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)

RNA isolated by conventional guanidinium isothiocyanate methods from tissues of a mollusc (red abalone: Haliotis rufescens) is largely degraded and discolored by contaminants. These contaminants are associated with inhibition of reverse transcriptase, prevent accurate spectrophotometric determination of RNA concentration, and impart undesirable viscosity to the preparations. A cold two-step method of RNA isolation was devised which provides high yields of full-length RNA templates from these tissues and eliminates the discolored contaminant. Immediately following homogenization of tissues at ca. 5 degrees C, which proved crucial for the recovery of high-molecular weight species, the RNA is isolated from the bulk of the RNase by a single acid-phenol-chloroform extraction at 0 degrees C. The inhibitor of reverse transcriptase, suspected to be a proteoglycan (or a similar high-molecular-weight polyanion) component of the intestinal mucus, is eliminated only by a second purification step employing ultracentrifugation through a dense cushion of CsCl. This cold two-step method should prove useful for providing full-length RNA templates relatively free of polysaccharide, a common contaminant of RNA preparations, from both plant and animal tissues.
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PMID:Isolation of full-length RNA templates for reverse transcription from tissues rich in RNase and proteoglycans. 768 67

The cellular distribution of hepatitis C virus was examined in formalin-fixed, paraffin-embedded tissues by in situ localization of the polymerase chain reaction (PCR)-amplified viral cDNA. Of nine liver biopsies studied, five were from patients seropositive for hepatitis C, one was from a seronegative patient who had detectable hepatitis C cDNA by standard reverse transcriptase (RT) PCR, and the remaining three were from patients without evidence of hepatitis C infection. Sequences homologous to viral RNA were rarely identified in the hepatitis C cases by standard RNA-cDNA in situ hybridization, but PCR-amplified viral cDNA was detectable in many hepatocytes in a panlobular distribution and in scattered Kupffer cells in each of the six hepatitis C cases and none of the controls. The pattern was equivalent whether intracellular localization was done by in situ hybridization after the RT and PCR steps or if digoxigenin-labeled nucleotide was incorporated into the amplified product. No signal was evident in the positive biopsies if the RT step was omitted, if "nonsense" primers were used, or if RT in situ PCR was preceded by RNase digestion. The RT in situ PCR technique allows for the rapid detection of any RNA virus, even if it is present in low copy number, and permits direct morphological correlation.
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PMID:Intracellular localization of polymerase chain reaction (PCR)-amplified hepatitis C cDNA. 768 49

RNases H are traditionally thought to degrade RNA only in RNA-DNA hybrid form. We found that the wild-type Moloney murine leukemia virus (M-MuLV) reverse transcriptase (RT) was capable of degrading RNA in RNA-RNA duplexes as well as in RNA-DNA hybrids, as assayed by in situ gel techniques. Escherichia coli RNase H does not degrade the RNA-RNA duplex in this assay, while E. coli RNase III, a double-strand-specific ribonuclease, does. The apparent specific activity of M-MuLV RT on RNA-RNA duplexes is similar to that on RNA-DNA hybrids. Neither the DNA polymerase domain nor the RNase H domain of RT expressed individually exhibited this RNA-RNA activity. We have generated a series of mutations in the RNase H domain of M-MuLV RT, expressed the mutant enzymes in E. coli, and assayed these mutants for various activities. All RTs were as active as the wild type in the oligo(dT):poly(rA) DNA polymerase assay, and many retained both nuclease activities. Two enzymes with mutations at the carboxyl terminus of the RNase H domain retained RNA-DNA activity, but not RNA-RNA activity. Another mutant enzyme showed the opposite phenotype, retaining RNA-RNA, but not RNA-DNA, nuclease activity. Thus, we were able to genetically separate the two activities. These results may be helpful in defining enzyme-substrate interactions.
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PMID:Nuclease activities of Moloney murine leukemia virus reverse transcriptase. Mutants with altered substrate specificities. 769 92

