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 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

DNA sequencing of the subgroup F human adenovirus serotype 41 (TAK, Ad41) fiber gene revealed the presence of two adjacent open reading frames encoding information for proteins with molecular weights of 60.6 kDa and 41.4 kDa (Pieniazek, et al; Nucleic Acids Res. 18: p. 1901, 1990). In this paper, various approaches were used to characterize the two proteins and determine whether both fibers were expressed in infected cells as well as on viral particles. We initially used a reverse transcriptase-polymerase chain reaction with primers for the short and long fiber genes to amplify mRNA from Ad41 infected HEp-2 cells at 48 h post-infection. Two distinct DNA bands; one slightly larger than 1.1 kbp and the other at about 1.7 kbp were identified. Second, we used polyclonal anti-Ad41 virion and monoclonal anti-Ad5 fiber antibodies to demonstrate that at both 24 and 36 h post-infection, Ad41 expressed two fiber proteins of the expected size. Specifically, by SDS-PAGE, one fiber (short) had a molecular weight of 40 kDa, while the other (long) had a molecular weight of 60 kDa. Third, by electron microscopy, two sizes of fibers were released from CsCl purified virions, both having a characteristic adenovirus morphology, with a knob at one end. The long fiber measured 315A in length and the short fiber was 250A long. These measurements are consistent with the two Ad41 fibers being encoded by the above open reading frames. We also performed a computer search to compare fiber sequences from other human adenovirus serotypes with that of the Ad41 short and long fiber proteins. The primary structure of both Ad41 fibers were found to be similar in that they contained tail, shaft and knob regions. Further, the tail region of both fibers (amino acids 1-42) showed a 74% overall homology to each other and contained the Ad conserved sequence NH2-F-N-P-V-Y-P-Y-COOH. An interesting difference, however, was observed in the shaft region where the long fiber (amino acids 43-389) had twenty-two 16-amino acid repeat motifs, while the short fiber (amino acids 43-233) had only twelve. Finally, we noted that the long fiber knob region was about 15% longer than that of the short fiber, and showed little overall homology. In conclusion, human adenovirus subgroup F (type 41) virions appear to differ from those of all other human adenoviruses (subgenera A-E) in that they contain two fiber genes and correspondingly, two different sized fibers.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Human adenovirus type 41 contains two fibers. 797 82

Human gamma-enolase cDNA prepared by reverse transcriptase-polymerase chain reaction was cloned into the Escherichia coli expression vector pKK223-3. The resulting plasmid, pHTK503, expressed human gamma-enolase as a 46-kDa protein in SDS-PAGE, and in the cells as the active gamma gamma form (designated as recombinant human NSE; R-NSE). R-NSE was purified from E. coli by several chromatographic elutions. Finally, 6.0 mg of R-NSE from 8.1 g cells was purified with a specific activity of 86 units/mg protein. The structural properties of R-NSE were compared with the NSE purified from human brain tissue (B-NSE). The biochemical and enzymatic characteristics were essentially the same, except for the isoelectric point (4.5 for B-NSE and 4.7 for R-NSE). In an NSE immunoassay system, R-NSE and standard NSE were almost equal in reactivity to the anti-NSE antibody. These results indicate that R-NSE can be used as standard assay material.
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PMID:Characterization of recombinant human neuron-specific enolase and its application to enzyme immunoassay. 823 6

Dp71, a C-terminal isoform of dystrophin, has been identified as the major DMD gene product in many nonmuscle tissues. In this report, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to clone and characterize four alternatively spliced Dp71 transcripts from cultured human amniocytes. The cDNAs encoding these Dp71 transcripts were shown to be alternatively spliced for exons 71 and/or 78. RT-PCR analysis also revealed that Dp71 transcripts alternatively spliced for exons 71 and/or 78 were expressed at varying levels in a number of adult human tissues, including muscle, heart, brain, kidney, lung, testis and liver. To investigate size heterogeneity at the translational level, Dp71 cDNAs isolated from amniocytes were expressed in E.coli to generate recombinant Dp71 fusion proteins. These fusion proteins were identified on immunoblots using antibodies specific for the C-terminal sequences of dystrophin that either included (antibody 1461) or excluded exon 78 (antibody 462B). The molecular masses of the Dp71 fusion proteins ranged from 71-75 kDa on SDS-PAGE, consistent with their predicted values. Immunoblot analysis using antibodies 1461 and 462B identified multiple Dp71 isoforms of approximately 70-75 kDa on SDS-PAGE in total protein lysates from amniocytes and various adult human tissues. This variation in molecular mass is consistent with the expression of Dp71 isoforms derived from transcripts alternatively spliced for exons 71 and/or 78. Total protein lysates from normal skeletal muscle, DMD muscle, amniocytes and brain were shown to contain beta-dystroglycan, a component of the dystrophin-associated glycoprotein complex (DGC).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cloning and characterization of alternatively spliced isoforms of Dp71. 854 29


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