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At least 1,243 records · Page 69Linked to original sources
PubMed1993

Characterization of novel reverse transcriptase encoding human endogenous retroviral sequences similar to type A and type B retroviruses: differential transcription in normal human tissues.

The polymerase chain reaction was used to amplify genomic DNA and reverse-transcribed RNA from human lymphocytes, using primers derived from conserved regions within the retroviral reverse transcriptase. Sequencing of 33 cloned amplification products revealed that a variety of sequences with similarity to mouse mammary tumor virus, mouse intracisternal A particle, and human endogenous retrovirus K10 were detected with this primer pair. The sequences were divided into six subgroups, with a nucleotide sequence dissimilarity of about 25% between the subgroups. Members within five of the subgroups were most closely related to human endogenous retrovirus K10 and mouse mammary tumor virus, whereas sequences of the sixth subgroup also showed similarity to mouse intracisternal A particle. Ten of the sequences had open reading frames with preference for silent mutations at conserved sites. Southern blot analysis showed that some HML (human endogenous MMTV-like) subgroups (HML-4 and HML-5) were present in a few copies (about 5), whereas others (HML-1 to HML-3 and HML-6) were present in at least 10 to 20 copies per genome. Northern (RNA) blot analysis revealed that several of the subgroups are differentially expressed in human normal tissues. A complex pattern of transcripts from about 12 to 1.4 kb was found in several of the tissues tested. However, the most abundant expression was detected in lung (all subgroups), skeletal muscle (HML-4 and HML-5), placenta (HML-2 and HML-5), and kidney (HML-2, HML-3 and HML-5). Expression of reverse transcriptase sequences in human tissues may have biological consequences. The described sequences are similar to elements which cause carcinoma and are immunoregulatory in mice. It remains to be seen whether human sequences also have such functions.

Base Sequence↗
PubMed1994

Isolation and characterization of simian T-cell leukemia virus type II from New World monkeys.

Since the description of human T-cell leukemia virus type I (HTLV-I) and its simian counterpart, simian T-cell leukemia virus type I (STLV-I), the possible existence of other related simian retroviruses has been raised. Here, we report a new retrovirus, STLV-II, which we have identified in spider monkeys (Ateles fusciceps), a New World primate species. Initially, a recombinant HTLV-II envelope protein (RP-IIB) was used to identify anti-STLV-II antibodies in New World monkeys by Western blot (immunoblot) assays. Subsequently, the virus was characterized by Southern blot hybridization, which showed that STLV-II and HTLV-II have a high degree of nucleotide sequence homology but have different restriction enzyme patterns. Nucleotide sequence analysis of the pX-II region of STLV-II provirus revealed 3% variation with the corresponding region of HTLV-II. Electron micrographic studies revealed HTLV-like, type C retrovirus particles outside the cell membranes of STLV-II-infected cells. This study describes the first link between HTLV-II and a simian reservoir in the New World. Further molecular studies of STLV-II infection in different species of New World monkeys, especially from the wild, may provide valuable information about the origin and intragroup relationships of South American monkeys. Spider monkeys infected with STLV-II may serve as an important animal model for HTLV-II infection in humans.

Animals↗
PubMed1997

Human endogenous retrovirus type I-related viruses have an apparently widespread distribution within vertebrates.

Retroviruses from lower vertebrate hosts have been poorly characterized to date. Few sequences have been isolated, and those which have been reported are all highly divergent when compared to the retroviruses known to be harbored by mammals and birds. Here we show that retroviruses with significant homology to the human endogenous retrovirus type I (HERV-I) are present within the genomes of fish, reptiles, birds, and mammals and that they may well be widespread within many vertebrates. Phylogenetic analysis of nucleotide sequences strongly supported the inclusion of viruses from each of these vertebrate classes into one monophyletic group. This analysis also demonstrated that the HERV-I-related viruses are more closely related to retroviruses belonging to the murine leukemia virus genus than to members of the other retroviral genera. The presence of HERV-I-related retroviruses in so many disparate vertebrate hosts suggests that other endogenous human retroviruses may also have a much wider distribution than is currently appreciated.

