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)

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

The red deer is a seasonally breeding mammal with a circannual cycle of prolactin secretion which reaches its peak during the non-breeding season. This study investigated expression of the prolactin receptor gene in red deer tissues collected in the breeding and non-breeding seasons. A 562 bp fragment of the extracellular domain of the red deer prolactin receptor cDNA was amplified from red deer liver poly(A)+ RNA by reverse transcriptase-polymerase chain reaction (RT-PCR) using primers designed from the human sequence. Northern blots were prepared using 10-20 micrograms poly(A)+ RNA. The blots were hybridized to the 562 bp cDNA labelled by random priming with alpha 32P-dCTP. A main transcript of 3.5 kb was expressed in liver, heart, kidney and testis throughout the year and in epididymis during the breeding season only. In the testis an additional major transcript of 1.7 kb was present during the breeding and non-breeding seasons. Competitive binding assays using 125I-ovine prolactin (125I-oPRL) were performed on microsomal membrane fractions prepared from liver. Scatchard analyses confirmed the presence of a single class of lactogen-binding receptor with a mean Ka of 0.87 +/- 0.12 x 10(9) M-1 and a Bmax of 73.6 +/- 9.8 fmol/mg protein (n = 5). Cross-linking of 125I-oPRL to liver microsomes with 0.5 mM disuccinimidyl suberate followed by SDS-PAGE revealed a major band of molecular mass 56 kDa which was displaced by ovine prolactin, suggesting a specific lactogen-binding entity of 33 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Expression of the prolactin receptor gene during the breeding and non-breeding seasons in red deer (Cervus elaphus): evidence for the expression of two forms in the testis. 756 44

Human CD6 is a monomeric 105/130-kDa T cell surface glycoprotein that is involved in T cell activation. The apparent discrepancy between the size of the cytoplasmic domain in human (44 amino acids) and mouse (243 amino acids) CD6, led us to use reverse transcriptase-polymerase chain reaction of human peripheral blood lymphocyte mRNA to isolate cDNA clones that include the carboxyl-terminal coding region of human CD6. The nucleotide sequence of the longest human cDNA clone, CD6-PB1, predicts a protein of 668 amino acids with a 244-amino acid cytoplasmic domain similar in size to and possessing 71.5% amino acid sequence identity with the cytoplasmic domain of mouse CD6. This previously unrecognized 244-amino acid cytoplasmic domain does not have significant homology to any other known protein (except mouse CD6), but does possess two proline-rich motifs containing the SH3 domain-binding consensus sequence, a serine-threonine-rich motif repeated three times, three protein kinase C phosphorylation-site motifs, and 10 casein kinase-2 phosphorylation-site motifs. These sequences are likely to play a role in the ability of CD6-specific monoclonal antibodies to stimulate T cell proliferation. Full-length CD6 cDNA containing this cytoplasmic domain sequence encodes a monomeric 105/130-kDa protein that can be immunoprecipitated from the surface of transfected cells and comigrates upon SDS-PAGE with wild-type CD6 immunoprecipitated from PBL. We also isolated two alternatively spliced forms of human CD6 cDNA lacking sequences encoding membrane-proximal regions of the cytoplasmic domain which maintain the same reading frame as CD6-PB1. The short cytoplasmic domain of the previously reported human CD6-15 cDNA clone results from a deletion of a 20-bp segment through use of an alternative 3' splice site, resulting in a frame shift and premature termination of translation relative to the clones we have isolated. These data demonstrate that human CD6 possesses a large cytoplasmic domain containing sequence motifs that are likely to be involved in signal transduction upon stimulation of T cells through CD6 ligation.
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PMID:Human CD6 possesses a large, alternatively spliced cytoplasmic domain. 758 69

mRNA was isolated and purified from human liver, and it was also used as templet for cDNA synthesis under the existence of reverse transcriptase. Two primers were designed and synthesized according to GST gene sequence which has been reported, GST gene was obtained using cDNA as templet and PCR technique. The sequencing result indicated that the GST gene is reliable, it was subcloned into NdeI and Bg1 II sites of plasmid pIJ6021, and then introduced into Streptomyces lividans TK54. Proteins were isolated from transformants (TK54/pIJ4486 and TK54/pIJ6021) respectively, SDS-PAGE result showed that the GST over-expressed and its yield is about 15% in soluble proteins in Streptomyces.
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PMID:[Over-expression of glutathione S-transferase in Streptomyces]. 763 3

