Terminal Hexanucleotide Research Articles

5’ and 3’ splicing signals evolution in vertebrates: Analysis in a conserved gene family

The mitochondrial solute carrier genes (SLC25) are highly conserved during vertebrate evolution. In most SLC25 genes of zebrafish, chicken, mouse, and human, the introns are located at exactly superimposable positions. In these topographically corresponding introns we studied the composition of the initial and terminal hexanucleotides (5’ss and 3’ss) which are instrumental in splicing signaling, focusing on the evolutionary conservation/mutation dynamics of these genetically related sequences. At each position, the per cent conservation of zebrafish individual nucleotides in chicken, mouse and human is proportional to their percent frequency in zebrafish; furthermore, nucleotide mutations are biased in favor of the more represented nucleotides, thus compensating for those highly represented zebrafish nucleotides which have not been conserved. As a result of these evolutionary dynamics, the general nucleotide composition at each position has remained relatively conserved throughout vertebrates. At 5’ss, following the canonical GT, A and G are largely prevailing at position +3, A at +4 and G at +5 (GT[A/G]AGx). At 3’ss, T and C are largely prevailing at positions −6, −5 and −3, preceding the canonical intron terminal AG ([C/T] [C/T]x[C/T]AG). However, the actual composition of the tetranucleotides at 5’ and 3’ often does not conform to the above scheme. At 5’ss the more canonical sequence is completely expressed in 63% of cases and partially (2 or 1 matches) in 37 % of cases. At 3’ss the more canonical sequence is completely expressed in 71 % of cases and partially (2 or 1 matches) in 29 % of cases. The nucleotide conservation loss (nucleotide mutation) is higher in the evolution from fish to the last common ancestor of birds and mammals (58 %), then diminishes in the successive evolution steps up to the mammalian common ancestor (10 %), and becomes still lower at the divergence of rodents and primates (5 %).

Conservation/Mutation in the Splice Sites of Mitochondrial Solute Carrier Genes of Vertebrates.

The "canonical" introns begin by the dinucleotide GT and end by the dinucleotide AG. GT, together with a few downstream nucleotides, and AG, with a few of the immediately preceding nucleotides, are thought to be the strongest splicing signals (5'ss and 3'ss, respectively). We examined the composition of the intronic initial and terminal hexanucleotides of the mitochondrial solute carrier genes (SLC25A's) of zebrafish, chicken, mouse, and human. These genes are orthologous and we selected the transcripts in which the arrangement of exons and introns was superimposable in the species considered. Both 5'ss and 3'ss were highly polymorphic, with 104 and 126 different configurations, respectively, in our sample. In the line of evolution from zebrafish to chicken, as well as in that from zebrafish to mammals, the average nucleotide conservation in the four variable nucleotides was about 50% at 5' and 40% at 3'. In the divergent evolution of mouse and human, the conservation was about 80% at 5' and 70% at 3'. Despite these changes, the splicing signals remain strong enough to operate at the same site. At both 5' and 3', the frequency of a nucleotide at a given position in the zebrafish sequence is positively correlated with its conservation in chicken and mammals, suggesting that selection continued to operate in birds and mammals along similar lines.

Conservation/Mutation in the Splice Sites of Cytokine Receptor Genes of Mouse and Human

Conservation/mutation in the intronic initial and terminal hexanucleotides was studied in 26 orthologous cytokine receptor genes of Mouse and Human. Introns began and ended with the canonical dinucleotides GT and AG, respectively. Identical configurations were found in 57% of the 5′ hexanucleotides and 28% of the 3′ hexanucleotides. The actual conservation percentages of the individual variable nucleotides at each position in the hexanucleotides were determined, and the theoretical rates of conservation of groups of three nucleotides were calculated under the hypothesis of a mutual evolutionary independence of the neighboring nucleotides (random association). Analysis of the actual conservation of groups of variable nucleotides showed that, at 5′, GTGAGx was significantly more expressed and GTAAGx was significantly less expressed, as compared to the random association. At 3′, TTTxAG and xTGCAG were overexpressed as compared to a random association. Study of Mouse and Human transcript variants involving the splice sites showed that most variants were not inherited from the common ancestor but emerged during the process of speciation. In some variants the silencing of a terminal hexanucleotide determined skipping of the downstream exon; in other variants the constitutive splicing hexanucleotide was replaced by another potential, in-frame, splicing hexanucleotide, leading to alterations of exon lengths.

