Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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 peripheral myelin protein PMP22 gene has been described as a growth arrest-specific gene gas3 and has been identified as disease gene of various demyelinating neuropathies. The gene consists of two highly conserved alternative noncoding 5'-exons la (CD25) and 1b (SR13), respectively. Differential expression patterns of these transcripts in vivo and in vitro suggest a very complex mode of PMP22 gene regulation, which cannot be explained merely by transcriptional control. In fact, the PMP22 gene is regulated on different post-transcriptional levels. While reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed no alterations in stability for both PMP22 transcripts in randomly growing Schwann cell cultures of rat sciatic nerve for at least 8 hours, in serum-induced synchronized cultures of resting cells we observed a specific cell cycle-regulated degradation of both transcripts. We further prepared diverse PMP22/CAT fusion genes to study the influence of the alternative 5'UTRs on PMP22 translation. Transient transfection of NIH3T3-fibroblasts and rat Schwann cells demonstrated that the alternative 5'UTRs (CD25 and SR13) and the 3'UTR exert differential regulatory influences on the translation efficiency.
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PMID:Post-transcriptional regulation of the peripheral myelin protein gene PMP22/gas3. 997 19

In the silkworm, Bombyx mori, a non-long terminal repeat (non-LTR) retrotransposon, BMC1, is considered to be a LINE (long interspersed nuclear element)-like element. So far, a BMC1 containing two intact open reading frames (ORFs) has not been found. However, we discovered a complete full-length BMC1 on the W chromosome. This BMC1 is 5091 bp and contains a 5' untranslated region (5'-UTR), two intact ORFs, and 3'-UTR which terminates in a poly(A) tail. ORF1 encodes a putative nucleic acid-binding protein, while ORF2 encodes a protein containing an endonuclease domain and a reverse transcriptase domain.
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PMID:A complete full-length non-LTR retrotransposon, BMC1, on the W chromosome of the silkworm, Bombyx mori. 1033 66

In this report, splice variants of human RAD50 (hRAD50) were cloned and characterized. A Northern blot survey identified two transcripts that hybridized to a hRAD50 cDNA clone, an upper faint band (5.9kb) and lower dense band (4.6kb). cDNA clones (hRAD50-2, 4.6kb) encompassing the entire hRAD50 transcript but having a shorter 3'-untranslated region (3'UTR) than the previously reported hRAD50-1 cDNA (5.9kb; Dolganov, G.M., Maser, R.S., Novikov, A., Tosto, L., Chong, S., Bressan, D.A., Petrini, J.H.J., 1996. Human Rad50 is physically associated with human Mre11: Identification of a conserved multiprotein complex implicated in recombinational DNA repair. Mol. Cell. Biol. 16, 4832-4841.) were isolated. The presence of AU-rich sequences in the 3'UTR of hRAD50-1, which define mRNA instability and Northern results, suggest that hRAD50-2 is the major transcript of hRAD50. A third alternative splice variant that lacks the ATP-binding domain was also identified (hRAD50-3, approximately 4.5kb). Expression of hRAD50-3 transcript was detected in all tissues examined by RT-PCR (reverse transcriptase-polymerase chain reaction) and nested DNA-PCR analyses. Expression of hRAD50 partially rescued the MMS (methyl methanesulfonate)-sensitive phenotype in rad50 mutant yeast, whereas hRAD50-3 did not show complementation. These data suggest that the hRAD50-3 does not repair DNA double-strand breaks most likely due to its inability to bind ATP, and to bind damaged DNA. The existence of these alternative splice forms is potentially important in regulation of the biological activity of the DNA recombinational repair gene, hRAD50.
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PMID:Molecular cloning and characterization of splice variants of human RAD50 gene. 1041 33

The mouse growth hormone receptor/growth hormone-binding protein (GHR/BP) gene produces several distinct mRNA forms through alternative splicing, including mRNAs encoding the membrane-bound growth hormone receptor (GHR) and the soluble growth hormone-binding protein (GHBP). Transcripts are also heterogeneous in their 5' regions due to alternative selection of two major 5' untranslated region (5'UTR) sequences, designated L1 and L2. Here we report the cloning of all mouse GHR/BP coding exons as well as the exon encoding 5'UTR L2, the most widely expressed 5'UTR. The mouse GHR/GHBP gene contains 11 coding exons, 9 of which are homologous in size and sequence to human GHR exons 2-10. The two mouse exons that do not have homologs in the human gene are designated exons 4B and 8A. Exon 4B, located between exons 4 and 5, encodes an 8-amino acid segment of the ligand binding domain that is unique to mouse GHR and GHBP. Analysis by reverse transcriptase-polymerase chain reaction indicated that exon 4B is constitutively present in mouse GHR and GHBP mRNA. Exon 8A encodes the GHBP hydrophilic tail and 3'UTR sequence. 5'UTR L2 is encoded by a single exon located at least 27 kb upstream of exon 2 and at least 12 kb upstream of the exon encoding 5'UTR L1. The transcription start sites of UTR L2 were mapped and the 5' flanking region sequenced. The exon and proximal promoter region are GC rich, and share a high level of conservation with the equivalent exons in the sheep, bovine and human GHR genes. A CCAAT motif and several putative Sp1 motifs are present, and there is no TATA box. Homology between the mouse sequence and other species is limited to a region of 450 bp upstream of the exon due to the insertion of a fragment of a LINE-1 element upstream of the mouse L2 exon. Ribonuclease protection assays were used to confirm that 5'UTR L2 is widely expressed in multiple tissues and is the predominant form of transcript except in the liver during pregnancy, in which 5'UTR L1 is the major form.
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PMID:Structure and expression of the mouse growth hormone receptor/growth hormone binding protein gene. 1042 45

