were validated in this manner, few of the currently annotated long noncoding RNAs appeared to encode stable polypeptides. RiboTaper is a powerful method for comprehensive de novo identification of actively used ORFs from Ribo-seq data. PMID:26657557

  11. Structural Insights Into Ribosome Recycling Factor Interactions with the 70S Ribosome

    PubMed Central

    Pai, Raj D.; Zhang, Wen; Schuwirth, Barbara S.; Hirokawa, Go; Kaji, Hideko; Kaji, Akira; Cate, Jamie H.D.

    2009-01-01

    SUMMARY At the end of translation in bacteria, ribosome recycling factor (RRF) is used together with Elongation Factor G (EF-G) to recycle the 30S and 50S ribosomal subunits for the next round of translation. In x-ray crystal structures of RRF with the Escherichia coli 70S ribosome, RRF binds to the large ribosomal subunit in the cleft that contains the peptidyl transferase center (PTC). Upon binding of either E. coli or T. thermophilus RRF to the E. coli ribosome, the tip of ribosomal RNA helix H69 in the large subunit moves away from the small subunit toward RRF by 8 Å, thereby disrupting a key contact between the small and large ribosomal subunits, termed bridge B2a. In the ribosome crystals, the ability of RRF to destabilize bridge B2a is influenced by crystal packing forces. Movement of H69 involves an ordered to disordered transition upon binding of RRF to the ribosome. The disruption of bridge B2a upon RRF binding to the ribosome seen in the present structures reveals one of the key roles that RRF plays in ribosome recycling, the dissociation of 70S ribosomes into subunits. The structures also reveal contacts between Domain II of RRF and protein S12 in the 30S subunit that may also play a role in ribosome recycling. PMID:18234219

  12. Mechanisms for ribotoxin-induced ribosomal RNA cleavage

    SciTech Connect

    He, Kaiyu; Zhou, Hui-Ren; Pestka, James J.

    2012-11-15

    The Type B trichothecene deoxynivalenol (DON), a ribotoxic mycotoxin known to contaminate cereal-based foods, induces ribosomal RNA (rRNA) cleavage in the macrophage via p38-directed activation of caspases. Here we employed the RAW 264.7 murine macrophage model to test the hypothesis that this rRNA cleavage pathway is similarly induced by other ribotoxins. Capillary electrophoresis confirmed that the antibiotic anisomycin (≥ 25 ng/ml), the macrocylic trichothecene satratoxin G (SG) (≥ 10 ng/ml) and ribosome-inactivating protein ricin (≥ 300 ng/ml) induced 18s and 28s rRNA fragmentation patterns identical to that observed for DON. Also, as found for DON, inhibition of p38, double-stranded RNA-activated kinase (PKR) and hematopoietic cell kinase (Hck) suppressed MAPK anisomycin-induced rRNA cleavage, while, in contrast, their inhibition did not affect SG- and ricin-induced rRNA fragmentation. The p53 inhibitor pifithrin-μ and pan caspase inhibitor Z-VAD-FMK suppressed rRNA cleavage induced by anisomycin, SG and ricin, indicating that these ribotoxins shared with DON a conserved downstream pathway. Activation of caspases 8, 9 and 3 concurrently with apoptosis further suggested that rRNA cleavage occurred in parallel with both extrinsic and intrinsic pathways of programmed cell death. When specific inhibitors of cathepsins L and B (lysosomal cysteine cathepsins active at cytosolic neutral pH) were tested, only the former impaired anisomycin-, SG-, ricin- and DON-induced rRNA cleavage. Taken together, the data suggest that (1) all four ribotoxins induced p53-dependent rRNA cleavage via activation of cathepsin L and caspase 3, and (2) activation of p53 by DON and anisomycin involved p38 whereas SG and ricin activated p53 by an alternative mechanism. Highlights: ► Deoxynivalenol (DON) anisomycin, satratoxin G (SG) and ricin are ribotoxins. ► Ribotoxins induce 18s and 28s rRNA cleavage in the RAW 264.7 macrophage model. ► Ribotoxins induce rRNA cleavage via

  13. Control of ribosome formation in rat heart

    SciTech Connect

    Russo, L.A.

    1987-01-01

    Diabetes of 9 days duration produced a 17% diminution in the rate of total protein synthesis in rat hearts perfused as Langendorff preparations supplied with glucose, plasma levels of amino acids, and 400 ..mu..U/ml insulin. This reduction was attributable to a decrease in efficiency of protein synthesis and total RNA content. Total messenger RNA content decreased in diabetic hearts in proportion to the reduction in total RNA. Diabetes also resulted in diminished ribosome content as reflected by the induction in total RNA. Ribosome production was investigated by monitoring incorporation of (/sup 3/H)phenylalanine into the proteins of cytoplasmic ribosomes. Rates of ribosome formation in diabetic hearts were as fast as control rates in the presence of insulin, and were faster than control rates in the absence of the hormone. These results indicated that ribosome content fell in diabetic hearts despite unchanged or faster rates of ribosome formation.

