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)

Somatic cell hybrids were constructed between BALB/c-RAG mouse cells and feline lymphoma cells by the hypoxanthine-aminopterin-thymidine selection scheme. RAG cells spontaneously produce an endogenous B-tropic type C virus. Cat-mouse hybrids preferentially segregate feline chromosomes and retain murine chromosomes-demonstrable by karyotypic and isozyme analyses. Despite the presence of the complete mouse genome, including the viral genome, virus production was diminished to 1-5% of the levels observed in RAG parents based upon particle-associated RNA-dependent DNA polymerase (reverse transcriptase) activity in the culture fluid. Thirty-seven hybrids made on four different occasions had suppressed virus levels, and no hybrids expressed parental virus levels. Reverse selection experiments on 6-thioguanine demonstrated that a restriction gene, tentatively named Bvr-1, was linked to the feline structural genes for hypoxanthine phosphoribosyltransferase (IMP:pyrophosphate phosphoribosyltransferase; EC 2.4.4.8) and glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase; EC 1.1.1.49) in cats, probably on the X-chromosome. The genetic mode of action of Bvr-1 is trans dominant in restriction of murine leukemia virus. The restriction locus results in a block late in virus maturation but prior to release, since expression of antigens for viral structural proteins and matrue budding particles is apparent on surfaces of restriced hybrid cells but not in high-speed pellets from culture fluid of restricted cells.
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PMID:Bvr-1, a restriction locus of a type C RNA virus in the feline cellular genome: identification, location, and phenotypic characterization in cat X mouse somatic cell hybrids. 6 49

Strong evidence points to mutation induction as one mechanism by which changes are introduced into normal cells to convert them into cancer cells. To understand the mechanisms by which mutations are induced in human cells by carcinogens, we are determining the kinds and spectra of mutations induced in the coding region of the hypoxanthine(guanine)phosphoribosyltransferase (hprt) gene. This region, composed of 654 bp, represents nine exons from a 44 kbp gene. To be able to analyze a large number of independent mutants rapidly and economically, we have optimized the conditions for copying mRNA directly from lysates of a small number of cells (e.g., 100) from a 6-thioguanine-resistant clone using reverse transcriptase and oligo(dT)12-18 primers. Then two 20-mer primers, specific for the cDNA of the hprt gene, are used to amplify the first and second strand cDNA 5 x 10(7)-fold during 30 cycles of polymerase chain reaction (PCR). The product (2 to 10 ng) is then purified by ultrafiltration, diluted 1:10, and subjected to an additional 30 cycles of PCR, using two 20-mer primers located just interior to the first set. The amplification product, 5 to 10 ug, is sequenced directly using three other end-labeled primers and Sequenase. To date, we have analyzed 26 mutants induced by (+-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha,epoxy-7,8,9,10-tetrahydrobenzo [a]pyrene and found that 24/26 involved base substitutions. 97% of these involved G.C, predominantly G.C----T.A, distributed over seven exons, with many of the substitutions located in exon 3.
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PMID:Amplification of mRNA of the hprt gene from lysates of mutant human cells and direct DNA sequencing to determine the spectrum of mutations induced by (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha, epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene. 211 60

A method has been developed for the specific amplification and cloning of human hprt cDNA which can be used for mutant sequence analysis. Messenger RNA is isolated from TK6 lymphoblasts and is used to produce a first strand cDNA with reverse transcriptase primed with oligo dT. Second strand synthesis and subsequent amplification of hprt sequences is accomplished using Thermus aquaticus DNA polymerase and hprt-specific primers in the polymerase chain reaction (PCR) procedure. Convenient restriction enzyme sites have been built into the 5' ends of the PCR primers to allow cloning of the hprt fragments in M13mp19. Dideoxy sequencing of hprt with specific primers can be carried out using either the PCR reaction product or fragments cloned in M13mp19 as substrate. This general cloning/sequencing method can be used to analyze hprt mutation in human cells obtained both in vitro and in vivo.
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PMID:A method for specific cloning and sequencing of human hprt cDNA for mutation analysis. 334 90