Amino Acid Sequence↗
PubMed1997

Attenuation of a human rotavirus vaccine candidate did not correlate with mutations in the NSP4 protein gene.

The NSP4 protein of a simian rotavirus was reported to induce diarrhea following inoculation of mice. If NSP4 is responsible for rotavirus diarrhea in humans, attenuation of a human rotavirus may be reflected in concomitant mutations in the NSP4 gene. After 33 passages in cultured monkey kidney cells, a virulent human rotavirus (strain 89-12) was found to be attenuated in adults, children, and infants. Nucleotide sequence analysis of the NSP4 protein gene revealed only one base pair change between the virulent (unpassaged) and attenuated 89-12 viruses, which resulted from a substitution of alanine for threonine at amino acid 45 of the encoded NSP4 protein. Because both threonine and alanine have been found at position 45 of NSP4 in symptomatic and asymptomatic human rotaviruses, neither amino acid in this position could be established as a marker of virulence. Therefore, attenuation of rotavirus strain 89-12 appears to be unrelated to mutations in the NSP4 gene.

Adult↗
PubMed1996

Downstream 28S gene sequences on the RNA template affect the choice of primer and the accuracy of initiation by the R2 reverse transcriptase.

R2 non-long terminal repeat retrotransposable elements insert at a unique site in the 28S rRNA genes of insects. The protein encoded by the single open reading frame of R2 is capable of conducting the initial steps of its integration in vitro. The protein nicks the noncoding strand of the 28S target DNA (the strand which serves as a template for RNA synthesis) and uses the 3' hydroxyl group exposed by this nick to prime reverse transcription of the R2 RNA template. This target-primed reverse transcription (TPRT) reaction requires that the RNA template contains the 250-nucleotide 3' untranslated region of the R2 element. If this RNA template ends at the precise 3' end of the R2 element, then extra nucleotides, which we refer to as nontemplated nucleotides, are added to the target before cDNA synthesis. The presence of downstream 28S gene sequences on the RNA template reduces the total efficiency but eliminates these nontemplated additions, resulting in nearly 90% of all TPRT products reproducing the 3' junctions seen in vivo. Templates with 5 to 10 nucleotides of the 28S sequence are used most efficiently in this in vitro TPRT reaction. The requirement for downstream 28S rRNA sequences probably explains why the R2 elements of most insects differ from the majority of non-long terminal repeat retrotransposons in that they do not contain an A-rich repeat at their 3' junction with the target DNA. The presence of downstream sequences on these in vitro R2 templates also revealed that the R2 reverse transcriptase can prime cDNA synthesis by using the 3' end of another RNA molecule. This RNA-primed cDNA synthesis is not based on sequence complementarity between the RNA primer and the R2 template. The ability to use the 3' end of a noncomplementary RNA molecule has also been seen with the reverse transcriptase of the mitochondrial Mauriceville plasmid of Neurospora crassa.

Animals↗
PubMed2003

Divergent long-terminal-repeat retrotransposon families in the genome of Paragonimus westermani.

To gain information on retrotransposons in the genome of Paragonimus westermani, PCR was carried out with degenerate primers, specific to protease and reverse transcriptase (rt) genes of long-terminal-repeat (LTR) retrotransposons. The PCR products were cloned and sequenced, after which 12 different retrotransposon-related sequences were isolated from the trematode genome. These showed various degrees of identity to the polyprotein of divergent retrotransposon families. A phylogenetic analysis demonstrated that these sequences could be classified into three different families of LTR retrotransposons, namely, Xena, Bel, and Gypsy families. Of these, two mRNA transcripts were detected by reverse transcriptase-PCR, showing that these two elements preserved their mobile activities. The genomic distributions of these two sequences were found to be highly repetitive. These results suggest that there are diverse retrotransposons including the ancient Xena family in the genome of P. westermani, which may have been involved in the evolution of the host genome.

Amino Acid Sequence↗
PubMed1995

Identification of protein motifs using conserved amino acid properties and partitioning techniques.