  14. Seeing is Believing in Ribosome Assembly.

    PubMed

    Warner, Jonathan R

    2016-07-14

    Many proteins have been implicated genetically and biochemically in the assembly of eukaryotic ribosomes. Now, Kornprobst et al. show us how they are put together with a cryoEM structure of the 90S processome that initiates ribosome assembly, revealing the arrangement of U3 RNA and the several UTP complexes that form a chaperone-like structure around and within the developing 40S ribosomal subunit. PMID:27419867

  15. Ribosome biogenesis in replicating cells: Integration of experiment and theory.

    PubMed

    Earnest, Tyler M; Cole, John A; Peterson, Joseph R; Hallock, Michael J; Kuhlman, Thomas E; Luthey-Schulten, Zaida

    2016-10-01

    Ribosomes-the primary macromolecular machines responsible for translating the genetic code into proteins-are complexes of precisely folded RNA and proteins. The ways in which their production and assembly are managed by the living cell is of deep biological importance. Here we extend a recent spatially resolved whole-cell model of ribosome biogenesis in a fixed volume [Earnest et al., Biophys J 2015, 109, 1117-1135] to include the effects of growth, DNA replication, and cell division. All biological processes are described in terms of reaction-diffusion master equations and solved stochastically using the Lattice Microbes simulation software. In order to determine the replication parameters, we construct and analyze a series of Escherichia coli strains with fluorescently labeled genes distributed evenly throughout their chromosomes. By measuring these cells' lengths and number of gene copies at the single-cell level, we could fit a statistical model of the initiation and duration of chromosome replication. We found that for our slow-growing (120 min doubling time) E. coli cells, replication was initiated 42 min into the cell cycle and completed after an additional 42 min. While simulations of the biogenesis model produce the correct ribosome and mRNA counts over the cell cycle, the kinetic parameters for transcription and degradation are lower than anticipated from a recent analytical time dependent model of in vivo mRNA production. Describing expression in terms of a simple chemical master equation, we show that the discrepancies are due to the lack of nonribosomal genes in the extended biogenesis model which effects the competition of mRNA for ribosome binding, and suggest corrections to parameters to be used in the whole-cell model when modeling expression of the entire transcriptome. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 735-751, 2016. PMID:27294303

  16. Ribosome biogenesis in the yeast Saccharomyces cerevisiae.

    PubMed

    Woolford, John L; Baserga, Susan J

    2013-11-01

    Ribosomes are highly conserved ribonucleoprotein nanomachines that translate information in the genome to create the proteome in all cells. In yeast these complex particles contain four RNAs (>5400 nucleotides) and 79 different proteins. During the past 25 years, studies in yeast have led the way to understanding how these molecules are assembled into ribosomes in vivo. Assembly begins with transcription of ribosomal RNA in the nucleolus, where the RNA then undergoes complex pathways of folding, coupled with nucleotide modification, removal of spacer sequences, and binding to ribosomal proteins. More than 200 assembly factors and 76 small nucleolar RNAs transiently associate with assembling ribosomes, to enable their accurate and efficient construction. Following export of preribosomes from the nucleus to the cytoplasm, they undergo final stages of maturation before entering the pool of functioning ribosomes. Elaborate mechanisms exist to monitor the formation of correct structural and functional neighborhoods within ribosomes and to destroy preribosomes that fail to assemble properly. Studies of yeast ribosome biogenesis provide useful models for ribosomopathies, diseases in humans that result from failure to properly assemble ribosomes. PMID:24190922

  17. Tricks an IRES uses to enslave ribosomes

    PubMed Central

    2012-01-01

    In eukaryotes, mRNAs are primarily translated through a cap-dependent mechanism whereby initiation factors recruit the 40S ribosomal subunit to a cap structure at the 5’ end of the mRNA. However, some viral and cellular messages initiate protein synthesis without a cap. They use a structured RNA element termed an internal ribosome entry site (IRES) to recruit the 40S ribosomal subunit. IRESs were discovered over 20 years ago but only recently have studies using a model IRES from dicistroviruses expanded our understanding of how a three dimensional RNA structure can capture and manipulate the ribosome to initiate translation. PMID:22944245

  18. Scattering studies on ribosomes in solution

    NASA Astrophysics Data System (ADS)

    Ramakrishnan, V.

    1986-02-01

    Ribosomes are organelles that play a central role in protein synthesis. They are complexes of protein and nucleic acid, and can be analysed as two-component systems by neutron scattering. Moreover, ribosomes can be biochemically prepared that have specific proteins deuterated. Both these properties have been exploited to study the structure of the ribosome by neutron scattering. This article reviews the studies carried out on the small ribosomal subunit, and describes a recent study that has resolved a conflict between the results of two classes of experiments.

