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At least 487 records · Page 27Linked to original sources
PubMed1992

Foret1, a reverse transcriptase-like sequence in the filamentous fungus Fusarium oxysporum.

We report here the isolation of Foret1, a repeated DNA sequence cloned from the fungal plant pathogen Fusarium oxysporum. This clone exhibits a high degree of sequence similarity with the retroviral pol genes. Sequences homologous to protease, reverse transcriptase, ribonuclease H are found in that order. The overall structure is homologous to the 'gypsy' class of LTR-retrotransposons. Its similarity to elements present in widely different organisms may result from its horizontal transmission in recent evolutionary time.

Amino Acid Sequence↗
PubMed1989

Detection of multiple, novel reverse transcriptase coding sequences in human nucleic acids: relation to primate retroviruses.

A variety of chemically synthesized oligonucleotides designed on the basis of amino acid and/or nucleotide sequence data were used to detect a large number of novel reverse transcriptase coding sequences in human and mouse DNAs. Procedures involving Southern blotting, library screening, and the polymerase chain reaction were all used to detect such sequences; the polymerase chain reaction was the most rapid and productive approach. In the polymerase chain reaction, oligonucleotide mixtures based on consensus sequence homologies to reverse transcriptase coding sequences and unique oligonucleotides containing perfect homology to the coding sequences of human T-cell leukemia virus types I and II were both effective in amplifying reverse transcriptase-related DNA. It is shown that human DNA contains a wide spectrum of retrovirus-related reverse transcriptase coding sequences, including some that are clearly related to human T-cell leukemia virus types I and II, some that are related to the L-1 family of long interspersed nucleotide sequences, and others that are related to previously described human endogenous proviral DNAs. In addition, human T-cell leukemia virus type I-related sequences appear to be transcribed in both normal human T cells and in a cell line derived from a human teratocarcinoma.

Amino Acid Sequence↗
PubMed1992

Worldwide prevalence of lentivirus infection in wild feline species: epidemiologic and phylogenetic aspects.

The natural occurrence of lentiviruses closely related to feline immunodeficiency virus (FIV) in nondomestic felid species is shown here to be worldwide. Cross-reactive antibodies to FIV were common in several free-ranging populations of large cats, including East African lions and cheetahs of the Serengeti ecosystem and in puma (also called cougar or mountain lion) populations throughout North America. Infectious puma lentivirus (PLV) was isolated from several Florida panthers, a severely endangered relict puma subspecies inhabiting the Big Cypress Swamp and Everglades ecosystems in southern Florida. Phylogenetic analysis of PLV genomic sequences from disparate geographic isolates revealed appreciable divergence from domestic cat FIV sequences as well as between PLV sequences found in different North American locales. The level of sequence divergence between PLV and FIV was greater than the level of divergence between human and certain simian immunodeficiency viruses, suggesting that the transmission of FIV between feline species is infrequent and parallels in time the emergence of HIV from simian ancestors.

Amino Acid Sequence↗
PubMed1994

Two regions of an avian hepadnavirus RNA pregenome are required in cis for encapsidation.

We have constructed a series of deletion mutants spanning the genome of duck hepatitis B virus in order to determine which regions of the viral genome are required in cis for packaging of the pregenome into capsid particles. Deletion of sequences within either of two nonadjacent regions prevented replication of the mutant viral genomes expressed in a permissive avian hepatoma cell line in the presence of functionally active viral core and P proteins. Extraction of RNA from cells transfected with these replication-defective mutants showed that the mutants retained the capacity to be transcribed into a pregenomic-size viral RNA, but that these RNA species were not packaged into viral capsids. The two regions defined by these deletions are located 36 to 126 (region I) and 1046 to 1214 (region II) nucleotides downstream of the 5' end of the pregenome and contain sequences which are required in cis for encapsidation of the duck hepatitis B virus pregenome.

Base Sequence↗
PubMed1994

High rates of frameshift mutations within homo-oligomeric runs during a single cycle of retroviral replication.