Analyzing a set of protein sequences involves a fundamental relationship between the coherency of the set and the specificity of the motif that describes it. Motifs may be obscured by training sets that contain incoherent sequences, in part due to protein subclasses, contamination, or errors. We develop an algorithm for motif identification that systematically explores possible patterns of coherency within a set of protein sequences. Our algorithm constructs alternative partitions of the training set data, where one subset of each partition is presumed to contain coherent data and is used for forming a motif. The motif is represented by multiple overlapping amino acid groups based on evolutionary, biochemical, or physical properties. We demonstrate our method on a training set of reverse transcriptases that contains subclasses, sequence errors, misalignments, and contaminating sequences. Despite these complications, our program identifies a novel motif for the subclass of retroviral and retrovirus-related reverse transcriptases. This motif has a much higher specificity than previously reported motifs and suggests the importance of conserved hydrophilic and hydrophobic residues in the structure of reverse transcriptases.

Algorithms↗
PubMed2002

Changes in conserved region 3 of Escherichia coli sigma 70 reduce abortive transcription and enhance promoter escape.

Mutations within the Escherichia coli rpoD gene encoding amino acid substitutions in conserved region 3 of the sigma(70) subunit of E. coli RNA polymerase restore normal stress responsiveness to strains devoid of the stress alarmone, guanosine-3',5'-(bis)pyrophosphate (ppGpp). The presence of a mutant protein, either sigma(70)(P504L) or sigma(70)(S506F), suppresses the physiological defects in strains devoid of ppGpp. In vitro, when reconstituted into RNA polymerase holoenzyme, these sigma mutants confer unique transcriptional properties, namely they reduce the probabilities of forming abortive RNAs. Here we investigated the behavior of these mutant enzymes during transcription of the highly abortive cellular promoter, gal P2. No differences between mutant and wild-type enzymes were observed prior to and including open complex formation. Remarkably, the mutant enzymes produced drastically reduced levels of gal P2 abortive RNAs and increased production of full-length gal P2 RNAs relative to the wild-type enzyme, leading to greatly reduced ratios of abortive to productive RNAs. These results are attributed mainly to a decreased formation of unproductive initial transcribing complexes with the mutant polymerases and increased rates of promoter escape. Altered transcription properties of these mutant polymerases arise from an alternative structure of the sigma(70) region 3.2 segment that permits efficient positioning of the nascent RNA into the RNA exit channel displacing sigma and facilitating sigma release.

Amino Acid Sequence↗
PubMed1992

Chloroplast RNA polymerase genes of Chlamydomonas reinhardtii exhibit an unusual structure and arrangement.

Nucleotide sequence analysis of a 17043 base-pair (bp) region of the Chlamydomonas reinhardtii plastome indicates the presence of three open reading frames (ORFs) similar to RNA polymerase subunit genes. Two, termed rpoB1 and rpoB2, are homologous to the 5'- and 3'-halves of the Escherichia coli beta subunit gene, respectively. A third, termed rpoC2, is similar to the 3'-half of the bacterial beta' subunit gene. These genes exhibit several unusual features: (1) all three represent chimeric structures in which RNA polymerase gene sequences are juxtaposed in-frame with long sequences of unknown identity; (2) unlike their counterparts in plants and eubacteria, rpoB1 and rpoB2 are separated from rpoC2 by a long (7 kilobase-pair, kbp) region containing genes unrelated to RNA polymerase; (3) DNA homologous to the 5' half of rpoC (termed rpoC1 in other species) is not present at the 5' end of rpoC2 and could not be detected in C. reinhardtii chloroplast DNA. RNA expression could not be detected for any of the RNA polymerase genes, suggesting that they are pseudogenes or genes expressed at stages of the C. reinhardtii life-cycle not investigated. The three genes are flanked by GC-rich repeat elements. We suggest that repeat DNA-mediated chloroplast recombination events may have contributed to their unusual arrangement.

Amino Acid Sequence↗
PubMed2001

Mg2+ does not induce isomerization of the open transcription complex of Escherichia coli RNA polymerase at the model Pa promoter bearing consensus -10 and -35 hexamers.

