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Query: EC:2.7.7.49 (reverse transcriptase)
 31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have used the technique of in vitro selection to generate variants of human immunodeficiency virus type 1 (HIV-1) that are resistant to 2',3'-dideoxyinosine (ddI) and cross-resistant to 2',3'-dideoxycytidine (ddC). The complete reverse transcriptase (RT)-coding regions, plus portions of flanking sequences, of viruses possessing a ddI-resistant phenotype were cloned and sequenced by polymerase chain reaction (PCR)-based methods. We observed that several of these viruses possessed mutations at amino acid sites 184 (Met-->Val; ATG-->GTG) and 294 (Pro-->Ser; CCA-->TCA). These mutations were introduced in the pol gene of infectious, cloned HXB2-D DNA by site-directed mutagenesis. Viral replication assays confirmed the importance of site 184 with regard to resistance to ddI. The recombinant viruses thus generated displayed more than fivefold-greater resistance to ddI than parental HXB2-D did. Moreover, more than fivefold-greater resistance to ddC was also documented; however, the recombinant viruses continued to be inhibited by zidovudine (AZT). No resistance to ddI, ddC, or AZT was introduced by inclusion of mutation site 294 in the pol gene of HXB2-D. PCR analysis performed on viral samples obtained from patients receiving long-term ddI therapy confirmed the presence of mutation site 184 in five of seven cases tested. In three of these five positive cases, the wild-type codon was also detected, indicating that mixtures of viral quasispecies were apparently present. Viruses possessing a ddI resistance phenotype were isolated from both subjects whose viruses contained only the mutated rather than wild-type codon at position 184 as well as from a third individual, whose viruses appeared to be mostly of the mutated variety.
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PMID:Novel mutation in the human immunodeficiency virus type 1 reverse transcriptase gene that encodes cross-resistance to 2',3'-dideoxyinosine and 2',3'-dideoxycytidine. 127 98

We have examined the specificity of human immunodeficiency virus-1 (HIV-1) reverse transcriptase-associated RNase H in removing the tRNA(Lys3) (-)-strand primer in vitro using a model substrate. This substrate represents an intermediate in the reverse transcription process where the tRNA(Lys3) primer has not yet been removed after (+)-strand strong stop DNA synthesis. The substrate consists of an RNA oligonucleotide corresponding to the 3'-terminal 17 nucleotides of the tRNA(Lys3) linked to U5 DNA and annealed to single-stranded DNA containing the U5 and the primer-binding site. Upon incubation with HIV-1 reverse transcriptase p66/p51 heterodimer, the minus-strand DNA product resulting from RNase H cleavage retained the 3'-rA from the model tRNA primer. Changing the 3'-terminal AMP of the model tRNA primer from rA to dA did not alter the RNase H cleavage site. Further, the retention of AMP was not dependent on recognition of adjacent U5 sequences or the CCA terminus of the model tRNA(Lys3). The synthetic RNA primer was released as an intact species by a single endonucleolytic cleavage 5' of the rA. The cleavage patterns of Moloney murine leukemia virus and avian myoblastosis virus RNase H activities on the HIV-1 model substrate were more heterogeneous compared to HIV-1 RNase H. This specificity of HIV-1 RNase H would result in linear DNA molecules with a single rA at the U5 terminus and would provide two bases adjacent to the conserved CA dinucleotide to be cleaved away during the integration process.
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PMID:Specificity of human immunodeficiency virus-1 reverse transcriptase-associated ribonuclease H in removal of the minus-strand primer, tRNA(Lys3). 137 44