Homo-oligomeric runs were inserted into a spleen necrosis virus-based retrovirus vector to determine the nature and rate of mutations within runs of 10 to 12 identical nucleotides during a single replication cycle. Clones of helper cells containing integrated copies of retroviral vectors were used to produce virus for infection of target (nonhelper) cells. Proviral sequences from target cell clones were compared with proviral sequences from helper cell clones to study mutations that occurred during a single cycle of replication. In addition to the internal region spanning the homo-oligomeric inserts, a naturally occurring run of 10 T's in the long terminal repeat (LTR) also was sequenced. Rates of mutation ranged from < 0.01 to 0.38 frameshift mutations per run per cycle for different nucleotide runs. Frameshift mutations ranged from deletions of 2 bases to additions of 5 bases; the most common mutations were +1 and -1. Frameshift mutation rates did not increase as the run length increased from 10 to 12 bases. Rates of frameshift mutation for runs of T's and A's were significantly higher than rates for runs of C's and G's, and rates for runs of pyrimidines were significantly higher than those for runs of purines. Interestingly, the vast majority of frameshift mutations in the internal region (95%) were positive, suggesting that the primer strand tends to slip backward on the template in this region. LTR runs had a significantly lower number of positive frameshift mutations than the internal runs. By analyzing the types of frameshift mutations within runs and by comparing the patterns of frameshift mutations in the 5' and 3' LTRs of individual proviruses, we conclude that the majority of mutations observed in our system occurred during minus-strand DNA synthesis of reverse transcription.

Animals↗
PubMed2000

Ty1-copia-like retrotransposons of tomato (Lycopersicon esculentum Mill.).

We have used a PCR and cloning strategy to identify Ty1-copia-like retrotransposons in tomato, Lycopersicon esculentum Mill. Using degenerate oligonucleotide primers corresponding to conserved domains of the Ty1-copia retrotransposon reverse transcriptase (RT), fragments of about 260 bp were obtained by PCR amplification. Sequences of 20 cloned amplification fragments showed similarity to retrotransposon sequences. The copy number for total tomato Ty1-copia-like RT population was estimated to be approximately 2500 and may account for about 1.5% of the tomato genome. Copy numbers for four of the individual RT clones ranged from 20 to 1400 copies. A comparison of the conceptual translations of the RT sequences identified four clusters as well as three sequences which were ungrouped. When compared to RT sequences reported from several other sources, the tomato RT population was found to be widely dispersed with the majority of the RT sequences from Lycopersicon species delineated by the four tomato cluster groups. The gag region of a tomato retrotransposon was cloned from PCRs with primers based on the Tnt1 retrotransposon of tobacco. The tomato clone (pTom1.1) had 81% sequence similarity to the Tntl gag region. Several pTom1.1 sequences are present in other solanaceous species as indicated by Southern hybridization.

Amino Acid Sequence↗
PubMed1990

Regulation of c-myc transcription in vitro: dependence on the guanine-rich promoter element ME1a1.

P2 is the major promoter for the murine c-myc proto-oncogene. The cis-acting elements that are required for initiation of transcription from P2 have not been well defined. In this report elements involved in initiating transcription from P2 were analysed in an in vitro system. In addition to a consensus TATA element at position -28, a guanine-rich element exists at position -48. This element, termed ME1a1, increases transcription initiation when inserted into a deletion mutant that lacks it. When mutations are engineered into ME1a1 it no longer acts to increase the level of initiation. Gel-shift and DNAase I footprinting analysis indicate that Me1a1 binds a protein. ME1a1 does not show any striking similarity to other promoter elements and therefore may be a novel cis-acting element.

Base Sequence↗
PubMed1999

Isolation and characterization of the fission yeast gene rpa42+, which encodes a subunit shared by RNA polymerases I and III.

Eukaryotic RNA polymerases I and III share two distinct alpha-related subunits that show limited homology to the alpha subunit of Escherichia coli RNA polymerase, which forms a homodimer to nucleate the assembly of prokaryotic RNA polymerase. To gain insight into the functions of alpha-related subunits in eukaryotes, we have previously identified the alpha-related small subunit RPA17 of RNA polymerase I (and III) in Schizosaccharomyces pombe, and have shown that it is a functional homolog of Saccharomyces cerevisiae AC19. In an extension of that study, we have now isolated and characterized rpa42+, which encodes the alpha-related large subunit RPA42 of S. pombe RNA polymerase I, by virtue of the fact that its product interacts with RPA17 in the yeast two-hybrid system. We have found that rpa42+ encodes a polypeptide with an apparent molecular mass of 42 kDa, which shows 58% identity to the AC40 subunit shared by RNA polymerases I and III in S. cerevisiae. Furthermore, we have shown that rpa42+ complements a temperature-sensitive mutation in RPC40 the gene that encodes AC40 in S. cerevisiae and which is essential for cell growth. Finally, we have shown that neither RPA42 nor RPA17 can self-associate. These results provide evidence that the two distinct alpha-related subunits, RPA42 and RPA17, of RNA polymerases I and III are functionally conserved between S. pombe and S. cerevisiae, and suggest that heterodimer formation between them is essential for the assembly of RNA polymerases I and III in eukaryotes.

