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)

Cytochrome P-450 mRNA has been partially purified from membrane-bound polysomes of the livers of phenobarbital-treated rats by SDS-phenol-chloroform extraction, followed by poly(U)-Sepharose chromatography and by centrifugation through a sucrose density gradient. Cytochrome P-450 mRNA activity was detected near 18S in the sucrose density gradient, accounting for approximately 5% of total mRNA activity on the basis of [3H]leucine incorporation in an in vitro translation system of wheat germ. Complementary DNA (cDNA) which had been synthesized on the partially purified mRNA by AMV reverse transcriptase was inserted into the Pst I site of pBR 322. After bacterial transformation, and in situ colony hybridization using [32P]cDNA as a probe, a colony carrying cytochrome P-50 cDNA sequence was identified by a hybridization-arrested translation assay. Sequence complementarity of the inserted DNA sequence to cytochrome P-450 mRNA was further confirmed by a positive hybridization-translation assay. The mRNA isolated from the partially purified mRNA preparation by hybridizing it with the recombinant DNA (III-8-10) showed enriched synthesis of a protein product whose apparent molecular weight was consistent with that of cytochrome P-450, and which was immunoprecipitable with anti-cytochrome P-450 antibody.
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PMID:Construction and identification of a hybrid plasmid containing DNA sequence complementary to phenobarbital-inducible cytochrome P-450 messenger RNA from rat liver. 616 9

The avian RNA tumour virus structural protein p12 was purified from avian myeloblastosis virus (AMV) by nucleic acid affinity chromatography to apparent homogeneity as judged from SDS--polyacrylamide gel electrophoresis. A filter binding assay was used for the identification of p12. High concentrations of p12 precipitated nucleic acids out of solution in the absence of MgCl2. Binding of p12 to single-stranded nucleic acids protected them from digestion with nucleases and resulted in a hyperchromic effect. These phenomena were reversible in the presence of salt. The affinity of p12 to nucleic acids was determined by competing for the binding of p12 to denatured radioactive DNA by various other nuclei acids. It was found that p12 bound preferentially to single-stranded nucleic acids and showed a higher affinity to poly(rI) than to poly(rC) and poly(rA). Purified RNA-dependent DNA polymerase activity from AMV was stimulated up to sixfold by p12, depending on the template. Solubilization of RNA in RNA--DNA hybrids by RNase H was inhibited in the presence of p12.
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PMID:Properties of the avian viral protein p12. 616 96

The three enzyme forms (alpha, alpha beta-, and beta-form) of the RNA-dependent DNA polymerase (the reverse transcriptase) from three strains of avian sarcoma virus (ASV B77, ASV tsLA334, and ASV QV2) and one exogenous (avian myeloblastosis virus (AMV)) and one endogenous avian leukosis virus (Rous-associated virus type-0 (RAV-0) were compared with each other in subunit structure and catalytic properties. Despite the gross similarity in the subunit molecular weight (Mr(alpha) = 65,000 and Mr(beta) = 92,000), minor differences were found in the molecular size of the subunit as determined by SDS-gel electrophoresis, the order being ASV tsLA334 less than or equal to ASV QV2 less than ASV B77 less than or equal to RAV-0 = AMV. The structural differences were supported by analysis of peptide fragments after treatment with S. aureus V8 protease. Although the general catalytic properties of the purified enzymes from the five virus strains were similar, the selectivety of template-primer differed in the RAV-0 enzymes. The template-primer selectivity of the reverse transcriptases from all five virus strains tested was also found to be greatly influenced by the reaction temperature for DNA synthesis, resulting in a temperature-dependent increase of poly(dG) synthesis over [(A)m] . [(dT)12-18]-dependent poly(dT) synthesis. The RAV-0 enzymes required a lower temperature for DNA synthesis, particularly for [(C)n] . [(dG)12-18]-dependent poly(dG) synthesis.
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PMID:Reverse transcriptase associated with avian sarcoma-leukosis viruses. II. Comparison of subunit structure and catalytic properties. 617 24