The 3' untranslated region (3' UTR) between the 3' end of env and the long terminal repeat is well conserved among avian retroviruses and is essential for efficient replication. Deletion of the dr1 element within the 3' UTR has been reported to have various effects, including reduced levels of unspliced RNA in the cytoplasm, decreased stability of unspliced RNA, decreased particle production, and decreased genomic RNA packaging. To probe the role of specific sequences within dr1 in virus replication, site-directed mutagenesis was utilized to perturb parts of the predicted secondary structure of dr1. Seven of thirteen mutations had no significant effect; the others resulted in an approximately 10- to 20-fold reduction in replication. These mutants were further characterized and found to impair cytoplasmic accumulation of unspliced RNA only slightly. Furthermore, no decreases were observed in the stability of the unspliced RNA or in the production of virus particles. Genomic RNA packaging, however, was reduced by about 10-fold. Similar amounts of particles were produced by cells containing the mutant and wild-type DNA, and all particles contained similar levels of reverse transcriptase activity. The results suggest that the region of the dr1 disrupted by the mutations plays a role in genomic RNA packaging, although that packaging may not be the only role for dr1.
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PMID:Point mutations in the avian sarcoma/leukosis virus 3' untranslated region result in a packaging defect. 1043 32

A noncytopathic type 1a bovine viral diarrhea virus (BVDV) was isolated from a free-ranging yearling female mule deer (Odocoileus hemionus) from northwestern Wyoming (USA). The mule deer was emaciated, weak, and salivating, and Arcanobacterium pyogenes was cultured from lung abscesses. Bovine viral diarrhea virus was isolated from lung, however, BVDV antigen was not detected by immunohistochemistry. The BVDV genotype was determined by reverse transcriptase polymerase chain reaction and the RNA sequences from the 5'UTR and E2 genes compared with sequences of a type 1a BVDV isolated from cattle from the same area as the deer. The sequences from the deer BVDV were distinct from those of the bovine type 1a BVDV, but similar to other bovine type 1a BVDVs. Seventy-four (60%) of 124 sera collected from mule deer in this area had serum neutralizing antibody titers to type 1a BVDV of > or = 1:32. The high prevalence of seropositive mule deer and isolation of BVDV suggests that this virus circulates in the mule deer population. The isolate described in this report is the second reported BVDV isolate from free-ranging deer in North America and the first from a mule deer.
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PMID:Isolation of bovine viral diarrhea virus from a free-ranging mule deer in Wyoming. 1131 Aug 81

Here, we describe a fast, simple method for constructing full-length cDNA libraries using SMART technology. This novel procedure uses the template-switching activity of Moloney murine leukemia virus (MMLV) reverse transcriptase to synthesize and anchor first-strand cDNA in one step. Following reverse transcription, three cycles of PCR are performed using a modified oligo(dT) primer and an anchor primer to enrich the cDNA population for full-length sequences. Starting with 1 microgram human skeletal muscle poly(A)+ RNA, a cDNA library was constructed that contained 3 x 10(6) independent clones with an average insert size of 2 kb. Sequence analysis of 172 randomly selected clones showed that 77% of cDNA clones corresponding to known genes contained intact open reading frames. The average length of complete open reading frames was 2.4 kb. Furthermore, 86% of the full-length clones retained longer 5' UTR sequences than the longest 5' end deposited in the GenBank database. cDNA libraries generated using this method will be useful for accelerating the collection of mRNA 5' end sequence information, which is currently very limited in GenBank.
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PMID:Reverse transcriptase template switching: a SMART approach for full-length cDNA library construction. 1131 72