Several epidemiological studies have demonstrated an association between familial adenomatous polyposis coli (FAP) and thyroid neoplasms. Predisposition to FAP is conferred by mutations in the APC gene, located on chromosome 5q21. Somatic mutations of APC are also observed in about 60% of sporadic colorectal adenomas and carcinomas, suggesting that disruption of this putative tumor suppressor gene may play a role in both familial as well as acquired colorectal tumorigenesis. The APC gene is expressed in normal human thyroid, thyroid adenomas, and differentiated carcinoma tissues as well as in four clonal human thyroid carcinoma cell lines, as demonstrated by reverse transcriptase-polymerase chain reaction of a 388-base APC messenger ribonucleic acid fragment spanning exons 14 and 15, followed by hybridization to an exon 15-specific complementary DNA probe. Eighty human thyroid neoplasms were examined for loss of heterozygosity of the APC locus, using primers flanking a hypervariable dinucleotide (CA) repeat (CB26) immediately adjacent to the APC gene. Of 71% informative samples, 2 showed allelic loss: a follicular adenoma (FA) and a nodule from a multinodular goiter (MNG). The DNA of 83 benign and malignant thyroid neoplasms and 4 thyroid carcinoma cell lines was examined for mutations within a 1200-basepair stretch of exon 15 by single strand conformation polymorphism. Five sets of overlapping primers were used for PCR. The anaplastic thyroid carcinoma cell line (ARO) had 1 APC allele with an adenine insertion at codon 1556 (ACTA to AACTA), leading to a premature stop codon at 1558. An anaplastic carcinoma had a mutation of codon 1346 (TCA-CCA; Ser to Pro). In summary, the APC gene is expressed in normal and neoplastic human thyroid tissue and is a target for inactivating mutations in some thyroid tumors.
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PMID:Mutations of the adenomatous polyposis coli gene in sporadic thyroid neoplasms. 796 23

The Mauriceville plasmid and the closely related Varkud plasmid of Neurospora spp. are retroelements that propagate in mitochondria. Replication appears to occur by a novel mechanism in which a monomer-length plasmid transcript having a 3' tRNA-like structure ending in CCA is reverse transcribed to give a full-length minus-strand cDNA beginning at or near the 3' end of the RNA. Here, we show that the plasmids are transcribed in vitro by the Neurospora mitochondrial RNA polymerase, with the major in vitro transcription start site approximately 260 bp upstream of the 5' end of the plasmid transcript. The location of the transcription start site suggests that the monomer-length transcripts are generated by transcription around the plasmid combined with a site-specific RNA cleavage after the 3'-CCA sequence. The 5' ends of minus-strand cDNAs in ribonucleoprotein particles were analyzed to obtain insight into the mechanism of initiation of reverse transcription in vivo. A major class of minus-strand cDNAs begins opposite C2 of the 3'-CCA sequence, the same site used for de novo initiation of cDNA synthesis by the plasmid reverse transcriptase in vitro. A second class of minus-strand cDNAs begins with putative primer sequences that correspond to cDNA copies of the plasmid or mitochondrial transcripts. These findings are consistent with the possibility that the plasmid reverse transcriptase initiates minus-strand cDNA synthesis in vivo both by de novo initiation and by a novel template-switching mechanism in which the 3' OH of a previously synthesized cDNA is used to prime the synthesis of a new minus-strand cDNA directly at the 3' end of the plasmid transcript.
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PMID:The Mauriceville plasmid of Neurospora spp. uses novel mechanisms for initiating reverse transcription in vivo. 816 65

The molecular defect of uroporphyrinogen decarboxylase (UROD) was examined in a patient with mild hepatoerythropoietic porphyria. To elucidate the UROD defect, we cloned UROD cDNAs from EBV-transformed lymphoblastoid cells of the proband using reverse transcriptase-polymerase chain reaction. Nucleotide sequence analysis of the cloned UROD cDNAs revealed two separate missense mutations, each occurring in a separate allele. One mutation was a Val134-->Gln transition, and was due to three sequential point mutations (T417G418T419-->CCA); the other mutation was a His220-->Pro transition (A677-->C). UROD phenotype studies demonstrated that the TGT-->CCA mutation was inherited from the father, and the A-->C mutation was inherited from the mother. In contrast to the null activity previously described for a mutant UROD from a patient with familial porphyria cutanea tarda, these mutant URODs had subnormal but substantial enzyme activities, when expressed in Chinese hamster ovary cells. This is the first demonstration of a mutation caused by three sequential base substitutions.
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PMID:Molecular defects of uroporphyrinogen decarboxylase in a patient with mild hepatoerythropoietic porphyria. 817 48