Amino Acid Sequence↗
PubMed2000

The roles of specific template nucleosides in the formation of stable transcription complexes by Escherichia coli RNA polymerase.

We have examined the effects of removing individual template nucleosides on promoter escape by Escherichia coli RNA polymerase in vitro. The ability of DNA templates containing random single nucleoside gaps generated by hydroxyl radical treatment to support the production of stable ternary transcription complexes was analyzed. On two templates containing different promoter and initial transcribed regions, we found that removal of nucleosides on the template strand in the region from -13 to at least +8 relative to the transcription start site interfered with ternary complex formation. The downstream border of this region varied for the two templates, suggesting an effect of the specific nucleotide sequence on the stability of intermediates in the promoter escape process. On the nontemplate strand, removal of nucleosides in the vicinity of the -10 consensus promoter element interfered with escape, whereas removal of nucleosides in the vicinity of the transcription start site actually enhanced the yield of ternary complexes. On one template, removal of nucleosides in an A-tract containing region upstream of the promoter caused a significant decrease in promoter escape, consistent with previous suggestions that contacts between this region and the RNA polymerase play a role in promoter binding and/or initiation.

Base Sequence↗
PubMed1990

Polymerase gene products of hepatitis B viruses are required for genomic RNA packaging as wel as for reverse transcription.

All reactions involving reverse transcription of RNA are segregated from the cytosol within a subviral particle or capsid composed of the major capsid protein, the polymerase and the RNA template. A key step in the formation of these particles is the selective encapsidation of the RNA template. Although an important general feature of the reverse transcription pathway, encapsidation has been carefully studied only for retroviruses. We have now examined the encapsidation reaction in a family of enveloped DNA viruses that replicate by reverse transcription--the hepatitis B viruses (hepadnaviruses). Our results indicate that the hepadnaviral polymerase (P) gene product is required for RNA packaging, and that the encapsidation function of the enzyme can be separated from its DNA polymerase activity. To our knowledge, this is the first description of a role for polymerase gene products in this step of the reverse transcription pathway.

Amino Acid Sequence↗
PubMed1986

Detection and sequence of plus-strand leader RNA of sonchus yellow net virus, a plant rhabdovirus.

Tobacco infected with the plant rhabdovirus sonchus yellow net virus (SYNV) contains short, 139- to 144-nucleotide (nt) transcripts complementary to the 3' terminus of the negative-strand genomic RNA. These transcripts are similar to the leader RNAs associated with several animal rhabdovirus infections in that they are encoded by the same region of the genome, but the SYNV transcripts are nearly 3 times longer than the animal rhabdovirus leader RNAs. The SYNV leader RNAs differ markedly in sequence from the leader RNAs associated with strains of vesicular stomatitis virus and rabies virus, although the first 30 nt of all three transcripts are rich in adenylate residues. The nucleotide sequence determined directly from SYNV RNA and from recombinant DNA clones derived from SYNV RNA reveals a possible initiation site for transcription of the N-protein mRNA that is located 147 nt from the 3' end of genomic RNA. The sequence (UUGU) at this site is complementary to the first 4 nt of the N-protein mRNAs of animal rhabdoviruses. In SYNV, the first AUG codon in the putative N-protein mRNA is located 57 nt downstream (at positions 203-205 in the viral genome) and is followed by an open reading frame for the remainder of the 1020 nt determined in these experiments.

Journal Article↗
PubMed2001

Identification of a conserved archaeal RNA polymerase subunit contacted by the basal transcription factor TFB.

Archaea possess two general transcription factors that are required to recruit RNA polymerase (RNAP) to promoters in vitro. These are TBP, the TATA-box-binding protein and TFB, the archaeal homologue of TFIIB. Thus, the archaeal and eucaryal transcription machineries are fundamentally related. In both RNAP II and archaeal transcription systems, direct contacts between TFB/TFIIB and the RNAP have been demonstrated to mediate recruitment of the polymerase to the promoter. However the subunit(s) directly contacted by these factors has not been identified. Using systematic yeast two-hybrid and biochemical analyses we have identified an interaction between the N-terminal domain of TFB and an evolutionarily conserved subunit of the RNA polymerase, RpoK. Intriguingly, homologues of RpoK are found in all three nuclear RNA polymerases (Rpb6) and also in the bacterial RNA polymerase (omega-subunit).

