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 c-Rmil/B-raf proto-oncogene is a member of the mil/raf family encoding serine/threonine protein kinases shown to be involved in signal transduction from the membrane to the nucleus. We isolated from a mouse brain library B-raf cDNAs containing a previously unidentified 36-base pair alternatively spliced exon located between exons 8 and 9 and, therefore, designated exon 8b. Human and mouse B-raf mRNAs also contain the 120-base pair alternatively spliced exon 10 previously described in the avian c-Rmil gene. Independent splicing of these two exons, located between the conserved region 2 (CR2) and the catalytic domain (CR3) gives rise to mRNAs potentially encoding four distinct proteins. By using specific sera generated against different portions of B-Raf, we identified at least 10 protein isoforms in adult mouse tissues. Some isoforms, in the range of 69-72 kDa, are not recognized by antisera directed against peptides encoded by exons 1 and 2, indicating the existence of B-Raf proteins with two different NH2 extremities. The other isoforms, in the range of 79-99 kDa, contain the amino acids encoded by exons 1 and 2, by either or both of the alternatively spliced exons, and, possibly, by another of the unidentified exon. Analysis of B-raf mRNA expression by reverse transcriptase-polymerase chain reaction and immunocharacterization of B-Raf proteins in different tissues of the adult mouse showed a tissue-specific pattern of B-Raf isoforms expression. Interestingly, isoforms containing amino acids encoded by exon 10 are specifically expressed in neural tissues. Taken together, these results suggest that distinct B-Raf proteins could be involved, in a tissue-specific manner, in signal transduction pathways.
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PMID:The mouse B-raf gene encodes multiple protein isoforms with tissue-specific expression. 755 96

An abundant 95-kDa protein belonging to the low density lipoprotein receptor supergene family is essential for chicken oocyte growth by mediating the uptake of multiple plasma-borne yolk precursors. This receptor harbors at the amino terminus a cluster of eight tandemly arranged repeats typical of the ligand binding domains of members of this family and is designated low density lipoprotein receptor relative with 8 repeats (LR8). Here, we demonstrate by reverse transcriptase-polymerase chain reaction, Northern, and Western blot analyses that the chicken expresses two forms of LR8, which are generated by differential splicing of an exon encoding a serine- and threonine-rich region characteristic of LRs, termed O-linked sugar domain. The female germ cell of the chicken expresses extremely high levels of the short form of LR8 (LR8-), i.e. the 95-kDa protein; in contrast, somatic cells express lower but detectable levels of the form containing the O-linked sugar domain (LR8+). The main sites of LR8+ expression in the chicken are the heart and skeletal muscle, i.e. the same tissues were LR8 mRNAs predominate in mammals; in addition, in situ hybridization demonstrates that a significant amount of LR8+ is produced in the hen's ovarian follicular granulosa cells. We found no apparent functional difference between the two receptor forms; however, cell type-specific targeting of the multiple ligands of these receptors possibly relates to their respective expression on the cell surface.
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PMID:Chicken oocytes and somatic cells express different splice variants of a multifunctional receptor. 755 19

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

Transforming growth factor-beta 1 (TGF-beta 1) induces angiogenesis in vivo and capillary morphogenesis in vitro. Two receptor serine/threonine kinases (types I and II) have been identified as signal transducing TGF-beta receptors. We explored the possibility of inhibiting TGF-beta-mediated events in glomerular capillary endothelial cells using a TGF-beta type II receptor (T beta R-II) transdominant negative mutant. A mutant TGF-beta type II receptor (T beta R-IIM), lacking the cytoplasmic serine/threonine kinase domain, was produced by polymerase chain reaction using rat T beta R-II cDNA as template. Since T beta R-II and TGF-beta type I receptor (T beta R-I) heterodimerize for signal transduction, the mutant receptor competes for binding to wild-type T beta R-I, hence acting in a dominant negative fashion. Glomerular capillary endothelial cells were stably transfected with T beta R-IIM, and four independent clones were expanded. That the T beta R-IIM mRNA was expressed was shown by reverse transcriptase-polymerase chain reaction, RNase protection assay, and Northern analysis. Presence of cell surface T beta R-IIM protein was shown by affinity cross-linking with 125I-TGF-beta 1. In wild-type endothelial cells, TGF-beta 1 (2 ng/ml) significantly inhibited [3H]thymidine incorporation to 63 +/- 10% of control (n = 4). In transfected endothelial cells carrying T beta R-IIM, TGF-beta 1 stimulated [3H]thymidine incorporation to 131 +/- 9% of control (n = 4, p < 0.005). Also, in wild-type endothelial cells, endogenous and exogenous TGF-beta 1 induced apoptosis and associated capillary formation. Both apoptosis and capillary formation were uniformly and entirely absent in transfected endothelial cells carrying T beta R-IIM. This represents the first demonstration that capillary morphogenesis in vitro is associated with apoptosis, and that interference with T beta R-II signaling inhibits this process in glomerular capillary endothelial cells.
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PMID:Inhibition of capillary morphogenesis and associated apoptosis by dominant negative mutant transforming growth factor-beta receptors. 767 46