  19. Most RNAs regulating ribosomal protein biosynthesis in Escherichia coli are narrowly distributed to Gammaproteobacteria

    PubMed Central

    Fu, Yang; Deiorio-Haggar, Kaila; Anthony, Jon; Meyer, Michelle M.

    2013-01-01

    In Escherichia coli, 12 distinct RNA structures within the transcripts encoding ribosomal proteins interact with specific ribosomal proteins to allow autogenous regulation of expression from large multi-gene operons, thus coordinating ribosomal protein biosynthesis across multiple operons. However, these RNA structures are typically not represented in the RNA Families Database or annotated in genomic sequences databases, and their phylogenetic distribution is largely unknown. To investigate the extent to which these RNA structures are conserved across eubacterial phyla, we created multiple sequence alignments representing 10 of these messenger RNA (mRNA) structures in E. coli. We find that while three RNA structures are widely distributed across many phyla of bacteria, seven of the RNAs are narrowly distributed to a few orders of Gammaproteobacteria. To experimentally validate our computational predictions, we biochemically confirmed dual L1-binding sites identified in many Firmicute species. This work reveals that RNA-based regulation of ribosomal protein biosynthesis is used in nearly all eubacterial phyla, but the specific RNA structures that regulate ribosomal protein biosynthesis in E. coli are narrowly distributed. These results highlight the limits of our knowledge regarding ribosomal protein biosynthesis regulation outside of E. coli, and the potential for alternative RNA structures responsible for regulating ribosomal proteins in other eubacteria. PMID:23396277

  20. Cap-dependent translation is mediated by 'RNA looping' rather than 'ribosome scanning'.

    PubMed

    Jang, Sung Key; Paek, Ki Young

    2016-01-01

    The 40S ribosomal subunit cannot directly recognize the start codon of eukaryotic mRNAs. Instead, it recognizes the start codon after its association with the 5'-cap structure via translation initiation factors. Base-by-base inspection of the 5'UTR by a scanning ribosome is the generally accepted hypothesis of start codon selection. As part of an effort to confirm the underlying mechanism of start codon selection by the 40S ribosome, we investigated the role of eIF4G, which participates in the recruitment of 40S ribosomes to various translation enhancers, such as 5'-cap structure, poly(A) tail, and several internal ribosome entry sites. We found that an artificial translation factor composed of recombinant eIF4G fused with MS2 greatly enhanced translation of an upstream reporter gene when it was tethered to the 3'UTR. These data suggest that the 40S ribosome recruited to a translation enhancer can find the start codon by looping of the intervening RNA segment. The 'RNA-looping' hypothesis of translation start codon recognition was further supported by an analysis of the effect of 5'UTR length on translation efficiency and the mathematically predicted probability of RNA-loop-mediated interactions between the start codon and the 40S ribosome associated at the 5'-end. PMID:26515582

  1. Mechanisms for ribotoxin-induced ribosomal RNA cleavage.

    PubMed

    He, Kaiyu; Zhou, Hui-Ren; Pestka, James J

    2012-11-15

    The Type B trichothecene deoxynivalenol (DON), a ribotoxic mycotoxin known to contaminate cereal-based foods, induces ribosomal RNA (rRNA) cleavage in the macrophage via p38-directed activation of caspases. Here we employed the RAW 264.7 murine macrophage model to test the hypothesis that this rRNA cleavage pathway is similarly induced by other ribotoxins. Capillary electrophoresis confirmed that the antibiotic anisomycin (≥25ng/ml), the macrocylic trichothecene satratoxin G (SG) (≥10ng/ml) and ribosome-inactivating protein ricin (≥300ng/ml) induced 18s and 28s rRNA fragmentation patterns identical to that observed for DON. Also, as found for DON, inhibition of p38, double-stranded RNA-activated kinase (PKR) and hematopoietic cell kinase (Hck) suppressed MAPK anisomycin-induced rRNA cleavage, while, in contrast, their inhibition did not affect SG- and ricin-induced rRNA fragmentation. The p53 inhibitor pifithrin-μ and pan caspase inhibitor Z-VAD-FMK suppressed rRNA cleavage induced by anisomycin, SG and ricin, indicating that these ribotoxins shared with DON a conserved downstream pathway. Activation of caspases 8, 9 and 3 concurrently with apoptosis further suggested that rRNA cleavage occurred in parallel with both extrinsic and intrinsic pathways of programmed cell death. When specific inhibitors of cathepsins L and B (lysosomal cysteine cathepsins active at cytosolic neutral pH) were tested, only the former impaired anisomycin-, SG-, ricin- and DON-induced rRNA cleavage. Taken together, the data suggest that (1) all four ribotoxins induced p53-dependent rRNA cleavage via activation of cathepsin L and caspase 3, and (2) activation of p53 by DON and anisomycin involved p38 whereas SG and ricin activated p53 by an alternative mechanism. PMID:23022514

  2. Mechanisms for Ribotoxin-induced Ribosomal RNA Cleavage

    PubMed Central

    He, Kaiyu; Zhou, Hui-Ren; Pestka, James J.