We exposed experimental animals to a series of alkylating agents that induced mutations at the X-linked hprt gene of T lymphocytes. We then isolated the mutant cells and analyzed the molecular nature of the mutations by amplification of hprt cDNA sequences with the use of reverse transcriptase PCR followed by DNA sequence analysis, and then correlated the mutational spectra obtained to the spectra of DNA adducts caused by the alkylating agents used. The nature of the base-pair changes causing the mutations was characteristic for the reaction pattern of the genotoxic agent with DNA. However, we also found a clear influence of DNA repair processes; i.e., in those cells that were able to remove certain types of DNA damage, the class of mutations expected from that type of damage was reduced.
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PMID:Detection of point mutations in T lymphocytes. 749 42

The cadherins are a family of calcium-binding membrane glycoproteins. Most cadherins are capable of acting as cell adhesion molecules (CAMs). In order to begin a thorough analysis of the roles of these CAMs in the testis, we employed a reverse transcriptase-polymerase chain reaction (RT-PCR) strategy to identify the cadherins expressed in this tissue at various stages of development. Oligonucleotides encoding amino acid sequences that are conserved among all of the known cadherins were used as primers in the RT-PCR, with cDNA preparations of fetal, newborn, 7-day, 21-day, and adult mouse testes employed as templates. The PCR products were subcloned into a plasmid vector and sequenced. On the basis of the nucleotide sequences of these PCR products, we have determined that five previously characterized cadherins (E-cadherin, N-cadherin, P-cadherin, K-cadherin, and OB-cadherin), as well as two novel cadherins (T1-cadherin and T2-cadherin), are expressed at various stages during testicular development. In order to determine the expression patterns of these cadherins, we ascertained the mRNA levels of each cadherin normalized to the levels of hypoxanthine phosphoribosyltransferase mRNA in fetal, newborn, 7-day, 21-day, and adult mouse testes. We observed that N-cadherin mRNA is expressed at all stages of testicular development, with maximal levels being present in the testes of 21-day-old mice. Furthermore, we found that E-, P-, K-, OB-, and T2-cadherin mRNAs are all expressed in the fetal gonad. The testicular levels of these cadherin mRNAs decreased dramatically after birth. Conversely, T1-cadherin mRNA was not detected in the fetal, newborn, and 7-day-old testes but was present in 21-day-old and adult testes. T1-cadherin levels were 10-fold higher in the testes of adult mice, compared to the levels found in the testes of 21-day-old mice. We speculate that these cadherins will be found to be intimately involved in mediating cell interactions during testicular development.
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PMID:A comprehensive survey of the cadherins expressed in the testes of fetal, immature, and adult mice utilizing the polymerase chain reaction. 887 95

The genome of the Australian marsupial Macropus robustus contains a highly conserved processed hypoxanthine phosphoribosyltransferase homologue, HPRT-2. Using the techniques of reverse transcriptase-polymerase chain reaction (RT-PCR) and protein isoelectric focusing (IEF) we have shown this processed gene to be fully functional, but liver specific. In contrast, the unprocessed X-linked parent gene HPRT-1 was expressed in all somatic tissues. Expression of the HPRT-2 gene effectively doubles the total HPRT enzyme activity in liver compared to other tissues. Analysis of the 5'-flanking sequence of HPRT-2 revealed regions with homology to the liver-specific regulatory motifs C/EBP, NF-IL6, LF-A1 and LF-B1, although the functional significance of these regions remains unknown. Consistent with X chromosome inactivation in female mammals, transcript levels of the unprocessed X-linked gene HPRT-1 were similar in males and females in all tissues examined. No HPRT-2 activity was detected in testes, indicating that this gene does not compensate for sex chromosome inactivation during spermatogenesis. Moreover, the demonstration of very high HPRT-1 enzyme levels in testes indicated that such a compensatory mechanism may not be required. Phylogenetic analyses attribute considerable antiquity to the processed gene and PCR with conserved primers spanning exons 4-8 of genomic DNA from several different kangaroo species inferring the existence of a conserved processed HPRT-2 homologue in these marsupial species. However, no such conserved PCR product was obtained with DNA from eutherian species, suggesting that integration of HPRT-2 occurred after the separation of the metatherian and eutherian lineages.
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PMID:Identification of a novel tissue-specific processed HPRT gene and comparison with X-linked gene transcription in the Australian marsupial Macropus robustus. 904 50