Site-directed mutagenesis has been used to assess the importance of lysine 263 in substrate binding of human immunodeficiency virus-1 (HIV-1) reverse transcriptase. Previous studies have indicated that lysine 263 functions in the binding of 2'-deoxynucleoside 5'-triphosphate (dNTP) substrates (Basu, A., Tirumalai, R. S., and Modak, M. J. (1989) J. Biol. Chem. 264, 8746-8752). We studied this interaction directly by using site-specific mutagenesis to change lysine 263 to a serine. Highly purified mutant enzyme K263S bound natural dNTP substrates and primed polynucleic acid substrates with equal affinity when compared to the wild type reverse transcriptase. No difference was observed in the binding of 3'-azido-2',3'-dideoxythymidine 5'-triphosphate to the mutant reverse transcriptase on the basis of Km and Ki determinations. The serine substitution had no effect on RNase H activity. These results indicate that lysine 263 is not essential in the binding of substrates to HIV-1 reverse transcriptase.
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PMID:Biochemical analysis of human immunodeficiency virus-1 reverse transcriptase containing a mutation at position lysine 263. 767 98

Illimaquinone, a natural marine product, was shown by us to inhibit preferentially the ribonuclease H (RNase H) activity of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). We have also shown that illimaquinone inhibits the RNase H activity of HIV-2 RT in addition to that of HIV-1 RT, murine leukemia virus RT, and Escherichia coli RNase H. Chemical modifications of HIV-1 RT by sulfhydryl-specific reagents, such as N-ethylmaleimide (NEM) have been demonstrated to specifically inhibit the RNase H activity of the enzyme. Since our previous studies have suggested that cysteine 280 in HIV-1 RT interacts with the sulfhydryl reagents, we have examined the possibility that illimaquinone interacts with the RT molecules via amino acid residues located in the vicinity of cysteine 280 in both HIV-1 and HIV-2 RTs. In the combined effect studies of illimaquinone and NEM, the two structurally unrelated compounds were shown to be mutually exclusive, exhibiting an antagonistic interaction with both HIV-1 and murine leukemia virus-associated RNase H activities. This implicates cysteine 280, in both HIV-1 and HIV-2 RTs, to be in close proximity to the putative binding site of the enzyme to illimaquinone. The above conclusion is further supported by the fact that the RNase H activity of an enzymatically active mutant of HIV-1 RT, in which cysteine 280 was replaced by serine, was substantially more resistant to illimaquinone than the corresponding activity of the wild-type enzyme. The fact that NEM failed to inhibit E. coli RNase H as opposed to illimaquinone highlights a major difference between the retroviral and bacterial RNase H.
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PMID:The interaction of illimaquinone, a selective inhibitor of the RNase H activity, with the reverse transcriptases of human immunodeficiency and murine leukemia retroviruses. 768 48

The translocation between chromosomes 8 and 21, t(8;21)(q22;q22), is the most frequent abnormality seen in approximately 46% of patients with acute myeloid leukemia with French-America-British (FAB)-M2 morphology and an aneuploid karyotype. The breakpoints in this translocation have been characterized at the molecular level, and the genes involved are AML1 on chromosome 21 and ETO (eight twenty one) on chromosome 8. AML1 has homology to the alpha subunit of the murine polyoma enhancer binding protein, pebp2, and to the segmentation gene, runt, of Drosophila melanogaster. ETO, also called MTG8 (myeloid translocation gene on 8) has no overall homology to known proteins, but it contains two DNA-binding zinc finger motifs and several regions that are proline- and serine-rich. Both AML1 and ETO are thought to be transcription factors because the motifs they contain are found in other transcription factors. Both genes are transcribed from telomere to centromere, and cytogenetic analysis of variant translocations has shown that the critical junction always conserved is on the derivative 8 chromosome. The rearrangement between the two chromosomes results in a fusion gene that contains the 5' region of AML1 including that homologous to runt fused to almost all of ETO. The fusion transcript from the der(8) chromosome is consistently detected in patients with the t(8;21). The translocation can be detected at the molecular level with selected genomic DNA probes from chromosome 21 and from chromosome 8 near the breakpoint in 80-100% of the t(8;21) patients at diagnosis and in relapse, and with reverse transcriptase-polymerase chain reaction (RT-PCR) in all of the patients at diagnosis and in long-term remission. These results indicate that leukemic clones are still circulating in patients who have been in remission for as long as 8 years.
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PMID:The AML1 and ETO genes in acute myeloid leukemia with a t(8;21). 781 94