    2012-01-01

    The Type B trichothecene deoxynivalenol (DON), a ribotoxic mycotoxin known to contaminate cereal-based foods, induces ribosomal RNA (rRNA) cleavage in the macrophage via p38-directed activation of caspases. Here we employed the RAW 264.7 murine macrophage model to test the hypothesis that this rRNA cleavage pathway is similarly induced by other ribotoxins. Capillary electrophoresis confirmed that the antibiotic anisomycin (≥25 ng/ml), the macrocylic trichothecene satratoxin G (SG) (≥10 ng/ml) and ribosome-inactivating protein ricin (≥300 ng/ml) induced 18s and 28s rRNA fragmentation patterns identical to that observed for DON. Also, as found for DON, inhibition of p38, double-stranded RNA-activated kinase (PKR) and hematopoietic cell kinase (Hck) suppressed MAPK anisomycin-induced rRNA cleavage, while, in contrast, their inhibition did not affect SG- and ricin-induced rRNA fragmentation. The p53 inhibitor pifithrin-μ and pan caspase inhibitor Z-VAD-FMK suppressed rRNA cleavage induced by anisomycin, SG and ricin, indicating that these ribotoxins shared with DON a conserved downstream pathway. Activation of caspase 8, 9 and 3 concurrently with apoptosis further suggested rRNA cleavage occurred in parallel with both extrinsic and intrinsic pathways of programmed cell death. When specific inhibitors cathepsin L and B (lysosomal cysteine cathepsins active at cytosolic neutral pH) were tested, only the former impaired anisomycin-, SG-, ricin- and DON-induced rRNA cleavage. Taken together, the data suggest that (1) all four ribotoxins induced p53-dependent rRNA cleavage via activation of cathepsin L and caspase 3, and (2) activation of p53 by DON and anisomycin involved p38 whereas SG and ricin activated p53 by an alternative mechanism. PMID:23022514

  3. A model for competition for ribosomes in the cell.

    PubMed

    Raveh, Alon; Margaliot, Michael; Sontag, Eduardo D; Tuller, Tamir

    2016-03-01

    A single mammalian cell includes an order of 10(4)-10(5) mRNA molecules and as many as 10(5)-10(6) ribosomes. Large-scale simultaneous mRNA translation induces correlations between the mRNA molecules, as they all compete for the finite pool of available ribosomes. This has important implications for the cell's functioning and evolution. Developing a better understanding of the intricate correlations between these simultaneous processes, rather than focusing on the translation of a single isolated transcript, should help in gaining a better understanding of mRNA translation regulation and the way elongation rates affect organismal fitness. A model of simultaneous translation is specifically important when dealing with highly expressed genes, as these consume more resources. In addition, such a model can lead to more accurate predictions that are needed in the interconnection of translational modules in synthetic biology. We develop and analyse a general dynamical model for large-scale simultaneous mRNA translation and competition for ribosomes. This is based on combining several ribosome flow models (RFMs) interconnected via a pool of free ribosomes. We use this model to explore the interactions between the various mRNA molecules and ribosomes at steady state. We show that the compound system always converges to a steady state and that it always entrains or phase locks to periodically time-varying transition rates in any of the mRNA molecules. We then study the effect of changing the transition rates in one mRNA molecule on the steady-state translation rates of the other mRNAs that results from the competition for ribosomes. We show that increasing any of the codon translation rates in a specific mRNA molecule yields a local effect, an increase in the translation rate of this mRNA, and also a global effect, the translation rates in the other mRNA molecules all increase or all decrease. These results suggest that the effect of codon decoding rates of endogenous and

  4. Coupling of mRNA Structure Rearrangement to Ribosome Movement during Bypassing of Non-coding Regions.

    PubMed

    Chen, Jin; Coakley, Arthur; O'Connor, Michelle; Petrov, Alexey; O'Leary, Seán E; Atkins, John F; Puglisi, Joseph D

    2015-11-19

    Nearly half of the ribosomes translating a particular bacteriophage T4 mRNA bypass a region of 50 nt, resuming translation 3' of this gap. How this large-scale, specific hop occurs and what determines whether a ribosome bypasses remain unclear. We apply single-molecule fluorescence with zero-mode waveguides to track individual Escherichia coli ribosomes during translation of T4's gene 60 mRNA. Ribosomes that bypass are characterized by a 10- to 20-fold longer pause in a non-canonical rotated state at the take-off codon. During the pause, mRNA secondary structure rearrangements are coupled to ribosome forward movement, facilitated by nascent peptide interactions that disengage the ribosome anticodon-codon interactions for slippage. Close to the landing site, the ribosome then scans mRNA in search of optimal base-pairing interactions. Our results provide a mechanistic and conformational framework for bypassing, highlighting a non-canonical ribosomal state to allow for mRNA structure refolding to drive large-scale ribosome movements. PMID:26590426

  5. Ribosomal protein methyltransferases in the yeast Saccharomyces cerevisiae: Roles in ribosome biogenesis and translation.