A full-length inducible nitric oxide synthase (iNOS) gene has been sequenced for the first time outside the mammals, and the gene organization compared with that already determined for human iNOS. While there are some differences from the human gene, overall the exons show remarkable conservation in sequence and organization. As in human, the trout iNOS gene has 27 exons, with 18 of the trout exons being identical in size with the equivalent human exons. The cofactor-binding domains are found in the same exons and in some cases are absolutely conserved. Differences include the start of the ORF in exon 3 instead of exon 2, resulting in a deletion at the 5' end of the trout iNOS protein. Exon 27 also shows a large difference in size and although the trout exon is larger this is due to the length of the 3'-UTR. Several non-mammalian features are notable, and include a conserved potential glycosylation site in chicken and fish, and an insertion at the boundary of exons 20 and 21 in fish. The intron sizes in trout were generally much smaller than in human iNOS, making the trout iNOS gene approximately half the size of the human gene. Analysis of RNA secondary structure revealed two regions with complementarity, which could interfere with reverse transcription. Using a trout fibroblast cell line (RTG-2 cells), it was shown by reverse transcriptase (RT)-PCR that virus infection was a good inducer of iNOS expression. However, when using a combination of Superscripttrade mark II for reverse transcription and primers at the 5' end of the gene only very weak products were amplified, in contrast with the situation when primers at the 3' end of the gene were used, or ThermoScripttrade mark-derived cDNA was used. The impact of such results on RT-PCR analysis of iNOS expression in trout is discussed.
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PMID:Molecular cloning, gene organization and expression of rainbow trout (Oncorhynchus mykiss) inducible nitric oxide synthase (iNOS) gene. 1153 35

We have previously demonstrated that the proximal tubular cell may contribute to the pathogenesis of renal interstitial fibrosis in diabetes. Transforming growth factor (TGF)-beta1 is one of a group of pro-fibrotic cytokines and growth factors, which have been associated with the development of interstitial fibrosis. The aim of the current study was to examine the effect of insulin on the generation of TGF-beta1 by proximal tubular cells. HK-2 cells were grown to confluence in the absence of insulin, and serum deprived for 48 hours before all experimental manipulations. Addition of insulin (5 microg/ml) to the culture medium led to a time-dependent increase in TGF-beta1 concentration in the cell culture supernatant, and increased incorporation of radiolabeled amino acids into TGF-beta1 suggestive of de novo TGF-beta1 protein synthesis. Addition of insulin did not alter TGF-beta1 mRNA expression as assessed by reverse transcriptase-polymerase chain reaction or Northern analysis. Insulin-induced increase in TGF-beta1 concentration was not abrogated by actinomycin D, however, stimulation by insulin, in the presence of cycloheximide led to a dose-dependent decrease in TGF-beta1 production. Addition of insulin had no effect on TGF-beta1 mRNA stability as assessed by actinomycin D chase, but led to increased binding of a cytoplasmic protein to a putative stem loop structure in the 5'-UTR of TGF-beta1 mRNA, previously implicated in the posttranscriptional control of TGF-beta1 synthesis. To address the functional significance of insulin-induced alteration in TGF-beta1 synthesis, we examined its effect on matrix turnover. Insulin stimulated type IV collagen gene expression and an increase in the concentrations of the type IV collagen laid down in the extracellular matrix. This increase in type IV collagen was abrogated when cells were stimulated by insulin in the presence of an anti-TGF-beta1-blocking antibody. In conclusion the data demonstrate that insulin may directly alter the production of TGF-beta1 by renal proximal tubular cells by a posttranscriptional mechanism, and that this may have implications for the increase in extracellular matrix that accompanies diabetic nephropathy.
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PMID:Translational regulation of renal proximal tubular epithelial cell transforming growth factor-beta1 generation by insulin. 1169 51

A reverse transcriptase PCR (RT-PCR) assay using conserved primers deduced from the core-envelope 1 (C-E1) region of the hepatitis C virus (HCV) genome was developed for subtyping purposes. The sensitivity and specificity of this assay tested against two HCV reference panels containing genotype 1 through 5 subtypes were similar to those of an RT-PCR assay from the 5'-untranslated region (5'-UTR). The sensitivity of the RT-PCR typing assay in the more variable C-E1 region was, however, lower than that of the RT-PCR in the highly conserved 5'-UTR when testing multiple clinical samples. Thus, 71 (88%) of 81 consecutive samples from hospitalized Danish patients positive for HCV antibodies and RNA (5'-UTR) were positive also in the C-E1 RT-PCR assay. Phylogenetic analysis of the E1 sequences obtained by direct sequencing of HCV from two reference panels and 71 Danish patients allowed us to readily distinguish the subtypes. In contrast, phylogenetic analysis of their corresponding 5'-UTR sequences was able to predict only major genotypes. Three different genotypes and four subtypes were identified among Danish samples: 1a (43%), 1b (11%), 2b (6%), and 3a (39%). An isolate from a Somalian refugee was identified as a new HCV type related to Somalian isolates described as subtype 3h. The most common genotype in Denmark is genotype 1 (53%), which is the most difficult to treat. However, Denmark had the highest prevalence in Europe of subtype 3a, which responds more favorably to treatment. The described C-E1 RT-PCR with sequencing is suggested as an easy routine assay for definitive genotyping and subtyping of HCV.
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PMID:Hepatitis C virus subtyping by a core-envelope 1-based reverse transcriptase PCR assay with sequencing and its use in determining subtype distribution among Danish patients. 1262 35


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