We constructed a human immunodeficiency virus (HIV) matrix (MA) deletion mutant by deletion of about 80% of the HIV type 1 Gag MA domain but retaining myristylation and proteolytic processing signals. The effects of this deletion matrix (dl.MA) mutant on HIV particle assembly, processing, and infectivity were analyzed. Surprisingly, the dl.MA mutant still could assemble and process virus particles, had a wild-type (wt) retrovirus particle density, and possessed wt reverse transcriptase activity. RNase protection experiments showed that dl.MA mutant particles preferentially packaged viral genomic RNA. When both mutant and wt particles were pseudotyped with an amphotropic murine leukemia virus envelope protein, mutant infectivity was about 10% of wt level. In contrast, infectivity of the dl.MA mutant was 1,000-fold less than that of wild-type when mutant and wt particles were pseudotyped with the HIV envelope protein. Protein analyses of pseudotyped virions indicated that there were no major differences between mutant and wt viruses in the efficiency of amphotropic murine leukemia virus envelope protein incorporation. In contrast, there was a reduction in the amount of mutant particle-associated HIV envelope protein gp120. Our results suggest that an intact HIV matrix domain is not absolutely required for reverse transcription, nuclear localization, or integration but is necessary for appropriate HIV envelope protein function.
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PMID:Conditional infectivity of a human immunodeficiency virus matrix domain deletion mutant. 769 66

The cardiac (versus retinal rod) Na/Ca exchanger gene has been cloned, sequenced and shown by RNA analysis to be present in diverse tissues. Analysis of published sequences shows that a single isoform is found in heart tissue from many species (NACA1 isoform). We provide evidence here by ribonuclease (RNase) protection assays and by reverse transcriptase-polymerase chain reaction (PCR) amplification with sequence analysis that a new isoform encoding the Na/Ca exchanger is present in renal tissue. This isoform (NACA3) reveals a 7-amino acid deletion in the tested region compared with the NACA2 isoform described by Reilly and Shugrue [Am. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): F1105-F1109, 1992] and is the dominant exchanger transcript in kidney. Analysis of the sequence of all isoforms indicates that the differences in the isoforms reside in the large intracellular loop region of the protein. Alternative splicing of a single Na/Ca exchanger message may be responsible for these tissue-specific transcripts.
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PMID:Na/Ca exchanger isoforms expressed in kidney. 769 82

Wild-type as well as variant oestrogen receptor (ER) mRNAs with exon 5 and 7 deleted were identified in a panel of human breast tumour cell lines by reverse transcriptase-polymerase chain reaction followed by dideoxynucleotide sequence analysis, and then quantitated by ribonuclease protection analysis. All cell lines categorised as ER+ by ligand-binding analysis expressed both wild-type and variant ER transcripts. Most cell lines classified as ER- did not express any ER transcript. However, three ER- cell lines (BT-20, MDA-MB-330 and T47Dco) expressed both wild-type and variant transcripts. A differential pattern of expression of wild type to variant was seen in both ER+ and ER- cell lines, however this pattern was not paralleled by differences in ligand-binding activity. Breast tumour cell lines previously classified as ER- expressed significantly lower levels of ER transcripts than did their ER+ counterparts. In view of these findings, as well as earlier reports that the exon 5 deletion ER variant encodes a dominant-positive receptor, it seems clear that some cell lines are misclassified as ER-, and express both wild-type and variant ER mRNAs, and that the overexpression of this variant may account, in part, for their oestrogen-independent phenotype.
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PMID:Coexpression of wild-type and variant oestrogen receptor mRNAs in a panel of human breast cancer cell lines. 773 23

The backbone dynamics of Escherichia coli ribonuclease HI (RNase HI) in the picosecond to nanosecond time scale were characterized by a combination of measurements of 15N-NMR relaxation (T1, T2, and NOE), analyzed by a model-free approach, and molecular dynamics (MD) simulation in water. The MD simulations in water were carried out with long-range Coulomb interactions to avoid the artificial fluctuation caused by the cutoff approximation. The model-free analysis of the 15N-NMR relaxation indicated that RNase HI has a rotational correlation time of 10.9 ns at 27 degrees C. The generalized order parameter (S2) for the internal motions varied from 0.15 to 1.0, with an average value of 0.85, which is much larger than that of the RNase H domain of HIV-1 reverse transcriptase (0.78). Large internal motions (small order parameters) were observed in the N-terminal region (Leu2-Lys3), the loop between beta-strands A and B (Cys13-Gly15), the turn between alpha-helix I and beta-strand D (Glu61, His62), the loop between beta-strand D and alpha-helix II (Asp70-Tyr71), the loop between alpha-helices III and IV (Ala93-Lys96), the loop between beta-strand E and alpha-helix V (Gly123-His127), and the C-terminal region (Gln152-Val155). The effective correlation time observed in these regions varied from 0.45 ns (Glu61, Lys96) to 2.2 ns (Leu14). The order parameters calculated from the MD agreed well with those from the NMR experiment, with a few exceptions. The distributions of most of the backbone N-H vectors obtained by MD are approximately consistent with the diffusion-in-a-cone model. These distributions, however, were elliptic, with a long axis perpendicular to the plane defined by the N-H and N-C alpha vectors. Distributions supporting the axial fluctuation model or the jump-between-two-cones model were also observed in the MD simulation.
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PMID:Characterization of the internal motions of Escherichia coli ribonuclease HI by a combination of 15N-NMR relaxation analysis and molecular dynamics simulation: examination of dynamic models. 775 90