    PubMed

    Al-Hadid, Qais; White, Jonelle; Clarke, Steven

    2016-02-12

    A significant percentage of the methyltransferasome in Saccharomyces cerevisiae and higher eukaryotes is devoted to methylation of the translational machinery. Methylation of the RNA components of the translational machinery has been studied extensively and is important for structure stability, ribosome biogenesis, and translational fidelity. However, the functional effects of ribosomal protein methylation by their cognate methyltransferases are still largely unknown. Previous work has shown that the ribosomal protein Rpl3 methyltransferase, histidine protein methyltransferase 1 (Hpm1), is important for ribosome biogenesis and translation elongation fidelity. In this study, yeast strains deficient in each of the ten ribosomal protein methyltransferases in S. cerevisiae were examined for potential defects in ribosome biogenesis and translation. Like Hpm1-deficient cells, loss of four of the nine other ribosomal protein methyltransferases resulted in defects in ribosomal subunit synthesis. All of the mutant strains exhibited resistance to the ribosome inhibitors anisomycin and/or cycloheximide in plate assays, but not in liquid culture. Translational fidelity assays measuring stop codon readthrough, amino acid misincorporation, and programmed -1 ribosomal frameshifting, revealed that eight of the ten enzymes are important for translation elongation fidelity and the remaining two are necessary for translation termination efficiency. Altogether, these results demonstrate that ribosomal protein methyltransferases in S. cerevisiae play important roles in ribosome biogenesis and translation. PMID:26801560

  6. Ribosome Mechanics Informs about Mechanism.

    PubMed

    Zimmermann, Michael T; Jia, Kejue; Jernigan, Robert L

    2016-02-27

    The essential aspects of the ribosome's mechanism can be extracted from coarse-grained simulations, including the ratchet motion, the movement together of critical bases at the decoding center, and movements of the peptide tunnel lining that assist in the expulsion of the synthesized peptide. Because of its large size, coarse graining helps to simplify and to aid in the understanding of its mechanism. Results presented here utilize coarse-grained elastic network modeling to extract the dynamics, and both RNAs and proteins are coarse grained. We review our previous results, showing the well-known ratchet motions and the motions in the peptide tunnel and in the mRNA tunnel. The motions of the lining of the peptide tunnel appear to assist in the expulsion of the growing peptide chain, and clamps at the ends of the mRNA tunnel with three proteins ensure that the mRNA is held tightly during decoding and essential for the helicase activity at the entrance. The entry clamp may also assist in base recognition to ensure proper selection of the incoming tRNA. The overall precision of the ribosome machine-like motions is remarkable. PMID:26687034

  7. Evaluation of phylogenetic relationships in Lemnaceae using nuclear ribosomal data.

    PubMed

    Tippery, N P; Les, D H; Crawford, D J

    2015-01-01

    Nuclear DNA sequence data are essential for obtaining a complete understanding of plant species relationships, yet these data have been conspicuously absent from phylogenetic analyses of Lemnaceae (duckweeds). Using a modified Sanger sequencing protocol, we obtained DNA sequences of duckweed nuclear ribosomal regions, including 18S and 26S rDNA genes, the external transcribed spacer (ETS) and the frequently used internal transcribed spacer (ITS). After obtaining sequence data for all Lemnaceae species, we ascertained that prior difficulty in sequencing the ITS regions likely resulted from extremely rigid secondary structures, precipitated by a high proportion of G/C nucleotides. In phylogenetic analyses, nuclear ribosomal data largely supported relationships that had been inferred using chloroplast DNA sequence data. PMID:24942778

  8. Three distinct ribosome assemblies modulated by translation are the building blocks of polysomes.

    PubMed

    Viero, Gabriella; Lunelli, Lorenzo; Passerini, Andrea; Bianchini, Paolo; Gilbert, Robert J; Bernabò, Paola; Tebaldi, Toma; Diaspro, Alberto; Pederzolli, Cecilia; Quattrone, Alessandro

    2015-03-01

    Translation is increasingly recognized as a central control layer of gene expression in eukaryotic cells. The overall organization of mRNA and ribosomes within polysomes, as well as the possible role of this organization in translation are poorly understood. Here we show that polysomes are primarily formed by three distinct classes of ribosome assemblies. We observe that these assemblies can be connected by naked RNA regions of the transcript. We show that the relative proportions of the three classes of ribosome assemblies reflect, and probably dictate, the level of translational activity. These results reveal the existence of recurrent supra-ribosomal building blocks forming polysomes and suggest the presence of unexplored translational controls embedded in the polysome structure. PMID:25713412