Actin is the major extractable protein component from the tube feet of four different species of sea urchin: Arbacia punctulata, Strongylocentrotus purpuratus, Strongylocentrotus droebachiensis, and Diadema setosum. Actin made up as much as 60% of the total Coomassie Blue-staining material after SDS polyacrylamide gel electrophoresis and densitometer analysis. Two-dimensional gel electrophoresis resolved two, and possible three, species of actin for each sea urchin of which the dominant component was analogous to the beta form in vertebrates. In a cell-free system from rabbit reticulocytes, total RNA from tube feet stimulated the synthesis of one protein that represented 80% of the total methionine incorporation, migrated with the properties characteristic of actin in a two-dimensional gel system, and on proteolysis yielded fragments identical to purified rabbit actin. The mRNAs from the tube feet of two divergent species of sea urchin, Arbacia punctulata and Strongylocentrotus purpuratus, synthesized actins differing by less than 0.02 pH unit for each isospecies 90% of the DNA copied from tube foot RNA by reverse transcriptase represented a highly abundant sequence class judged by copy DNA(cDNA)-RNA excess hybridization. At least two-thirds of this class represented a low-complexity component, with a Rot1/2 about three times that expected for actin messenger RNA. The remarkable degree of conservation of the actin protein is reflected in concomitant conservation of the protein-coding nucleotide sequences of the messenger RNA, which has allowed the use of a cDNA probe to isolate actin sequences from a human phage library.
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PMID:Sea urchin tube feet: unique structures that allow a cytological and molecular approach to the study of actin and its gene expression. 689 47

We report here a human-immunodeficiency-virus-type-1 (HIV-1) recombinant reverse transcriptase (RT) engineered to contain a 26-amino-acid linker insertion from the tether domain of feline leukaemia virus (FLV) RT. The chimaeric protein was expressed in Escherichia coli and migrated on SDS/PAGE as a 68 kDa band. A monomeric form of the chimaeric HIV-1 RT has been prepared by the coordinated applications of immobilized-metal-affinity chromatography and gel filtration on Superose 12 columns. The monomeric nature of this chimaeric HIV-I RT was further characterized by cross-linking studies using disuccinimidyl suberate. The RNA-dependent DNA polymerase activity of the monomeric chimaeric HIV-1 RT was 35% that of the heterodimeric (p66/p51) HIV-1 RT. These results support our recent studies on the monomeric polymerase domain (p51 RT) which exhibited an RNA-dependent DNA polymerase activity equal to 33% of that of the p66/p51 heterodimeric HIV-1 RT (Evans, Kezdy, Tarpley and Sharma [1993] Biotechnol. Appl. Biochem. 17, 91-102). The inability of the monomeric chimaeric HIV-1 RT to display polymerase activity like that of the heterodimeric HIV-1 RT is attributed to a decrease in the processive rate of DNA synthesis (75%) and DNA binding (65%). However, the monomeric chimaeric HIV-1 RT (p68) exhibited RNAase H activity like that of the heterodimeric form (p66/p51) of HIV-1 RT. These results suggest that the linker insertion from FLV RT does not interfere with the RNAase H activity associated with the monomeric HIV-1 RT.
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PMID:Engineering of the human-immunodeficiency-virus-type-1 (HIV-1) reverse transcriptase gene to prevent dimerization of the expressed chimaeric protein: purification and characterization of a monomeric HIV-1 reverse transcriptase. 751 79