The isolation of a cDNA corresponding to a portion (amino acid 943 to 1073) of the cytoplasmic domain of the mouse EGF receptor surrounding the auto phosphorylation sites was obtained by using the reverse transcriptase polymerase chain reaction (RT-PCR) approach. Deduced amino acid sequence of mouse EGF receptor (EGFr) shows a 92% and 76% homology to corresponding regions in the human and the chicken EGFr, respectively. This cDNA was used to develop a sensitive RNase protection assay to investigate EGF receptor mRNA expression in mouse C3H teratoma derived cell lines with increased tumorigenic properties which display a progressive decrease of EGF binding and response. The results show that increased tumorigenicity was not accompanied by a change in EGF receptor mRNA expression. Moreover, they indicate that the RNase protection assay developed using the probe described here is a sensitive approach to investigate EGF receptor expression in murine cells.
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PMID:Nucleotide sequence of the C-terminal region of the mouse epidermal growth factor receptor and expression in teratoma-derived cell lines with increased tumorigenic properties. 776 4

To study the differential expression of the murine VLA-4 (alpha 4 beta 1) integrin, the 5'-flanking region of the gene for the alpha subunit (alpha 4m) was isolated and a cDNA for alpha 4m was obtained with reverse transcriptase polymerase chain reaction (RT-PCR). The cDNA sequence contained a difference in the signal peptide region compared to the previously described cDNA (Neuhaus et al., 1991). As a consequence, another start codon is predicted, resulting in a decrease in size of the signal peptide. This was confirmed by genomic sequencing. The promoter region was delimited by ribonuclease protection assay (RPA) and transfection experiments fusing 5'-upstream fragments to the luciferase gene. A fragment extending from -936 to +221 was capable of controlling the expected cell-type-specific expression. Sequence comparison of the mouse alpha 4m promoter region with the human alpha 4h promoter revealed little homology. Like most integrin subunits, alpha 4m lacks TATA anc CCAAT boxes. Putative recognition sites for DNA-binding nuclear factors (AP1, AP2, Sp1, and PU1) were identified. The characterization of the promoter region and further identification of the transcription regulatory elements should provide insight in the regulation of alpha 4m integrin gene expression.
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PMID:Cloning and characterization of the promoter region of the murine alpha-4 integrin subunit. 777 55

Insulin-like growth factor I (IGF-I) is a widely expressed abundant autocrine and paracrine factor that regulates the proliferation and differentiation of a variety of cell types. Prostaglandin E2 (PGE2) is a potent stimulator of IGF-I synthesis in bone. We examined the regulation of IGF-I synthesis by PGE2 in osteoblast-enriched (Ob) cells from fetal rat calvaria. PGE2 treatment of Ob cells at 1 microM for 2 h resulted in a 5-fold increase in heterogeneous nuclear RNA levels, as measured by a reverse transcriptase-polymerase chain reaction assay, suggesting an increase in IGF-I gene transcription. RNase protection analysis was used to map the transcriptional start sites in the IGF-I gene that are used in Ob cells. Consistent with other extrahepatic tissues, initiation of transcription occurs primarily at three sites within the 5'-regions of exon 1 of the IGF-I gene. PGE2 treatment did not alter start site usage. The regions upstream of these transcriptional start sites were analyzed by transiently transfecting Ob cells with putative rat IGF-I promoter sequences ligated to a luciferase reporter gene. Constructs containing 1.4 kilobases of the 5'-regions regions of exons 1 and 2 had significant promoter activity. PGE2 treatment of transfected Ob cells increased luciferase activity 5-fold when a 1.4-kilobase exon 1 promoter fragment was tested. This increase in luciferase activity was time and dose dependent. Smaller regions of the exon 1 promoter sequence gave higher basal activity and were less responsive to PGE2. We conclude that regions involved in IGF-I regulation by PGE2 are contained within the IGF-I promoter.
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PMID:Regulation of insulin-like growth factor I transcription by prostaglandin E2 in osteoblast cells. 782 49


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