  9. Three distinct ribosome assemblies modulated by translation are the building blocks of polysomes

    PubMed Central

    Lunelli, Lorenzo; Passerini, Andrea; Bianchini, Paolo; Gilbert, Robert J.; Bernabò, Paola; Tebaldi, Toma; Diaspro, Alberto; Pederzolli, Cecilia

    2015-01-01

    Translation is increasingly recognized as a central control layer of gene expression in eukaryotic cells. The overall organization of mRNA and ribosomes within polysomes, as well as the possible role of this organization in translation are poorly understood. Here we show that polysomes are primarily formed by three distinct classes of ribosome assemblies. We observe that these assemblies can be connected by naked RNA regions of the transcript. We show that the relative proportions of the three classes of ribosome assemblies reflect, and probably dictate, the level of translational activity. These results reveal the existence of recurrent supra-ribosomal building blocks forming polysomes and suggest the presence of unexplored translational controls embedded in the polysome structure. PMID:25713412

  10. Molecular basis for the ribosome functioning as an L-tryptophan sensor.

    PubMed

    Bischoff, Lukas; Berninghausen, Otto; Beckmann, Roland

    2014-10-23

    Elevated levels of the free amino acid L-tryptophan (L-Trp) trigger expression of the tryptophanase tnaCAB operon in E. coli. Activation depends on tryptophan-dependent ribosomal stalling during translation of the upstream TnaC peptide. Here, we present a cryoelectron microscopy (cryo-EM) reconstruction at 3.8 Å resolution of a ribosome stalled by the TnaC peptide. Unexpectedly, we observe two L-Trp molecules in the ribosomal exit tunnel coordinated within composite hydrophobic pockets formed by the nascent TnaC peptide and the tunnel wall. As a result, the peptidyl transferase center (PTC) adopts a distinct conformation that precludes productive accommodation of release factor 2 (RF2), thereby inducing translational stalling. Collectively, our results demonstrate how the translating ribosome can act as a small molecule sensor for gene regulation. PMID:25310980

  11. Kinetoplast DNA-encoded ribosomal protein S12

    PubMed Central

    Aphasizheva, Inna; Maslov, Dmitri A; Aphasizhev, Ruslan

    2013-01-01

    Mitochondrial ribosomes of Trypanosoma brucei are composed of 9S and 12S rRNAs, which are encoded by the kinetoplast genome, and more than 150 proteins encoded in the nucleus and imported from the cytoplasm. However, a single ribosomal protein RPS12 is encoded by the kinetoplast DNA (kDNA) in all trypanosomatid species examined. As typical for these organisms, the gene itself is cryptic and its transcript undergoes an extensive U-insertion/deletion editing. An evolutionary trend to reduce or eliminate RNA editing could be traced with other cryptogenes, but the invariably pan-edited RPS12 cryptogene is apparently spared. Here we inquired whether editing of RPS12 mRNA is essential for mitochondrial translation. By RNAi-mediated knockdowns of RNA editing complexes and inducible knock-in of a key editing enzyme in procyclic parasites, we could reversibly downregulate production of edited RPS12 mRNA and, by inference, synthesis of this protein. While inhibition of editing decreased edited mRNA levels, the translation of edited (Cyb) and unedited (COI) mRNAs was blocked. Furthermore, the population of SSU-related 45S complexes declined upon inactivation of editing and so did the amount of mRNA-bound ribosomes. In bloodstream parasites, which lack active electron transport chain but still require translation of ATP synthase subunit 6 mRNA (A6), both edited RPS12 and A6 mRNAs were detected in translation complexes. Collectively, our results indicate that a single ribosomal protein gene retained by the kinetoplast mitochondrion serves as a possible functional link between editing and translation processes and provide the rationale for the evolutionary conservation of RPS12 pan-editing. PMID:24270388

  12. Biochemical characterization of three mycobacterial ribosomal fractions.

    PubMed

    Portelance, V; Beaudet, R

    1983-02-01

    The induction of antituberculous immunity by crude ribosomal fractions isolated from Mycobacterium tuberculosis strain H37Ra, M. bovis strain BCG, and M. smegmatis was studied in CF-1 mice. Levels of antituberculous immunity similar to that induced by live BCG were induced by the BCG and H37Ra ribosomal fractions whereas that isolated from M. smegmatis was found to be inactive. Electrophoresis of the three ribosomal fractions in sodium dodecyl sulfate - polyacylamide gels followed by differential staining showed the two active ribosomal fractions to be similar in their proteins, carbohydrate-containing substances, and lipid profiles. The inactive smegmatis ribosomal fraction differed mainly from the active ones on the basis of its carbohydrate-containing substances profile and by the absence of lipids. The polysaccharides and the ribosomes present in the H37Ra ribosomal fractions were purified by affinity chromatography on concanavalin A - Sepharose 4B. Each purified preparation showed no or only low antituberculous activity when injected separately, but when mixed together a high protection was observed. The formation of complexes between the ribosomes and the polysaccharide fraction was suggested and appears to be necessary for the induction of antituberculous immunity. PMID:6189570