The kinetics and thermodynamics of the formation of the transcriptional open complex (RPo) by Escherichia coli RNA polymerase at the synthetic Pa promoter bearing consensus -10 and -35 recognition hexamers were studied in vitro. Previously, this promoter was used as a control one in studies on the effect of DNA bending by An x Tn sequences on transcription initiation and shown to be fully functional in E. coli (Loziński et al., 1991, Nucleic Acids Res. 19, 2947; Loziński & Wierzchowski, 1996, Acta Biochim. Polon. 43, 265). The data now obtained demonstrate that the mechanism of Pa-RPo formation and dissociation conforms to the three-step reaction model: bind-nucleate-melt, commonly accepted for natural promoters. Measurements of the dissociation rate constant of Pa-RPo as a function of MgCl2 concentration allowed us to determine the number of Mg2+ ions, nMg approximately/= 4, being bound to the RPo in the course of renaturation of the melted DNA region. This number was found constant in the temperature range of 25-37 degrees C, which indicates that under these conditions the complex remaines fully open. This observation, taken together with the recent evidence from KMnO4 footprinting studies that the length of the melted region in Pa-RPo at 37 degrees C is independent of the presence of Mg2+ ions (Lozinski & Wierzchowski, 2001, Acta Biochim. Polon. 48, 495), testifies that binding of Mg2+ to RPo does not induce its further isomerization, which has been postulated for the lambdaP(R)-RPo complex (Suh et al., 1992, Biochemistry 31, 7815; 1993, Science 259, 358).

Base Sequence↗
PubMed2001

A Mutation in the C-terminal domain of the RNA polymerase alpha subunit that destabilizes the open complexes formed at the phage phi 29 late A3 promoter.

Regulatory protein p4 from Bacillus subtilis phage phi29 activates the viral late A3 promoter mainly by stabilizing the binding of RNA polymerase (RNAP) to it as a closed complex. This requires an interaction between protein p4 residue Arg120 and the C-terminal domain (CTD) of the RNAP alpha subunit. Several acidic residues of the alpha-CTD, considered as plausible targets for p4 residue Arg120, were individually changed into alanine. In addition, a truncated alpha subunit lacking the last four residues, two of which are acidic, was obtained. The modified alpha subunits were purified and reconstituted into RNAP holoenzyme in vitro. Protein p4 was found to be unable to activate the late A3 promoter when residue Glu297 of the alpha subunit was changed to Ala, a modification that did not impair transcription from several other promoters. Interestingly, protein p4 could stabilize the modified RNAP at the A3 promoter as a closed complex, although the open complexes formed were unstable and did not proceed to elongation complexes. Our results indicate that the change of the alpha residue Glu297 into Ala destabilizes the open complexes formed at this promoter, but not at other promoters. Considered in the context of earlier findings indicating that the RNAP alpha-CTD may participate in the transition from closed to intermediate complexes at some other promoters, the new results expand and clarify our view of its role in transcription initiation.

Amino Acid Sequence↗
PubMed1986

U6 small nuclear RNA is transcribed by RNA polymerase III.

A DNA fragment homologous to U6 small nuclear RNA was isolated from a human genomic library and sequenced. The immediate 5'-flanking region of the U6 DNA clone had significant homology with a potential mouse U6 gene, including a "TATA box" at a position 26-29 nucleotides upstream from the transcription start site. Although this sequence element is characteristic of RNA polymerase II promoters, the U6 gene also contained a polymerase III "box A" intragenic control region and a typical run of five thymines at the 3' terminus (noncoding strand). The human U6 DNA clone was accurately transcribed in a HeLa cell S100 extract lacking polymerase II activity. U6 RNA transcription in the S100 extract was resistant to alpha-amanitin at 1 microgram/ml but was completely inhibited at 200 micrograms/ml. A comparison of fingerprints of the in vitro transcript and of U6 RNA synthesized in vivo revealed sequence congruence. U6 RNA synthesis in isolated HeLa cell nuclei also displayed low sensitivity to alpha-amanitin, in contrast to U1 and U2 RNA transcription, which was inhibited greater than 90% at 1 microgram/ml. In addition, U6 RNA synthesized in isolated nuclei was efficiently immunoprecipitated by an antibody against the La antigen, a protein known to bind most other RNA polymerase III transcripts. These results establish that, in contrast to the polymerase II-directed transcription of mammalian genes for U1-U5 small nuclear RNAs, human U6 RNA is transcribed by RNA polymerase III.