The Mauriceville retroplasmid of Neurospora mitochondria encodes a novel reverse transcriptase that initiates cDNA synthesis de novo (i.e., without a primer) at the 3' CCA of the plasmid transcript's 3' tRNA-like structure (H. Wang and A. M. Lambowitz, Cell 75:1071-1081, 1993). Here, we show that the plasmid reverse transcriptase also initiates cDNA synthesis de novo at the 3' end of tRNAs, leading to synthesis of a full-length cDNA copy of the tRNA. The use of tRNA templates in vivo was suggested previously by the structure of suppressive mutant plasmids that have incorporated mitochondrial tRNA sequences (R. A. Akins, R. L. Kelley, and A. M. Lambowitz, Cell 47:505-516, 1986). The in vitro experiments show that efficient de novo initiation on tRNA templates requires an unpaired 3' CCA and occurs predominantly opposite position C-2 of the 3' CCA sequence, the same position as in the plasmid transcript. In other reactions, the plasmid reverse transcriptase synthesizes cDNA dimers by template switching between two tRNA templates and initiates at an internal position in a tRNA by using the 3' end of the tRNA as a primer. Finally, we show that template switching between the tRNA and the plasmid transcript in vitro gives rise to hybrid cDNAs of the type predicted to be intermediates in the generation of the suppressive mutant plasmids. The ability of the plasmid reverse transcriptase to initiate at the 3' end of tRNAs presumably reflects the recognition of structural features similar to those of the 3' tRNA-like structure of the plasmid transcript. The recognition of tRNAs or tRNA-like structures as templates for cDNA synthesis may be characteristic of primitive reverse transcriptases that evolved from RNA-dependent RNA polymerases.
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PMID:The Mauriceville retroplasmid reverse transcriptase initiates cDNA synthesis de novo at the 3' end of tRNAs. 923 10

The Mauriceville mitochondrial retroplasmid of Neurospora encodes a novel reverse transcriptase that initiates cDNA synthesis at a 3' tRNA-like structure of the plasmid transcript, either de novo (i.e. without a primer) or by using the 3' OH group of a DNA primer. Both the de novo and primer-mediated initiations involve recognition of structural features at the 3' end of the retroplasmid transcript, which ends with a 3' CCACCA. Here, detailed biochemical characterization of the retroplasmid reverse transcriptase shows that the 3' CCA of the plasmid transcript is the major structural feature recognized by the reverse transcriptase for both the de novo and primer-mediated initiations. Complementarity between the DNA primer and RNA template is not required for the primer-mediated initiation, although short (1 to 3 nt) base-pairing interactions can influence both the efficiency and site of initiation near the 3' end of the transcript. Single nucleotide changes in the 3' CCA lead to less efficient initiation in the upstream CCA with an increased propensity to add extra "non-coded" nucleotides to the 5' end of the cDNA during de novo initiation or to the 3' end of the primer during primer-mediated initiation. Secondary structure features upstream of the 3' CCA also influence the efficiency of initiation, but are not stringently required in vitro. Finally, we find that the retroplasmid reverse transcriptase does not efficiently use DNA primers that are base-paired to internal positions in the RNA template, nor does it use analogs of natural substrates used by non-long terminal repeat retrotransposon or retroviral reverse transcriptases. Our results indicate that the retroplasmid reverse transcriptase is uniquely adapted to initiate cDNA synthesis by recognizing a 3' CCA sequence. The ability to recognize a specific template sequence is common for RNA polymerases, but unprecedented for a reverse transcriptase.
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PMID:De novo and DNA primer-mediated initiation of cDNA synthesis by the mauriceville retroplasmid reverse transcriptase involve recognition of a 3' CCA sequence. 926 61