Our laboratory has characterized several hundred mutant hprt cDNAs produced using Moloney murine leukemia reverse transcriptase to convert mRNA to cDNA. During the characterization of these mutants we have detected six T-lymphocyte mutants that demonstrate multiple G:C --> A:T transitions along the hprt cDNA coding sequence. Attempts to repeat the mRNA to cDNA conversion and subsequent characterization have demonstrated that the multiple transitions are likely artifacts. We suggest that reverse transcriptase is directly responsible for these multiple base substitutions and as such, that multiple mutations be viewed as suspect requiring confirmation at the genomic level.
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PMID:Moloney murine leukemia reverse transcriptase suspect in the production of multiple misincorporations during hprt cDNA synthesis. 906 24

Expression and androgen regulation of the gene for neuronal nitric oxide synthase (NOS I) were examined in neurons of the major pelvic ganglia in male rats. Some of these postganglionic neurons innervate the penis and produce nitric oxide, which is believed to play a major role in penile erection. Rats were either castrated or sham operated and implanted with SILASTIC brand capsules filled with powdered testosterone (T) or 5alpha-dihydrotestosterone (5alphaDHT) or left empty. After 4 days, the number of neurons intensely stained for NADPH-diaphorase as well as those giving a NOS I signal in in situ hybridization experiments increased in castrated rats treated with testosterone by 31% and 42%, respectively, relative to those in untreated castrated rats. This suggests that the increase in NADPH-diaphorase activity resulted from enzyme synthesis and was due to a modification of NOS I messenger RNA (mRNA) accumulation. After 7 days, Northern blot analysis showed that castration produced a decrease in the amount of NOS I mRNA relative to that of ribosomal RNA. This decrease was almost prevented by T treatment. No significant differences were observed by reverse transcriptase-PCR between 7-day and 28-day treatments. However, in 7-day castrated rats treated with 5alphaDHT, NOS I signals relative to those of hypoxanthine phosphoribosyltransferase, taken as reference, were significantly higher than those in castrated rats and resembled those in sham-castrated rats, suggesting that 5alphaDHT was probably more potent than testosterone in preventing the decrease in NOS I mRNA levels elicited by castration. These results show that NOS I can be positively regulated by androgens and are consistent with the suggestion that these steroids play a role in the physiological processes of penile erection.
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PMID:Androgens modulate nitric oxide synthase messenger ribonucleic acid expression in neurons of the major pelvic ganglion in the rat. 923 55

We measured the frequency of mutant (MF) lymphocytes at the hprt locus in a population of 43 coke-oven workers exposed to PAH and in a group of 26 non-exposed workers. A non-significant increase in MF in the exposed group (19.0 +/- 16.3) compared to the non-exposed group (15.8 +/- 14.6) was observed. Moreover, when we considered smoking habits for the overall population, the MF values were higher, although not significantly, in smokers than in non-smokers. For some T-cell mutant clone structural alterations, splicing and coding errors were detected by PCR-based methods. We analysed 161 HPRT- clones, derived from exposed and non-exposed workers by multiplex-PCR and 56 HPRT- clones by reverse transcriptase-PCR. Overall, the percentages of the different types of gene alterations were similar in exposed and non-exposed subjects. Only the frequency of splice mutations in mutant clones derived from coke-oven workers was higher (22%) than in non-exposed donors (11%).
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PMID:Determination of HPRT mutant frequency and molecular analysis of T-lymphocyte mutants derived from coke-oven workers. 953 72

Various "housekeeping" genes are often used as endogenous controls in gene expression experiments. We have cloned from swine, three genes commonly used as endogenous controls in other species and have characterized their relative levels of expression in various porcine tissues and their response to various cell activators. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin were readily detected by northern hybridization in various tissues and in alveolar macrophages. The expression of hypoxanthine phosphoribosyltransferase (HPRT) was detected only by northern hybridization of poly-A+ enriched RNA and by reverse transcriptase-polymerase chain reaction (RT-PCR), making it more suitable for highly sensitive detection methods. Expression of GAPDH varied less among tissues than did beta-actin, making it more useful control for comparisons of gene expression between tissues with northern hybridizations. Various treatments of cultured alveolar macrophages differentially affected levels of beta-actin and GAPDH, while HPRT expression was unchanged in alveolar macrophages or spleen cells similarly treated. Therefore, while HPRT can be used as the endogenous control with sensitive detection methods such as RT-PCR, less sensitive detection methods require a more abundant gene such as GAPDH.
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PMID:Regulation of hypoxanthine phosphoribosyltransferase, glyceraldehyde-3-phosphate dehydrogenase and beta-actin mRNA expression in porcine immune cells and tissues. 967 36


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