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)

Decorin is a chondroitin/dermatan sulfate proteoglycan expressed by most vascular and avascular connective tissues and, because of its ability to interact with collagen and growth factors, has been implicated in the control of matrix assembly and cellular growth. To understand the molecular mechanisms involved in regulating its tissue expression, we have isolated a number of genomic clones encoding the complete decorin gene. The human decorin gene spans over 38 kb of continuous DNA sequence and contains eight exons and very large introns, two of which are 5.4 and > 13.2 kb. We have discovered two alternatively spliced leader exons, exons Ia and Ib, in the 5' untranslated region. These exons were identified by cloning and sequencing cDNAs obtained by polymerase chain reaction amplification of a fibroblast cDNA library. Using Northern blotting or reverse transcriptase PCR, we detected the two leader exons in a variety of mRNAs isolated from human cell lines and tissues. Interestingly, sequences highly (74-87%) homologous to exons Ia and Ib are found in the 5' untranslated region of avian and bovine decorin, respectively. This high degree of conservation among species suggests regulatory functions for these leader exons. In the 3' untranslated region there are several polyadenylation sites, and at least two of these sites could give rise to the transcripts of approximately 1.6 and approximately 1.9 kb, typically detected in a variety of tissues and cells. Using a genomic clone as the labeled probe and in situ hybridization of human metaphase chromosomes, we have mapped the decorin gene to the discrete region of human chromosome 12q23. This study provides the molecular basis for discerning the transcriptional control of the decorin gene and offers the opportunity to investigate genetic disorders linked to this important human gene.
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PMID:The human decorin gene: intron-exon organization, discovery of two alternatively spliced exons in the 5' untranslated region, and mapping of the gene to chromosome 12q23. 843 26

Alternative splicing of ovine insulin-like growth factor-I (IGF-I) transcripts generates three different mRNAs. Class 1 and class 2 transcripts contain exons 1 and 2 spliced to exon 3, respectively. A novel IGF-I mRNA containing exon W is spliced to exon 3 and has been located upstream of exon 1. No in-frame methionine codon was present in exon W and therefore translation is proposed to initiate at the methionine codon present in exon 3. Using primer extension, transcription initiation sites were found 179, 336, and 368 nucleotides upstream of exon 1 and 86, 96, 131, and approximately 850 nucleotides upstream of exon 2. The locations of these transcription initiation sites are well conserved among mammalian and avian IGF-I genes. Expression of exon 1-, 2-, and W-specific transcripts was examined in brain, heart, kidney, liver, lung, skeletal muscle, and spleen from adult ewes or 75-day fetal lambs using a reverse transcriptase-polymerase chain reaction assay. Exon 1 transcripts were the most abundant and found in all fetal and adult tissues. Exon 2 transcripts were found in all tissues and were generally expressed the highest in adult liver. Exon W transcripts were also found to be expressed in all tissues examined. Thus, the three alternatively spliced ovine IGF-I transcripts were expressed in a variety of fetal and adult tissues.
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PMID:Characterization of multiple transcription initiation sites of the ovine insulin-like growth factor-I gene and expression profiles of three alternatively spliced transcripts. 846 47

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

HOX11 is identified from the breakpoint of human T cell acute lymphoblastic leukemias with t(10;14). Since overexpression of HOX11 in T cells caused leukemias in transgenic mice, the endogenous HOX11 may play a role in proliferation and differentiation of T cells. In order to elucidate the role, we examined the expression of Hox11 in normal lymphocytes by a reverse transcriptase-polymerase chain reaction analysis. Two alternatively spliced Hox11 mRNAs were expressed in fetal spleens. However, lymphocytes did not express Hox11 mRNA during differentiation. Furthermore, it was not induced in primary lymphocytes after activation. These results suggest that ectopic expression of HOX11 in T cells is responsible for leukemogenesis.
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PMID:Two forms of Hox11 a T cell leukemia oncogene, are expressed in fetal spleen but not in primary lymphocytes. 855 42