Complete sequence of a reovirus associated with necrotic focus formation in the liver and spleen of Muscovy ducklings

The complete sequence of a reovirus, strain 815-12 associated with necrotic focus formation in the liver and spleen of Muscovy ducklings in China, was determined and compared with sequences of other duck-, goose-, and chicken-origin reoviruses. The 815-12 genome comprised 22,969 bp with 10 dsRNA segments ranging from 1125 bp (S4) to 3958 bp (L1), all of which (except S4) were almost identical in length to the cognate segments of other waterfowl and chicken isolates. Detailed analyses revealed that 815-12 and other waterfowl isolates contained the conserved 3'-terminal pentanucleotide sequence (UCAUC-3') of the orthoreoviruses and 5'-terminal hexanucleotide sequence (5'-GCUUUU) of avian orthoreoviruses (ARVs), and conserved functional motifs previously identified in ARV proteins. Several notable differences, including organization of the polycistronic genome segments and genomic coding assignments of the S segments, existed between viruses represented by 815-12 and the waterfowl reoviruses emerging in China in recent years; the latter was somewhat similar to chicken isolates. Pairwise sequence comparisons demonstrated extensive sequence diversity among the various waterfowl isolates and between waterfowl and chicken isolates. Phylogenetic analyses identified two genetic groups for waterfowl reoviruses, and potential genetic reassortment of segment M2 between waterfowl and chicken reoviruses and segments encoding for λA, λB, μA, μNS and σA between waterfowl reoviruses. Taken together, it was suggested that common designation ARV-Wa should be used to represent ARV isolates from different waterfowl species and that the two ARV-Wa genotypes should be considered as two separate groups distinct from chicken isolates within the species Avian orthoreovirus.

Genetic and phylogenetic analysis of the non-structural proteins NS1, NS2 and NS3 of epizootic haemorrhagic disease virus (EHDV)

Three unique non-structural (NS) proteins are produced by Epizootic haemorrhagic disease virus (EHDV) during infection of a host cell; NS1, NS2 and NS3. This study presents a complete genetic and phylogenetic analysis of these proteins (and the genes that code for them) to allow comparison of the selective pressures acting on each. Accession numbers, gene and protein sizes, ORF positions, G + C contents, terminal hexanucleotides, start and stop codons and phylogenetic relationships are all presented. Unlike the core, or outer-coat proteins, there are no characteristic genetic or phylogenetic traits common to all of the EHDV NS proteins; indicating that each is evolving under different selection pressures. These differences are discussed. Evidence of genetic recombination in genome segment 8 (coding for NS2) is also presented, together with evidence of gene duplication and mutation, suggesting the EHDV genome may have evolved using mechanisms such as these.

Genetic and phylogenetic analysis of the core proteins VP1, VP3, VP4, VP6 and VP7 of epizootic haemorrhagic disease virus (EHDV)

The core proteins of epizootic haemorrhagic disease virus (EHDV) have important roles to perform in maintaining the structure and function of the virus. A complete genetic and phylogenetic analysis was therefore performed on these proteins (and the genes that code for them) to allow comparison of the selective pressures acting on each. Accession numbers, gene and protein sizes, ORF positions, G + C contents, terminal hexanucleotides, start and stop codons and phylogenetic relationships are all presented. The inner core proteins (VP1, VP3, VP4 and VP6) were characterised by high levels of sequence conservation, and the ability to topotype isolates very strongly into eastern or western groups. This is particularly evident in genome segment 9 (VP6) which exists as two different sized homologues. VP7 did not topotype, but rather exhibited a more random, radial phylogeny suggestive of genetic drift. With the exception of VP6, all of the core proteins also showed high numbers of synonymous mutations in the third base position, suggesting they have been evolving for a long period of time. Interestingly, VP6 did not show this, and possible reasons for this are discussed.

Genetic and phylogenetic analysis of the outer-coat proteins VP2 and VP5 of epizootic haemorrhagic disease virus (EHDV): Comparison of genetic and serological data to characterise the EHDV serogroup

The outer-coat proteins, VP2 and VP5, of epizootic haemorrhagic disease virus (EHDV) are important for host cell binding during the initiation of infection. They are also known to determine virus serotype. This study presents a complete genetic and phylogenetic analysis of these proteins (and the genes that code for them) to allow comparison of the selective pressures acting on each and the correlation of genetic sequence data with serotype. Accession numbers, gene and protein sizes, ORF positions, G+C contents, terminal hexanucleotides, start and stop codons and phylogenetic relationships are all presented. The results show that VP2 is highly variable, is under great pressure to adapt and can be correlated with serotype. While also variable, VP5 appears to be under less adaptive pressure than VP2 but still shows some correlation with serotype. Seven serotypes of EHDV have been defined in this study, although the results do show that some serotypes are extremely closely related—and highlight the benefit of using both molecular and serologic analyses. Analysis of the terminal hexanucleotides showed that the 5′ terminus is under greater purifying selection than the 3′. Evidence is also presented that both segments 2 and 6 (coding for VP2 and VP5 respectively) have grown via gene duplication and subsequent mutation.