A retrovirus, known as salmon leukemia virus (SLV), was purified from farm-reared chinook salmon (Oncorhynchus tshawytscha) with plasmacytoid leukemia (PL). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified SLV revealed the presence of 9 virus-associated polypeptides with molecular weights from 82 kDa to 15 kDa. Endoglycosidase digestion and alcian blue staining of viral polypeptides separated by SDS-PAGE, and immunoprecipitation experiments using hyperimmune antisera suggest that the non-glycosylated 27 kDa polypeptide may represent a capsid-associated protein and the 82 kDa glycoprotein may represent an envelope-associated protein, which appears to be composed of a 67 kDa protein moiety. Fish injected with PL-positive tissue homgenate developed a bimodal viremia, as indicated by the presence of cell-free, virus-associated reverse transcriptase activity and SLV in serum of fish from 1 to 3 wk post-injection and again from 7 wk on through the rest of the study. If horizontal transmission of SLV and PL occurs in infected chinook salmon, it is most likely to occur after the second viremic period begins.
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PMID:Preliminary analysis of the polypeptides of the salmon leukemia virus (SLV) and evidence for development of a bimodal viremia following SLV infection. 753 62

This study probes the regulation of cholesterol biosynthesis in the ocular lens by estimating the concentration and distribution of the messenger RNA for the rate-controlling enzyme for sterol synthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). Because the lens is dependent on biosynthesis for cholesterol, HMGR activity is crucial for the life-long growth of this organ. Young rat lenses were serially divided into several fractions by dissolution in an SDS-containing buffer and each fraction was equated to a percent of the lens radius based upon its protein content. HMGR enzyme activity and cholesterol synthesis has been shown to disappear from the lens cortex over a narrow arc of radius due to loss of enzyme protein. Using a published competitive reverse transcriptase-polymerase chain reaction method for amplifying HMGR mRNA (Powell, E. E., and P. A. Kroon. 1992. J. Lipid Res. 33: 609-614), an average of about 46,000 copies of this mRNA was estimated per lens at all rat ages examined (5-day-old to adult). However, copies/microgram total RNA decreased with aging. The distribution of HMGR mRNA across 95-60% of the lens radius was essentially uniform at 2000-3000 copies/mm3 tissue. But the very superficial cortex contained 5- to 7-times this concentration and accounted for about 35% of the total copies/lens. We estimated that cells in this region each contained 1 to 2 copies of message, a value similar to the estimated copy number of HMGR message in human lymphocytes (Powell and Kroon, ibid). This suggests that the translational efficiency and stability of lens HMGR mRNA must be very high.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Spatial distribution of 3-hydroxy-3-methylglutaryl coenzyme A reductase messenger RNA in the ocular lens: relationship to cholesterologenesis. 753 8

Proteoglycans metabolically labelled with [35S]sulphate and [3H]glucosamine or [3H]leucine were isolated from the incubation medium and cell layer of human adult mesangial cells and glomerular visceral epithelial cells using sequential DEAE chromatography purification steps followed by gel-filtration chromatography. The proteoglycan composition of each peak was analysed by treatment with HNO2, chondroitinase ABC or chondroitinase AC followed by chromatography on Sephadex G-50 columns. Heparan sulphate proteoglycan (HSPG) and dermatan sulphate proteoglycan were detected in both the culture medium and cell layer of mesangial cells. Culture medium of glomerular visceral epithelial cells contained HSPG and a second proteoglycan with the properties of a hybrid molecule containing HS and chondroitin sulphate (CS). The cell layer contained HSPG and CSPG. Detailed analysis of the hybrid molecule revealed that it had an apparent molecular mass of 400 kDa. SDS/PAGE of hybrid molecules, after treatment with heparitinase and chondroitinase ABC, revealed a core protein of 80 kDa. Using 1.8% polyacrylamide/0.6% agarose-gel electrophoresis, we deduced that the HS and CS were independently attached to one core protein. Because glomerular-basement-membrane HSPG is thought to be derived from mesangial cells and glomerular visceral epithelial cells and this molecule is involved in several kidney diseases, we investigated its synthesis in more detail. Anti-(rat glomerular-basement-membrane HSPG) monoclonal antibodies (JM403) and anti-(human glomerular-basement-membrane HSPG) polyclonal antibodies (both antibodies known to react with the large basement-membrane HSPG, perlecan) reacted strongly with HSPG obtained from both mesangial cells and glomerular visceral epithelial cells. However, the hybrid molecule did not react with these antibodies, suggesting that the HS side chain and the core protein were different from glomerular-basement-membrane HSPG. To quantify HS we performed an inhibition ELISA using mouse antibodies specific for glomerular-basement-membrane HS glycosaminoglycan side chains. Glomerular visceral epithelial cells produced significantly higher levels of HS (between 197.56 and 269.40 micrograms/72 h per 10(6) cells) than mesangial cells (between 29.8 and 45.5 micrograms/72 h per 10(6) cells) (three different cell lines; n = 3; P < 0.001). HS production by these cells was inhibited by cycloheximide, revealing that it was synthesized de novo. Expression of perlecan mRNA, demonstrated using reverse transcriptase PCR, was different in the two cell types. We conclude that glomerular visceral epithelial cells and mesangial cells have characteristic patterns of proteoglycan production. Glomerular visceral epithelial cells produced a hybrid proteoglycan containing CS and HS independently attached to its core protein.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Proteoglycan production by human glomerular visceral epithelial cells and mesangial cells in vitro. 753 59