Human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) and its domain fragments were used to map nucleic acid binding sites within the enzyme. Discrete domain fragments were produced after the digestion of three forms of RT (p66, p66/p51 heterodimer, and p51) with V8 protease or trypsin, and the primary structure of each domain fragment was mapped by both immunoblotting and N-terminal amino acid sequence analysis. These domain fragments represent N-terminal, middle, or C-terminal regions of RT. Using Northwestern or Southwestern blotting assays, the domain fragments were evaluated for nucleic acid binding. In this technique, RT proteins are electroblotted onto the membrane and renatured after SDS-PAGE; the proteins are then probed with the primer analogues 32P-labeled d(T)16 or 32P-labeled tRNA(Lys,3). A V8 protease domain fragment spanning residues 195 to approximately 300 (p12), which was found earlier to be UV cross-linked to the primer in intact RT [Sobol et al. (1991) Biochemistry 30, 10623-10631], showed binding to both nucleic acid probes. We first localized nucleic acid binding in p66 to an N-terminal domain fragment of residues 1 approximately equal to 300. By contrast, a C-terminal domain fragment termed p30(303 approximately equal to 560) did not show nucleic acid binding. To investigate the role of the region just N-terminal to residue 303, an expression vector named pRC-35 encoding residues 273-560 was constructed.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mapping of nucleic acid binding in proteolytic domains of HIV-1 reverse transcriptase. 768 75

We have successfully expressed and purified the human immunodeficiency virus type-1 reverse transcriptase (RT) using the baculovirus expression vector system. This expression system provides a eukaryotic environment in which post-translational modifications of foreign gene products can occur. After infection with recombinant virus, Western blot analysis confirmed the presence of an immunoreactive polypeptide of approximately 66 kDa from insect Sf9 cell lysates. RT was then purified from crude extracts of baculovirus-infected Sf9 cells; SDS-PAGE analysis of fractions obtain from partial purification showed that in contrast to the Escherichia coli-expressed RT, the baculovirus-expressed RT corresponded to a doublet of peptides at approximately 66 kDa. Further purification of the protein resulted in a p66 protein, judged to be more than 90% pure by SDS-PAGE and Coomassie blue stain. Following purification, the baculovirus derived RT had specific activity for DNA polymerase similar to that of the E. coli-derived RT. Therefore, RT purified from Sf9 cells appears to be suitable for structure-function studies of this enzyme.
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PMID:Expression and purification of the HIV-1 reverse transcriptase using the baculovirus expression vector system. 769 Jun 27

Poliovirus RNA polymerase (3Dpol) was cross-linked to [32P]ribonucleoside triphosphates (NTPs) by reduction of oxidized NTP-protein complexes. Cross-linked complexes were digested with cyanogen bromide, and resulting peptides were fractionated by reverse-phase HPLC. 32P-Labeled peptides were purified by secondary HPLC fractionation and/or additional digestion with endoproteinases Glu-C, TPCK-trypsin, or Asp-N followed by another HPLC fractionation. N-Terminal sequences of the major [32P]-peptides were determined, and approximate sizes of these peptides were obtained by SDS-polyacrylamide gel electrophoresis. Two major NTP binding sites in 3Dpol were found. One site was between Asp-266 and Met-286; possible binding residues in this fragment were Lys-276, Lys-278, or Lys-283. A second binding site was between Ala-57 and Met-74 with Lys-61 or Lys-66 as possible binding residues. Alignment of these regions on the known structure of HIV-1 reverse transcriptase allowed us to predict the position of the downstream nucleotide binding site in the conserved "fingers" subdomain present near the active site cleft of both RNA and DNA polymerases. The N-terminal nucleotide binding site is not contained within a region that is conserved among other polymerases.
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PMID:Identification of nucleotide binding sites in the poliovirus RNA polymerase. 775 55

Carp acclimated to 10 degrees C gave 69k, 66k, and 62kDa light meromyosin (LMM) fragments in SDS-PAGE, while fish acclimated to 30 degrees C gave 74k, 69k, 66k, and 62kDa fragments. The microsequence analysis revealed that the 69k and 66kDa components from the 10 degrees C-acclimated carp contained an N-terminal amino acid sequence different from that of 62kDa. The four fragments from the 30 degrees C-acclimated carp showed the same sequence as that of the 69k and 66kDa components from the 10 degrees C-acclimated carp, except that the 2nd amino acid, Ala, of the 10 degrees C-acclimated LMM was replaced by Thr. DNA fragments encoding an N-terminal region of LMM were amplified by PCR or reverse transcriptase-PCR, demonstrating that the two acclimated groups further contained several amino acids substituted.
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PMID:Temperature acclimation induces light meromyosin isoforms with different primary structures in carp fast skeletal muscle. 788 20


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