Caractérisation génétique des virus Tilligerry et Mitchell River

Viruses that are normally safely contained within their host spe­cies can emerge due to intense livestock farming, trade, travel, climate change and encroachment of human activities into new environments. The unexpected emergence of bluetongue virus (BTV), the prototype species of the genus Orbivirus, in economi­cally important livestock species (sheep and cattle) across the whole of Europe (since 1998), indicates that other orbiviruses represent a potential further threat to animal and human popula­tions in Europe and elsewhere. The genus Orbivirus is the largest within the family Reoviridae, containing 22 virus species, as well as 14 unclassified orbiviruses, some of which may repre­sent additional or novel species. The orbiviruses are transmitted primarily by arthropod vectors (e.g. Culicoides, mosquitoes or ticks). Viral genome sequence data provide a basis for virus taxonomy and diagnostic test development, and make it possible to address fundamental questions concerning virus biology, pathogenesis, virulence and evolution, that can be further explored in mutation and reverse genetics studies. Genome sequences also provide criteria for the classification of novel isolates within individual Orbivirus species, as well as the identification of different sero­types, topotypes, reassortants and even closely related but dis­tinct virus lineages. Full-length genome characterization of Tilligerry virus (TILV), a member of the Eubenangee virus species, and Mitchell River virus (MRV), a member of the Warrego virus species, have revealed highly conserved 5’ and 3’ terminal hexanucleotide sequences. Phylogenetic analyses of orbivirus T2 ‘sub-core-shell’ protein sequences reinforce the hypothesis that this protein is an important evolutionary marker for these viruses. The T2 protein shows high levels of amino acid (AA) sequence identity (> 91%) within a single Orbivirus species / serogroup, which can be used for species identification. The T2-protein gene has therefore been given priority in sequencing studies. The T2 protein of TILV is closely related to that of Eubenangee virus (~91% identity), con­firming that they are both members of the same Eubenangee virus species. Although TILV is reported to be related to BTV in serological assays, the TILV T2 protein shows only 68-70% AA identity to BTV. This supports its current classification within a different serogroup (Eubenangee). Warrego virus and MRV are currently classified as two distinct members (different serotypes) within the Warrego virus species. However, they show only about 79% AA identity in their T2 pro­tein (based on partial sequences). It is therefore considered likely that they could be reclassified as members of distinct Orbivirus species. The taxonomic classification of MRV will be reviewed after generating full length sequences for the entire genomes of both viruses. The taxonomic status of each of these viruses will also be tested further by co-infections and attempts to create reassortants between them (only viruses belonging to the same species can reassort their genome segments). TILV and MRV are the first viruses from their respective serogroups / virus species to be genetically fully characterized, and will provide a basis for the further characterization / identification of additional viruses within each group / species. These data will assist in the devel­opment of specific diagnostic assays and potentially in control of emerging diseases. The sequences generated will also help to evaluate current diagnostic [reverse transcriptase - polymerase chain reaction (RT-PCR)] tests for BTV, African horse sickness virus, epizootic haemorrhagic disease virus, etc., in silico, by identifying any possibility of cross reactivity.

Analysis and phylogenetic comparisons of full-length VP2 genes of the 24 bluetongue virus serotypes

The outer capsid protein VP2 of Bluetongue virus (BTV) is a target for the protective immune response generated by the mammalian host. VP2 contains the majority of epitopes that are recognized by neutralizing antibodies and is therefore also the primary determinant of BTV serotype. Full-length cDNA copies of genome segment 2 (Seg-2, which encodes VP2) from the reference strains of each of the 24 BTV serotypes were synthesized, cloned and sequenced. This represents the first complete set of full-length BTV VP2 genes (from the 24 serotypes) that has been analysed. Each Seg-2 has a single open reading frame, with short inverted repeats adjacent to conserved terminal hexanucleotide sequences. These data demonstrated overall inter-serotype variations in Seg-2 of 29 % (BTV-8 and BTV-18) to 59 % (BTV-16 and BTV-22), while the deduced amino acid sequence of VP2 varied from 22.4 % (BTV-4 and BTV-20) to 73 % (BTV-6 and BTV-22). Ten distinct Seg-2 lineages (nucleotypes) were detected, with greatest sequence similarities between those serotypes that had previously been reported as serologically 'related'. Fewer similarities were observed between different serotypes in regions of VP2 that have been reported as antigenically important, suggesting that they may play a role in the neutralizing antibody response. The data presented form an initial basis for BTV serotype identification by sequence analyses and comparison of Seg-2, and for development of molecular diagnostic assays for individual BTV serotypes (by RT-PCR).