GH3 pituitary tumor cells have surface receptors for transforming growth factors-beta (TGF-beta s) and activins/inhibins. GH3 cell mRNA was screened by a novel reverse transcriptase-polymerase chain reaction technique with primers for receptor serine-threonine kinases. We isolated rat homologs of previously identified clones for type I (ALK-2 and ALK-5) and type II (ActRII, TGF-beta RII) activin and TGF-beta receptors, together with a novel clone, whose full-length version was isolated from a GH3 cell cDNA library. Named B1, it encodes a 505-amino-acid protein belonging to the family of type I receptor serine/threonine kinases. The kinase domain of B1 exhibits 90% identity to that of the TGF-beta type I receptor. B1 mRNA is expressed not only in pituitary cells but also in all other cells and tissues examined. B1 protein can be expressed on the cell surface, but cannot bind ligand unless a type II receptor is also present. When coexpressed with the type II receptors specific for TGF-beta or activin, B1 can be efficiently cross-linked to either ligand, suggesting that it can form heteromeric complexes with both type II receptor subunits.
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PMID:Molecular characterization of a type I serine-threonine kinase receptor for TGF-beta and activin in the rat pituitary tumor cell line GH3. 781 22

The glandular or tissue kallikreins are a multigene family of serine proteases, of which 13 genes (rKLK1-13) have been identified in the rat and are expressed in a wide variety of tissues. Kallikrein-like enzyme activity has been detected during the periovulatory period in the gonadotropin-primed immature female rat ovary and suggested to play a role in the inflammatory-like response at ovulation. In this study, we examined whether this enzyme activity was due to local expression of a rat KLK gene family member. Ovarian RNA, prepared from gonadotropin-treated animals, was assessed for rKLK gene expression by reverse transcriptase-polymerase chain reaction (RT-PCR) with universal rKLK primers derived from highly conserved regions in the rat KLK genes. Southern blot analysis of the RT-PCR products, using oligonucleotide probes specific for the individual genes, indicated that five rKLK gene family members, rKLK1 (encoding true kallikrein), rKLK3, rKLK7, rKLK8, and rKLK9, were expressed at varying levels in the ovaries of both untreated control and gonadotropin-treated immature female rats. The identities of these five rKLK messenger RNAs were further confirmed by DNA sequence analysis of the PCR products. In situ hybridization of gonadotropin-treated ovaries localized rKLK3 and rKLK7 gene expression to the luteinizing granulosa cells of periovulatory follicles. In an enriched population of nonluteinizing granulosa cells prepared from estrogen-primed animals, we also demonstrated rKLK3, rKLK7, rKLK9, and rKLK12 (but not rKLK1 or rKLK8) expression, whereas all six genes were expressed in the ovaries of these animals. In summary, we have reported the expression of six KLK gene family members in the rat ovary and localized this expression primarily to the granulosa cell. The potential roles of these enzymes in ovulation or other aspects of ovarian, particularly granulosa cell, function are yet to be elucidated.
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PMID:Kallikrein gene family expression in the rat ovary: localization to the granulosa cell. 786 67

In Drosophila and Caenorhabditis, signal transduction pathways initiated by the activation of receptor-protein tyrosine kinases can mediate developmental fate decisions. In order to examine whether similar mechanisms are employed during mammalian embryogenesis, we undertook a search for novel protein kinases expressed during heart development in the mouse. The primitive mouse heart is formed between 7.75 and 8.5 days post coitum (dpc) and consists of myocardial and endocardial cells. A reverse transcriptase polymerase chain reaction-based approach was used to amplify protein kinase specific products from cDNAs obtained from 8.5 dpc heart tissue. Twenty independent PCR products corresponding to either protein serine/threonine or tyrosine kinases were identified. In this report, we describe the characterization of two of the genes corresponding to the novel PCR products (designated Hek2 and msk). Hek2 encodes the mouse ortholog of human HEK2, a recently identified member of the eph receptor-protein tyrosine kinase gene family. Prior to and at the time of heart formation (7.5-8.0 dpc), Hek2 is expressed in the cranial (rostral) region of the embryo from which a subpopulation of cells will give rise to the rudimentary heart. Between 8.0 and 9.5 dpc, Hek2 mRNA expression is observed in myocardial cells, head mesenchyme and paraxial mesoderm. Hek2 transcripts are not detected in endocardial cells. After 9.5 dpc, Hek2 expression is downregulated. msk (for myocardial SNF1-like kinase) encodes a putative protein serine/threonine kinase most similar to the yeast gene SNF1. msk mRNA expression is restricted to myocardial cells and their progenitors in the 7.75-8.5 dpc developing heart. Subsequently, msk mRNA expression is rapidly downregulated. The patterns of Hek2 and msk expression suggest that these protein kinases may function during development of the primitive heart.
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PMID:Identification of novel protein kinases expressed in the myocardium of the developing mouse heart. 789 99


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