Amanitins↗
PubMed1989

Molecular characterization of the Trypanosoma brucei RNA polymerase I and III largest subunit genes.

We have sequenced a 7.1-kilobase fragment of the Trypanosoma brucei RNA polymerase (pol) I largest subunit gene. The 6.9-kilobase transcript from this gene is of roughly equal abundance in bloodstream- and procyclic-form trypanosomes and has a 39-nucleotide trans-spliced leader 290 nucleotides upsteam of the putative initiation codon. The 1781-amino acid trypanosome pol I polypeptide is considerably less similar to its yeast homolog than the trypanosome pol II and pol III polypeptides are to their counterparts in yeast. The yeast and trypanosome pol I polypeptides are distinguished from pol II and pol III largest subunits by two inserts, 100-200 amino acids in length, which are found in the nonconserved portions of the polypeptides. We also report our sequence of the trypanosome pol III largest subunit gene, which agrees with that reported by Köck et al. (Köck, J., Evers, R., and Cornelissen, A.W.C.A. (1988) Nucleic Acids Res. 16, 8753-8772) except for 13 nucleotide differences. Four spliced leader addition sites between 60 and 99 nucleotides upstream of the AUG initiation codon were observed for the 5.4-kilobase transcript from this gene. Four short stretches of amino acid homology between the yeast and trypanosome pol III largest subunit polypeptides were identified which appear to be specific to this class of RNA polymerase; only one short pol I-specific sequence was identified in our comparison of the yeast and trypanosome pol I largest subunit polypeptides.

Amino Acid Sequence↗
PubMed1999

Intragenic suppression of trans-dominant lethal substitutions in the conserved GEME motif of the beta subunit of RNA polymerase: evidence for functional cooperativity within the C-terminus.

BACKGROUND: The ubiquitous multimeric RNA polymerases contain two large, conserved subunits, of which the beta subunit has been implicated in all three stages of transcription. We have previously described a genetic system involving random, PCR-mediated mutagenesis of the region of rpoB encoding the C-terminal 116 amino acids of the beta subunit of Escherichia coli RNA polymerase and the characterization of dominant-negative mutations. This study identified the invariant motif GEME (residues 1271-->1274; Cromie et al. 1999). Starting with three of these GEME-motif lethal mutations (G1271E, G1271V, M1273V), we have selected for intragenic suppressors, located within the same 3'-region, that prevent expression of the trans-dominant phenotype. RESULTS: We isolated a total of 24 missense mutants and a further 14 frameshift alleles (the latter generating a nested set of C-terminal deletions of the beta subunit) and studied the effect of the missense suppressors in vivo and in vitro. The majority of the second-site substitutions pinpoint highly conserved residues and were allele-specific. In contrast, one particular missense substitution (S1332P) acted on all three primary site mutations whilst not appreciably affecting assembly proficiency, suggesting motif-specific suppression. Two missense substitutions were found to perturb assembly of the beta subunit (M1232T and L1233P) and define a small conserved region (1228-->1233) adjacent to one of the active-site residues identified by affinity-labelling, H1237. The majority of primary mutations were located in three main clusters within the 116 amino acid region. CONCLUSIONS: The importance and functional co-operativity of the three main clusters pinpointed is supported by the present isolation of suppressors of three different GEME primary mutations in the same three regions (whereas the suppressors of G1271V and M1273V are located in all three clusters, those for G1271E are all C-terminal of this residue). Moreover, the location of the suppressors suggests that the GEME and HLVDDK regions are present as alpha-helices in holoenzyme, and that functional co-operativity is through one particular face of each helix.

Amino Acid Motifs↗
PubMed1998

Analysis of RNA-dependent RNA polymerase structure and function as guided by known polymerase structures and computer predictions of secondary structure.