Stable Protein–RNA Interaction Involves the Terminal Domains of Bluetongue Virus mRNA, but Not the Terminally Conserved Sequences

The interaction of bluetongue virus (BTV) proteins with viral RNA was investigatedin vitroby means of a biochemical approach. By subjecting cytoplasmic extracts from virus-infected baby hamster kidney cells andin vitrosynthesized radiolabeled RNA to ultraviolet cross-linking assays, we demonstrated that, of all the BTV proteins, NS2 becomes most intimately associated with the labeled viral RNA. Competition binding studies indicated that NS2 has the greatest affinity for the 3′ region of the viral transcripts. By analyzing the binding efficiency of NS2 to mutant RNA transcripts which lacked the fully conserved 5′- and/or 3′-terminal hexanucleotides, we have established that these sequences are not necessary for optimal binding. The specificity of the NS2–RNA interaction was investigated by competition experiments with unlabeled BTV-specific homologous and heterologous competitor RNAs as well as with viral double-stranded RNA (dsRNA). Although apparent differences in the ability of NS2 to bind to the different RNA transcripts were observed, it did not bind to the dsRNA.

Molecular analysis of rice dwarf phytoreovirus segment S11 corresponding to wound tumour phytoreovirus segment S12

The complete nucleotide sequence of rice dwarf phytoreovirus (RDV) genome segment S11 was determined. S11 is 1067 nucleotides long. There is an inverted repeat of 10 bp adjacent to the conserved 5'-terminal hexanucleotide (5' GGUAAA 3') and 3'-terminal tetranucleotide (5' UAGU 3') sequences. A single large open reading frame found in the plus strand of S11 begins with the first AUG codon (bases 6 to 8) and extends for 567 bases. Evolutionary relatedness between RDV S11 and wound tumour phytoreovirus S12 based on amino acid sequence similarity (25.8%) was found. In addition to the first AUG triplet, RDV S11 possesses a second in-phase AUG triplet (positions 30 to 32) nearby, which conforms to the Kozak consensus sequence. Two forms of the protein were identified by using an in vitro transcription and translation system in which a tailored full-length cDNA was the initial template. The abolition of the first AUG codon by site-directed mutagenesis resulted in disappearance of the larger translation product. These results strongly suggest that the two products are translated from the first and second AUG codons. Whether the two proteins are expressed in vivo is at present unclear.

A characterization of the nonstructural protein from which the virus-specified tubules in epizootic haemorrhagic disease virus-infected cells are composed

The complete nucleotide sequence of segment 6 of epizootic haemorrhagic disease virus serotype 2 (Alberta) which encodes nonstructural protein NS1 was determined from a cDNA clone containing a full-length copy of the gene. The gene was found to be 1806 bp in length, constituted by one open reading frame of 1656 bp which is flanked by 5' and 3' noncoding regions of 32 and 118 bp, respectively. The conserved 5' and 3' terminal hexanucleotide sequences were identical to those of BTV-10. The 5' noncoding nucleotide sequences of cognate genome segments 4, 6 and 8 were found to be highly conserved in EHDV-2 and BTV-10. The 3' noncoding regions are less conserved but share common characteristics. The predicted EHDV-2 NS1 gene product, a 552 amino acid polypeptide, is predominantly hydrophobic and has a net charge of + 5 at neutral pH. Comparison to its BTV-10 counterpart revealed a high degree of homology. Regions of high amino acid similarity were shown to correlate with hydrophobic domains on the proteins whilst regions of lower amino acid similarity corresponded with the hydrophilic domains. Thirteen conserved cysteine residues of which the majority occurred in hydrophobic regions with more than 80% amino acid similarity were identified.