HIV-1 reverse transcriptase from the HIV-1 strain WMF 1.13 was expressed in Escherichia coli JM 105 using a pKK233-2 vector. The bacteria were cultivated in a 20-l fermentor with 14-l net volume using M9ZB medium containing bactotryptone and yeast extract. After induction of reverse transcriptase (RT) expression by addition of isopropyl-beta-D-thiogalactopyranoside the enzyme concentration was monitored. Both soluble and inclusion-body deposited RT were detected by Western blots. Inclusion-body formation was confirmed by transmission electron microscopy. Further purification of soluble and insoluble RT was investigated. After cell desintegration by enzymatic treatment combined with osmotic shock and centrifugation, the supernatant was desalted by size-exclusion chromatography and further purified by DEAE-Sepharose FF, AF-Heparin Toyopearl 650 M and Fractogel EMD TMAE 650 (S). The results of the purification steps were monitored by SDS-PAGE with silver staining, non-radioactive RT assay and protein determination with Coomassie Blue. The sediment was extracted with 6 M GuHCl and after clarification and conventional refolding, treated in the same manner as soluble RT. This method is well suited for studying fermentation conditions as well as purification conditions. The RT is expressed in approximately equal amounts as soluble and insoluble enzyme.
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PMID:Laboratory-scale production and purification of recombinant HIV-1 reverse transcriptase. 753 52

We have mapped specific RNA-protein contacts between human immunodeficiency virus (HIV) type I reverse transcriptase (RT) and its natural primer, human tRNA(3Lys), using a site-specific crosslinking strategy. Four different tRNA(3Lys) constructs with a single 32P-labeled 4-thiouridine (4-thioU) residue at positions -1, 16, 36 or 41 were synthesized. After incubation with RT followed by irradiation, crosslinks were localized to either the p66 or p51 subunit of RT by digestion with nuclease and SDS gel fractionation. 4-thioU at position -1 or 16 transferred label to the p66 subunit almost exclusively (> 90%), whereas position 36 labeled both p66 and p51 (3:1). Position 41 yielded no detectable crosslinks. The region of p66 contacted by position -1 of tRNA(3Lys) was localized to the 203 C-terminal amino acids of RT by CNBr cleavage, whereas a 127 amino acid-CNBr peptide (residues 230-357) from both p66 and p51 was labeled by position 36. Functionality of the 4-thioU-modified tRNA(3Lys)(-1) crosslinked to RT in the presence of an RNA but not a DNA template was demonstrated by the ability of the tRNA to be extended. These results localize the 5' half of the tRNA on the interface between the two RT subunits, closer to the RNase H domain than to the polymerase active site, in accord with previous suggestions. They argue further that a specific binding site for the 5' end of the primer tRNA(3Lys) may exist within the C-terminal portion of the p66 subunit, which could be important for the initiation of reverse transcription.
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PMID:Site-specific crosslinking of 4-thiouridine-modified human tRNA(3Lys) to reverse transcriptase from human immunodeficiency virus type I. 754 Jan 37


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