Gene/Protein Disease Symptom Drug Enzyme Compound
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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)

The sequence of 129 nucleotides next to the poly(A) tail of encephalomyocarditis virus RNA has been determined by rapid gel sequencing of cDNA synthesized with DNA polymerase I or reverse transcriptase and a phasing primer, [5'-32P]p(dT)8dC. The sequence is in accord with (a) the pyrimidine tracts which were mapped in blocks along the cDNA, (B) the sequences of seven characteristic T1 RNase oligonucleotides in the RNA transcribed from the cDNA with RNA polymerase, and (c) a limited amount of sequence deduced by partial spleen phosphodiesterase digestion and depurination of endonuclease IV oligonucleotides. The 3' end shows little secondary structure on its own. Ten nonsense codons block all three reading frames such that at least 26 nucleotides do not code for protein. The possible function of a homology A-A-U-A-A-A with other polyadenylated RNAs is discussed.
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PMID:Sequence of 129 nucleotides at the 3'-terminus of encephalomyocarditis virus RNA. 7 85

The cytochrome P450 2C24 gene is characterized by the capability to generate, in rat kidney, a transcript containing exons 2 and 4 spliced at correct sites but having the donor site of exon 4 directly joined to the acceptor site of exon 2 (exon scrambling). By reverse transcriptase-PCR analysis, it is now shown that the only exons present in the scrambled transcript are exons 2, 3, and 4 and that this molecule lacks a poly(A)+ tail. Furthermore, the use of PCR primers in both orientations of either exon 2 or exon 4 revealed that the orders of the exons in the scrambled transcript are 2-3-4-2 and 4-2-3-4, respectively. These results, combined with the observation that P450 2C24 is a single-copy gene, with no duplication of the exon 2 to exon 4 segment, suggest that the scrambled transcript has properties consistent with that of a circular molecule. In line with this is the observation of an increased resistance of the transcript to phosphodiesterase I, a 3'-exonuclease. Moreover, an alternatively processed cytochrome P450 2C24 mRNA, lacking the three scrambled exons and having exon 1 directly joined to exon 5, has been identified in kidney and liver, tissues that express the scrambled transcript. This complete identity of the exons that are absent in the alternatively processed mRNA but present in the scrambled transcript is interpreted as indicative of the possibility that exon scrambling and exon skipping might be interrelated phenomena. It is therefore proposed that alternative pre-mRNA processing has the potential to generate not only mRNAs lacking one or more exons but also circular RNA molecules.
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PMID:Circular RNAs from transcripts of the rat cytochrome P450 2C24 gene: correlation with exon skipping. 869 51

Spatial orientations of bulky DNA adducts can influence the extent of resistance to digestion by exonucleases and translesion synthesis by HIV-1 reverse transcriptase (HIV-1 RT). In order to determine how different diastereomers of benzo[a]pyrene 7,8-dihydrodiol 9,10-epoxide (BPDE)-adducted DNAs influence the activity of these enzymes, 11-mer and 33-mer oligodeoxyribonucleotides were synthesized bearing site-specific and stereospecific BPDE adducts at adenine N6 on position two of the human N-ras codon 61. Phosphodiesterase I, which hydrolyzes DNA in the 3'-->5' direction, exhibited greater resistance opposite the lesion with C10-R BPDE-adducted templates than the corresponding C10-S adducts. However, the opposite stereoselective resistance to digestion was observed with phosphodiesterase II, which hydrolyzes DNA in the 5'-->3' direction. These results are complemented by the in vitro replication pattern exhibited with HIV-1 RT. Primer extension reactions under conditions defining single encounters between polymerase and substrate revealed adduct-dependent termination one base 3' to each of the lesions. When experimental conditions were altered to permit multiple encounters, HIV-1 RT was able to replicate past the damaged site on four of the six adducted templates, exhibiting little pausing opposite the lesion. Analyses of the replication pattern past these lesions revealed two general categories of replication blockage, which, like the exonucleolytic digestion data, were also based on the C10-R and C10-S configuration of the stereoisomers. Thus, the chirality of BPDE-dA adducts modulates enzymatic functions. Furthermore, the (+)- and (-)-anti-trans-BPDE-dA modified templates exhibited the most facile bypass, while the (+)- and (-)-anti-cis-BPDE adducts were most blocking.
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PMID:Impact of the stereochemistry of benzo[a]pyrene 7,8-dihydrodiol 9,10-epoxide-deoxyadenosine adducts on resistance to digestion by phosphodiesterases I and II and translesion synthesis with HIV-1 reverse transcriptase. 883 43