Amino Acid Sequence↗
PubMed2000

6S RNA regulates E. coli RNA polymerase activity.

The E. coli 6S RNA was discovered more than three decades ago, yet its function has remained elusive. Here, we demonstrate that 6S RNA associates with RNA polymerase in a highly specific and efficient manner. UV crosslinking experiments revealed that 6S RNA directly contacts the sigma70 and beta/beta' subunits of RNA polymerase. 6S RNA accumulates as cells reach the stationary phase of growth and mediates growth phase-specific changes in RNA polymerase. Stable association between sigma70 and core RNA polymerase in extracts is only observed in the presence of 6S RNA. We show 6S RNA represses expression from a sigma70-dependent promoter during stationary phase. Our results suggest that the interaction of 6S RNA with RNA polymerase modulates sigma70-holoenzyme activity.

Base Sequence↗
PubMed1998

In vitro synthesis of multicopy single-stranded DNA, using separate primer and template RNAs, by Escherichia coli reverse transcriptase.

A minor population of wild strains of Escherichia coli contains a retron, a retroelement responsible for the synthesis of multicopy single-stranded DNA (msDNA). The retron is a genetic element consisting of the gene for reverse transcriptase (RT) and the msr-msd region under a single promoter. A single RNA transcript from the msr-msd region serves not only as a template but also as a primer for msDNA synthesis. Here, using a cell-free system with purified RT from retron Ec73, we examined whether the reaction can occur in a bimolecular reaction with use of separately expressed msr and msd transcripts. DNA sequencing of the cell-free product revealed that the sequence of the 5'-end region was identical to that of msDNA-Ec73, indicating that the cDNA synthesis was primed from the 2'-OH group of the specific internal G residue of the primer RNA, identical to the branching G residue in the RNA molecule of msDNA-Ec73. The present results raise an intriguing possibility for a role of bacterial retrons in vivo, the possibility that cellular mRNAs can be converted into cDNAs in retron-harboring cells if the mRNAs contain a sequence complementary to the sequence directly upstream of the branching G residue of the msr RNA transcript.

DNA, Bacterial↗
PubMed1993

Sequence analysis of group B rotavirus gene 1 and definition of a rotavirus-specific sequence motif within the RNA polymerase gene.

The complete nucleic acid sequence was determined for the largest genomic segment of the IDIR strain of group B rotavirus and compared with RNA polymerase genes of rotavirus groups A and C as well as other RNA viruses. IDIR gene 1 contained 3509 bases with a single, long open reading frame which encoded a deduced polypeptide of 1159 amino acids (MW = 131.6 kDa; pl 8.851). The deduced amino acid sequence of IDIR gene 1 shared 50% similar sequences and 27.6% identical sequences with VP1 of the RF strain of group A rotavirus. IDIR gene 1 also contained 45.4% similar and 26.5% identical amino acid sequences in comparison with gene 1 of the Cowden strain of group C rotavirus. Amino acids 643-689 of IDIR gene 1 corresponded to the conserved viral RNA polymerase domains, "SG . . . T . . . NS . . N" and "GDD." Within these domains, group A, B, and C rotaviruses displayed substantial homologies that were not shared with other RNA viruses. These sequences indicated the presence of highly conserved structural or functional components among groups of rotaviruses which were otherwise quite heterogeneous. The identification of rotavirus-specific residues within RNA polymerase sequence may prove valuable in devising strategies aimed at the control of rotavirus replication and infection.

Amino Acid Sequence↗
PubMed1989

Tentative identification of RNA-dependent RNA polymerases of dsRNA viruses and their relationship to positive strand RNA viral polymerases.

Amino acid sequence stretches similar to the four most conserved segments of positive strand RNA viral RNA-dependent RNA polymerases have been identified in proteins of four dsRNA viruses belonging to three families, i.e. P2 protein of bacteriophage phi 6 (Cystoviridae), RNA 2 product of infectious bursa disease virus (Birnaviridae), lambda 3 protein of reovirus, and VP1 of bluetongue virus (Reoviridae). High statistical significance of the observed similarity was demonstrated, allowing identification of these proteins as likely candidates for RNA-dependent RNA polymerases. Based on these observations, and on the previously reported sequence similarity between the RNA polymerases of a yeast dsRNA virus and those of positive strand RNA viruses, a possible evolutionary relationship between the two virus classes is discussed.

Amino Acid Sequence↗