Retroviral reverse transcriptase (RT) is involved in the selection of a specific tRNA primer which initiates proviral DNA minus-strand synthesis. Studies of the interactions between human immunodeficiency virus type 1 (HIV-1) RT and primer tRNALys3 have shown that the dihydrouridine (diHU), anticodon, and pseudouridine regions of tRNA are highly protected in the RT-tRNA complex. The CCA 3' end of tRNA is also in close contact with the enzyme during the cDNA initiation step. Using synthetic oligoribonucleotides corresponding to the anticodon and diHU regions, we have previously shown a low but significant inhibition of HIV-1 RT activity. We extend this observation and show that primer tRNA-derived oligodeoxynucleotides (ODNs) carrying a phosphorothioate (PS) modification are strong inhibitors of HIV-1 RT. The affinity of PS-ODNs for the enzyme was monitored by gel mobility shift electrophoresis. Experiments with HIV-1-infected human cells (MT-2 cells) were performed with the latter ODNs. A PS-ODN corresponding to the 3' end of tRNALys3 (acceptor stem [AS]) was able to inhibit HIV-1 replication. No effect of the other modified ODNs was observed in infected cells. The analysis of HIV-1 RNase H activity in a cell-free system strongly suggests that the inhibitory effect of the PS-AS may be mediated via both a sense and an antisense mechanism.
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PMID:Phosphorothioate oligonucleotides derived from human immunodeficiency virus type 1 (HIV-1) primer tRNALys3 are strong inhibitors of HIV-1 reverse transcriptase and arrest viral replication in infected cells. 933 39

The complete nucleotide sequence of the Chlamydomonas eugametos (Chlamydomonadales, Chlorophyceae, sensu Mattox and Stewart) mitochondrial genome has been determined (22,897 bp, 34.6% G + C). The genes identified in this circular-mapping genome include those for apocytochrome b, subunit 1 of the cytochrome oxidase complex, subunits 1, 2, 4, 5, and 6 of the NADH dehydrogenase complex, discontinuous large and small subunit ribosomal rRNAs and three tRNAs whose anticodons CAU, CCA and UUG are specific for methionine, tryptophan and glutamine, respectively. The C. eugametos mitochondrial DNA (mtDNA), therefore, shares almost the same reduced set of coding functions and similar unusual features of rRNA gene organization with the linear 15.8 kb mtDNA of Chlamydomonas reinhardtii, the only other completely sequenced chlamydomonadalean mtDNA. However, sequence analysis of the C. eugametos mtDNA has revealed the following distinguishing features relative to those of C. reinhardtii: (1) the absence of a reverse transcriptase-like gene homologue, (2) the presence of an additional gene for tRNA(met) that may be a pseudogene, (3) a completely different gene order, (4) transcription of all genes from the same mtDNA strand, (5) a lower G + C content, (6) less pronounced bias in codon usage, and (7) nine group I introns, several of which contain open reading frames coding for potential maturases/endonucleases and two have a nucleotide at the 5' or 3' splice site of the deduced precursor RNAs that deviates from highly conserved nucleotides reported in other group I introns. The features of mitochondrial genome organization and gene content shared by C. eugametos and C. reinhardtii contrast with those of other green algal mtDNAs that have been characterized in detail. The deep evolutionary divergence between these two Chlamydomonas taxa within the Chlamydomonadales suggests that their shared features of mitochondrial genome organization evolved prior to the origin of this group.
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PMID:Complete sequence of the mitochondrial DNA of Chlamydomonas eugametos. 948 40

Degenerative processes in the filamentous fungus Podospora anserina are strongly correlated with the instability of the mitochondrial genome. Among the sources of instability is the mobile group-II intron COX1-i1, also called intron alpha, which encodes a protein with a reverse transcriptase activity. In this paper we characterize, through PCR experiments, mitochondrial recombinant DNA molecules joining the 5' end of intron alpha to the 3' end of tRNA sequences including the CCA motif. The structure of these junctions led us to propose that they were most probably initiated by a RNA template switching of the reverse transcriptase encoded in COX1-i1. This activity might be involved in a number of mitochondrial rearrangements occurring in degenerative syndromes and in some long-lived mutants.
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PMID:Recombinant mitochondrial DNA molecules suggest a template switching ability for group-II-intron reverse transcriptase. 1067 40


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