Reverse transcription of HIV-1 vRNA into the double-stranded DNA provirus involves initiation of plus-strand DNA synthesis at the polypurine tract (PPT) by reverse transcriptase (RT). The PPT is highly conserved among the known human immunodeficiency virus (HIV-1) strains and is a possible target for triplex formation. We show the effects of triple-helix formation by assays of primer extension inhibition in vitro, using a two-strand system (foldback triplex-forming oligonucleotides (FTFOs)) targeted to the PPT of HIV-1. The two-stranded composition of a triple-helix is thermodynamically and kinetically superior to the three-strand system. The FTFOs inhibited the RT activity in a sequence-specific manner, i.e. the triplex actually formed at the PPT and blocked the RT. The FTFOs containing the phosphorothioate groups at the antisense sequences showed greater 3'-exonuclease resistance. In HIV-1-infected MOLT-4 cells, the FTFOs containing the phosphorothioate groups at the antisense sequence sites and guanosine rich parts within the third Hoogsteen base-pairing sequence inhibit the replication of HIV-1 more effectively than the antisense oligonucleotides, indicating sequence-specific inhibition of HIV-1 replication.
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PMID:Inhibition of HIV-1 replication by a two-strand system (FTFOs) targeted to the polypurine tract. 1045 55

Reverse transcription of HIV-1 into double-stranded DNA involves initiation of plus-strand DNA synthesis at the polypurine tract, PPT, by reverse transcriptase (RT). The PPT is a possible target for triple-helix formation. We show the effects of triple-helix formation by assays of RNase H cleavage inhibition in vitro using two systems (two-strand-system (FTFOs) or three-strand-system (TFOs)) targeted to the polypurine tract (PPT) of HIV-1. The two-stranded composition of a triple-helix is thermodynamically and kinetically superior to the three-strand-system. The FTFOs inhibited the RNase H activity in a sequence-specific manner, i.e., the triplex actually formed at the PPT and blocked the RNase H. The FTFOs containing the phosphorothioate groups at the antisense strand showed greater 3'-exonuclease resistance. In HIV-1 infected MT-4 cells, the FTFOs containing the phosphorothioate groups at the antisense strand and guanosine rich parts within the third Hoogsteen base pairing sequence inhibit the replication of HIV-1 more effectively than the antisense phosphorothioate oligonucleotides, indicating sequence-specific inhibition of HIV-1 replication.
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PMID:Anti-HIV-1 activity by a triple-helix forming oligonucleotides targeted to polypurine tract on viral RNA. 1120 Feb 68

For many years, dengue viruses were among the most difficult flaviviruses to isolate and to identify, but technical advances in the past 20 years have facilitated this process. Dengue viruses are usually recovered from specimens by the infection of mosquito-cell cultures. The virus may be passaged several times in cell cultures until a sufficient infectivity titer is attained. The viral nucleocapsid consists of capsid protein and an RNA genome. The dengue genome is a single stranded messenger (positive) sense RNA of approximately 11 kb in length. The isolation of dengue genomic RNA from various sources requires precautions to avoid RNases. RNases are released during cell disruption, and their activity must be inhibited as quickly as possible by using guanidinium thiocyanate in the presence of 2-mercaptoethanol. There has recently been a revolution in molecular biology with the development of the powerful reverse transcriptase (RT) and polymerase chain reaction (PCR) technology. Advanced studies on RT technique lead to much further improvement of the reverse transcriptase enzyme by genetic engineering. The Superscript II RNase H- RT (GIBCO BRL, USA) is genetically engineered DNA polymerase that synthesizes a complementary DNA strand from single-stranded RNA. DNA or an RNA-DNA hybrid. This enzyme is produced from a cloned M-MLV RT gene constructed by the introduction of point mutation in the RNase H active center. The selective mutations within the RNase H domain maintain full polymerase activity. This structural modification eliminates degradation of RNA molecules during the first strand cDNA synthesis. The combination of thermostable DNA polymerase with and without proofreading activity (3'-exonuclease activity), improved buffer conditions and thermal cycling profiles overcome the length limitation of PCR. On the basis of these findings, we have developed a long RT-PCR system for preparing large cDNA fragments of dengue 3 virus (H-87) by using the Superscript II RNase H- RT for reverse transcription and a mixture of Taq and Pwo DNA polymerases for PCR. Three large cDNA fragments covered the full genomic RNA from the 5'-end to the 3'-end of dengue-3 virus (H-87; 10,696 bps) could be successfully prepared as the lengths of 2.437 bps, 3,980 bps and 4,337 bps respectively. The ability of our developed long RT-PCR will bring speed and simplicity to genomic mapping and sequencing and facilitate studies in molecular genetics of dengue viruses.
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PMID:A novel method for the preparation of large cDNA fragments from dengue-3 RNA genome by long RT-PCR amplification. 1141 41