  13. Ribosome flow model with positive feedback

    PubMed Central

    Margaliot, Michael; Tuller, Tamir

    2013-01-01

    Eukaryotic mRNAs usually form a circular structure; thus, ribosomes that terminatae translation at the 3′ end can diffuse with increased probability to the 5′ end of the transcript, initiating another cycle of translation. This phenomenon describes ribosomal flow with positive feedback—an increase in the flow of ribosomes terminating translating the open reading frame increases the ribosomal initiation rate. The aim of this paper is to model and rigorously analyse translation with feedback. We suggest a modified version of the ribosome flow model, called the ribosome flow model with input and output. In this model, the input is the initiation rate and the output is the translation rate. We analyse this model after closing the loop with a positive linear feedback. We show that the closed-loop system admits a unique globally asymptotically stable equilibrium point. From a biophysical point of view, this means that there exists a unique steady state of ribosome distributions along the mRNA, and thus a unique steady-state translation rate. The solution from any initial distribution will converge to this steady state. The steady-state distribution demonstrates a decrease in ribosome density along the coding sequence. For the case of constant elongation rates, we obtain expressions relating the model parameters to the equilibrium point. These results may perhaps be used to re-engineer the biological system in order to obtain a desired translation rate. PMID:23720534

  14. Evolution of the ribosome at atomic resolution

    PubMed Central

    Petrov, Anton S.; Bernier, Chad R.; Hsiao, Chiaolong; Norris, Ashlyn M.; Kovacs, Nicholas A.; Waterbury, Chris C.; Stepanov, Victor G.; Harvey, Stephen C.; Fox, George E.; Wartell, Roger M.; Hud, Nicholas V.; Williams, Loren Dean

    2014-01-01

    The origins and evolution of the ribosome, 3–4 billion years ago, remain imprinted in the biochemistry of extant life and in the structure of the ribosome. Processes of ribosomal RNA (rRNA) expansion can be “observed” by comparing 3D rRNA structures of bacteria (small), yeast (medium), and metazoans (large). rRNA size correlates well with species complexity. Differences in ribosomes across species reveal that rRNA expansion segments have been added to rRNAs without perturbing the preexisting core. Here we show that rRNA growth occurs by a limited number of processes that include inserting a branch helix onto a preexisting trunk helix and elongation of a helix. rRNA expansions can leave distinctive atomic resolution fingerprints, which we call “insertion fingerprints.” Observation of insertion fingerprints in the ribosomal common core allows identification of probable ancestral expansion segments. Conceptually reversing these expansions allows extrapolation backward in time to generate models of primordial ribosomes. The approach presented here provides insight to the structure of pre-last universal common ancestor rRNAs and the subsequent expansions that shaped the peptidyl transferase center and the conserved core. We infer distinct phases of ribosomal evolution through which ribosomal particles evolve, acquiring coding and translocation, and extending and elaborating the exit tunnel. PMID:24982194

  15. Effects of induction of rRNA overproduction on ribosomal protein synthesis and ribosome subunit assembly in Escherichia coli.

    PubMed Central

    Yamagishi, M; Nomura, M

    1988-01-01

    Overproduction of rRNA was artificially induced in Escherichia coli cells to test whether the synthesis of ribosomal protein (r-protein) is normally repressed by feedback regulation. When rRNA was overproduced more than twofold from a hybrid plasmid carrying the rrnB operon fused to the lambda pL promoter (pL-rrnB), synthesis of individual r-proteins increased by an average of about 60%. This demonstrates that the synthesis of r-proteins is repressed under normal conditions. The increase of r-protein production, however, for unknown reasons, was not as great as the increase in rRNA synthesis and resulted in an imbalance between the amounts of rRNA and r-protein synthesis. Therefore, only a small (less than 20%) increase in the synthesis of complete 30S and 50S ribosome subunits was detected, and a considerable fraction of the excess rRNA was degraded. Lack of complete cooperativity in the assembly of ribosome subunits in vivo is discussed as a possible explanation for the absence of a large stimulation of ribosome synthesis observed under these conditions. In addition to the induction of intact rRNA overproduction from the pL-rrnB operon, the effects of unbalanced overproduction of each of the two large rRNAs, 16S rRNA and 23S rRNA, on r-protein synthesis were examined using pL-rrnB derivatives carrying a large deletion in either the 23S rRNA gene or the 16S rRNA gene. Operon-specific derepression after 23S or 16S rRNA overproduction correlated with the overproduction of rRNA containing the target site for the operon-specific repressor r-protein. These results are discussed to explain the apparent coupling of the assembly of one ribosomal subunit with that of the other which was observed in earlier studies on conditionally lethal mutants with defects in ribosome assembly. PMID:3053641