RNA-dependent RNA polymerases (RdRps) function as the catalytic subunit of the viral replicase required for the replication of all positive strand RNA viruses. The vast majority of RdRps have been identified solely on the basis of sequence similarity. Structural studies of RdRps have lagged behind those of the DNA-dependent DNA polymerases, DNA-dependent RNA polymerases, and reverse transcriptases until the recent report of the partial crystal structure of the poliovirus RdRp, 3Dpol [Hansen, J. L., et al. (1997). Structure 5, 1109-1122]. We seek to address whether all RdRps will have structures similar to those found in the poliovirus polymerase structure. Therefore, the PHD method of Rost and Sander [Rost, B., and Sander, C. (1993a). J. Mol. Biol. 232, 584-599; Rost, B., and Sander, C. (1994). Protein 19, 55-77] was used to predict the secondary structure of the RdRps from six different viral families: bromoviruses, tobamoviruses, tombusvirus, leviviruses, hepatitis C-like viruses, and picornaviruses. These predictions were compared with the known crystal structure of the poliovirus polymerase. The PHD method was also used to predict picornavirus structures in places in which the poliovirus crystal structure was disordered. All five families and the picornaviruses share a similar order of secondary structure elements present in their polymerase proteins. All except the leviviruses have the unique region observed in the poliovirus 3Dpol that is suggested to be involved in polymerase oligomerization. These structural predictions are used to explain the phenotypes of a collection of mutations that exist in several RNA polymerases. This analysis will help to guide further characterization of RdRps.

Amino Acid Sequence↗
PubMed1993

An abundant LINE-like element amplified in the genome of Lilium speciosum.

The genomes of Lilium species are very large, containing 30-40 million kilobase pairs of DNA. An abundant fragment of 3.5 kb was released by BamHI digestion of genomic DNA of Lilium speciosum. Analysis of 20 genomic clones containing sequences homologous to the fragment showed it to be part of a 4.45 kb dispersed repeat, which was named del2. Sequence analysis of one full element and regions of four others revealed del2 to be a non-LTR (long terminal repeat) retrotransposon. It is flanked by short direct repeats of from 4 to 13 bp and a run of adenines occurs at one end (the proposed 3' end), 63 bp downstream from a polyadenylation signal. A possible RNA polymerase II promoter similar to that found in Drosophila I and F group elements is present internally 30 bp downstream from the 5' end. Two degenerate open reading frames (ORFs) are present, the 5' ORF containing a gag-related cysteine motif, and the 3' ORF containing a different cysteine motif also found in most non-LTR retrotransposons. The 3' ORF also has regions with homology to reverse transcriptase sequences, which are most similar to those in Cin4 of maize, the L1 LINE elements of humans and mice and the R2 ribosomal DNA inserts of insects. The majority of del2 elements occur as the full 4.45 kb element. They account for an estimated 4% of the L. speciosum genome and are present in approximately 250,000 copies. del2-related sequences were also detected in 12 other monocot species.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2003

Biochemical and genetic studies of the VPg uridylylation reaction catalyzed by the RNA polymerase of poliovirus.

The first step in poliovirus (PV) RNA synthesis is the covalent linkage of UMP to the terminal protein VPg. This reaction can be studied in vitro with two different assays. The simpler assay is based on a poly(A) template and requires synthetic VPg, purified RNA polymerase 3D(pol), UTP, and a divalent cation. The other assay uses specific viral sequences [cre(2C)] as a template for VPg uridylylation and requires the addition of proteinase 3CD(pro). Using one or both of these assays, we analyzed the VPg specificities and metal requirements of the uridylylation reactions. We determined the effects of single and double amino acid substitutions in VPg on the abilities of the peptides to serve as substrates for 3D(pol). Mutations in VPg, which interfered with uridylylation in vitro, were found to abolish viral growth. A chimeric PV containing the VPg of human rhinovirus 14 (HRV14) was viable, but substitutions of HRV2 and HRV89 VPgs for PV VPg were lethal. Of the three rhinoviral VPgs tested, only the HRV14 peptide was found to function as a substrate for PV1(M) 3D(pol) in vitro. We also examined the metal specificity of the VPg uridylylation reaction on a poly(A) template. Our results show a strong preference of the RNA polymerase for Mn(2+) as a cofactor compared to Mg(2+) or other divalent cations.

Amino Acid Sequence↗