In vitro RNA transcription by the New Jersey serotype of vesicular stomatitis virus. I. Characterization of the mRNA species

The in vitro RNA synthesis by the virion-associated RNA polymerase of vesicular stomatitis virus (VSV), New Jersey serotype, was compared with that of the serologically distinct Indiana serotype of VSV. The New Jersey serotype of VSV synthesized five distinct mRNA species in vitro, three of which were smaller than the corresponding species synthesized by the Indiana serotype of VSV. These included the mRNA's coding for the G, M, and NS proteins. By hybridization experiments, virtually no sequence homology was detected between the mRNA's of the two serotypes. Despite this lack of overall homology, the 12 to 18S mRNA species of both serotype contained a common 5'-terminal hexanucleotide sequence, G(5')ppp(5')A-A-C-A-G. The signicance of this finding in light of specific interactions between the two serotypes of VSV in vivo is discussed.

5′-Terminal Sequence of Vesicular Stomatitis Virus mRNA's Synthesized In Vitro

Unmethylated or methylated 12 to 18S mRNA's synthesized in vitro by the virion-associated RNA polymerase of vesicular stomatitis virus contain the 5'-terminal hexanucleotide sequence G(5')ppp(5')ApApCpApGp...or m 7G(5')-ppp(5')ApmApCpApGp..., respectively. The implication of these results in relation to the regulation of transcription in vesicular stomatitis virus is discussed.

Identical 3'-terminal octanucleotide sequence in 18S ribosomal ribonucleic acid from different eukaryotes. A proposed role for this sequence in the recognition of terminator codons.

The 3'-terminal sequence of 18S ribosomal RNA from Drosophila melanogaster and Saccharomyces cerevisiae was determined by stepwise degradation from the 3'-terminus and labelling with [(3)H]isoniazid. The sequence G-A-U-C-A-U-U-A(OH) was found at the 3'-terminus of both 18S rRNA species. Less extensive data for 18S RNA from a number of other eukaryotes are consistent with the same 3'-terminal sequence, and an identical sequence has previously been reported for the 3'-end of rabbit reticulocyte 18S rRNA (Hunt, 1970). These results suggest that the base sequence in this region is strongly conserved and may be identical in all eukaryotes. As the 3'-terminal hexanucleotide is complementary to eukaryotic terminator codons we discuss the possibility that the 3'-end of 18S rRNA may have a direct base-pairing role in the termination of protein synthesis.

Conserved terminal sequence in 18SrRNA may represent terminator anticodons.

WE report here that the 3′-terminal octanucleotide sequences of 18S rRNA from yeast, Drosophila and rabbit reticulocytes are identical. The 3′-terminal hexanucleotide is complementary to known eukaryotic terminator codons suggesting a role for the 3′-end of the small rRNA in the termination of protein synthesis.

Studies on transfer ribonucleic acids and related compounds. VI. Synthesis of yeast alanine transfer ribonucleic acid 3'-terminal nonanucleotides and 5'-terminal hexanucleotides.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTTransfer ribonucleic acids and related compounds. VI. Synthesis of yeast alanine transfer ribonucleic acid 3'-terminal nonanucleotides and 5'-terminal hexanucleotidesEiko. Ohtsuka, Masaru. Ubasawa, Shigeo. Morioka, and Morio. IkeharaCite this: J. Am. Chem. Soc. 1973, 95, 14, 4725–4733Publication Date (Print):July 1, 1973Publication History Published online1 May 2002Published inissue 1 July 1973https://doi.org/10.1021/ja00795a042RIGHTS & PERMISSIONSArticle Views32Altmetric-Citations15LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1011 KB) Get e-Alerts Get e-Alerts

The 3′-terminal hexanucleotide, CAACCA, is not essential for fragment recombination or cross-linking in E. coli tRNA fMet

FEBS LettersVolume 22, Issue 2 p. 169-174 Full-length articleFree Access The 3′-terminal hexanucleotide, CAACCA, is not essential for fragment recombination or cross-linking inE. coli tRNAfMet M.A.Q. Siddiqui, M.A.Q. Siddiqui Roche Institute of Molecular Biology, Nutley, New Jersey 07110, USASearch for more papers by this authorJames Ofengand, James Ofengand Roche Institute of Molecular Biology, Nutley, New Jersey 07110, USASearch for more papers by this author M.A.Q. Siddiqui, M.A.Q. Siddiqui Roche Institute of Molecular Biology, Nutley, New Jersey 07110, USASearch for more papers by this authorJames Ofengand, James Ofengand Roche Institute of Molecular Biology, Nutley, New Jersey 07110, USASearch for more papers by this author First published: May 01, 1972 https://doi.org/10.1016/0014-5793(72)80036-XCitations: 3AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume22, Issue2May 01, 1972Pages 169-174 ReferencesRelatedInformation