  16. Complementary roles of initiation factor 1 and ribosome recycling factor in 70S ribosome splitting

    PubMed Central

    Pavlov, Michael Y; Antoun, Ayman; Lovmar, Martin; Ehrenberg, Måns

    2008-01-01

    We demonstrate that ribosomes containing a messenger RNA (mRNA) with a strong Shine–Dalgarno sequence are rapidly split into subunits by initiation factors 1 (IF1) and 3 (IF3), but slowly split by ribosome recycling factor (RRF) and elongation factor G (EF-G). Post-termination-like (PTL) ribosomes containing mRNA and a P-site-bound deacylated transfer RNA (tRNA) are split very rapidly by RRF and EF-G, but extremely slowly by IF1 and IF3. Vacant ribosomes are split by RRF/EF-G much more slowly than PTL ribosomes and by IF1/IF3 much more slowly than mRNA-containing ribosomes. These observations reveal complementary splitting of different ribosomal complexes by IF1/IF3 and RRF/EF-G, and suggest the existence of two major pathways for ribosome splitting into subunits in the living cell. We show that the identity of the deacylated tRNA in the PTL ribosome strongly affects the rate by which it is split by RRF/EF-G and that IF3 is involved in the mechanism of ribosome splitting by IF1/IF3 but not by RRF/EF-G. With support from our experimental data, we discuss the principally different mechanisms of ribosome splitting by IF1/IF3 and by RRF/EF-G. PMID:18497739

  17. Viral IRES RNA structures and ribosome interactions.

    PubMed

    Kieft, Jeffrey S

    2008-06-01

    In eukaryotes, protein synthesis initiates primarily by a mechanism that requires a modified nucleotide 'cap' on the mRNA and also proteins that recruit and position the ribosome. Many pathogenic viruses use an alternative, cap-independent mechanism that substitutes RNA structure for the cap and many proteins. The RNAs driving this process are called internal ribosome-entry sites (IRESs) and some are able to bind the ribosome directly using a specific 3D RNA structure. Recent structures of IRES RNAs and IRES-ribosome complexes are revealing the structural basis of viral IRES' 'hijacking' of the protein-making machinery. It now seems that there are fundamental differences in the 3D structures used by different IRESs, although there are some common features in how they interact with ribosomes. PMID:18468443

  18. Ribosome defects in disorders of erythropoiesis.

    PubMed

    Narla, Anupama; Hurst, Slater N; Ebert, Benjamin L

    2011-02-01

    Over the past decade, genetic lesions that cause ribosome dysfunction have been identified in both congenital and acquired human disorders. These discoveries have established a new category of disorders, known as ribosomopathies, in which the primary pathophysiology is related to impaired ribosome function. The protoptypical disorders are Diamond-Blackfan anemia, a congenital bone marrow failure syndrome, and the 5q- syndrome, a subtype of myelodysplastic syndrome. In both of these disorders, impaired ribosome function causes a severe macrocytic anemia. In this review, we will discuss the evidence that defects in ribosomal biogenesis cause the hematologic phenotype of Diamond-Blackfan anemia and the 5q- syndrome. We will also explore the potential mechanisms by which a ribosomal defect, which would be expected to have widespread consequences, may lead to specific defects in erythropoiesis. PMID:21279816

  19. Viral IRES RNA structures and ribosome interactions

    PubMed Central

    Kieft, Jeffrey S.

    2009-01-01

    In eukaryotes, protein synthesis initiates primarily by a mechanism that requires a modified nucleotide ‘cap’ on the mRNA and also proteins that recruit and position the ribosome. Many pathogenic viruses use an alternative, cap-independent mechanism that substitutes RNA structure for the cap and many proteins. The RNAs driving this process are called internal ribosome-entry sites (IRESs) and some are able to bind the ribosome directly using a specific 3D RNA structure. Recent structures of IRES RNAs and IRES–ribosome complexes are revealing the structural basis of viral IRES’ ‘hijacking’ of the protein-making machinery. It now seems that there are fundamental differences in the 3D structures used by different IRESs, although there are some common features in how they interact with ribosomes. PMID:18468443

  20. Interaction of Chloramphenicol Tripeptide Analogs with Ribosomes.

    PubMed

    Tereshchenkov, A G; Shishkina, A V; Tashlitsky, V N; Korshunova, G A; Bogdanov, A A; Sumbatyan, N V

    2016-04-01

    Chloramphenicol amine peptide derivatives containing tripeptide fragments of regulatory "stop peptides" - MRL, IRA, IWP - were synthesized. The ability of the compounds to form ribosomal complexes was studied by displacement of the fluorescent erythromycin analog from its complex with E. coli ribosom