Neutral endopeptidase (NEP) may regulate peptide-induced inflammation in the respiratory tract. It is of interest to determine which respiratory resident cells express NEP. Trachea and bronchi from seven nonsmoking, nonasthmatic subjects were examined. NEP messenger ribonucleic acid (mRNA) was characterized by Northern blot hybridization of cultured human tracheobronchial epithelial and smooth muscle cells, and reverse transcriptase-polymerase chain reaction (RT-PCR) in trachea and bronchi. In situ hybridization with biotin- and 35S-labelled antisense complementary ribonucleic acid (cRNA) probes was used to determine the distribution of NEP mRNA in human bronchial mucosa. NEP-immunoreactive material was detected using MEK10 murine monoclonal antibodies and the immunogold method with silver enhancement. NEP mRNA was 4.5 kb in size in the cultured human smooth muscle and epithelial cells by Northern blot analysis. No evidence was found by RT-PCR for truncated, alternatively spliced NEP mRNAs, such as del exon 16 or del exons 5-18 in human bronchus. NEP mRNA was detected by in situ hybridization in epithelial cells, submucosal glands, bronchial smooth muscle and endothelium. NEP-immunoreactive material was identified in the epithelium, submucosal glands, bronchial smooth muscle, and endothelium, demonstrating an excellent correlation between the distribution of NEP mRNA and the cell surface protein. NEP mRNA and immunoreactive material were excluded from epithelial goblet cell and submucosal gland mucous cell vacuoles. We conclude that the various sites of NEP protein and mRNA expression correlate with the locations of peptide receptors and NEP enzyme function, and are consistent with the hypothesis that NEP may regulate peptide-induced inflammation in human bronchi.
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PMID:Localization of neutral endopeptidase (NEP) mRNA in human bronchi. 857 69

The granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) is composed of at least two chains (alpha and beta). The alpha chain binds GM-CSF specifically with low affinity, and the binding is converted to high affinity when the alpha chain is associated with the beta chain. To date, there are at least six isoforms described for the GM-CSFR alpha, all involving alternative splicing at the 3' end, which alters the coding region and hence the protein produced. To detect variants at the 5' end of the GM-CSFR alpha mRNA, RNAse protection and reverse transcriptase polymerase chain reaction (RT-PCR) assays were performed using a probe spanning nucleotides 102-392 and pairs of primers covering exons 1-4. in addition to the expected full-length transcript, two mRNAs were detected, one containing a deletion of 24 nucleotides by alternative splicing at the 3' end of exon 2 (exon 2b-deleted isoform) and another in which exon 2 was completely deleted (exon 2-deleted isoform). Together, the isoforms were more highly expressed form). Together, the isoforms were more highly expressed than the full-length sequence (TF-1 cells: full-length 36 +/- 2.8% vs. exon 2-deleted isoforms 64 +/- 5.5%). These isoforms were detected in primary hematopoietic cells, blasts from patients with acute myeloid leukemia (AML), and malignant cell lines and the relative mRNA expression for the isoforms, was always similar to that of TF-1 cells. As sequences in the 5'untranslated region can be involved in the modulation of translational efficiency, translation of constructs constructs corresponding to these exon 2 deleted isoforms was assessed using an in vitro reticulocyte lysate system. Deletion of exon 2 resulted in significantly lower in vitro translation of the receptor protein relative to the full-length sequence (53, 56, and 76% in three separate batches of reticulocytes), while deletion of exon 2b resulted in higher translation of the sequence (164, 128, and 305%; p = 0.01). These data suggest a mechanism by which expression of the GM-CSFR alpha protein may be regulated by alternatively spliced transcripts with different translational efficiencies.
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PMID:Expression of two alternatively spliced forms of the 5' untranslated region of the GM-CSF receptor alpha chain mRNA. 863 32

The human genome contains a wide variety of endogenous retrovirus-like sequences. The human endogenous retrovirus type K (HERV-K) family comprises 30-50 members per haploid genome in humans and is highly conserved in Old World monkeys and apes. Some proviruses are displaying open reading frames (ORF) with coding capacity for viral particles. HERV-K sequences most likely code for the previously described human teratocarcinoma-derived virus (HTDV) and correlated expression of HERV-K Gag has been demonstrated by immunoelectron microscopy studies. Protease, but not yet reverse transcriptase (RT), enzymatic activity was demonstrated for recombinant HERV-K proteins. However, an ultrasensitive RT assay revealed specific polymerase activity associated with the HTDV particles. HERV-K transcription is specifically regulated by viral long terminal repeats and RNA is expressed at low steady-state levels in a variety of human tissues and tumours. In teratocarcinoma cell lines, HERV-K is highly expressed in a complex pattern showing full-length as well as subgenomic envelope (env) and two alternatively spliced small transcripts. The doubly spliced 1.8-kb mRNA codes for cORF protein which resembles Rev of HIV-1 and is located in the nucleolus. In addition, the cORF sequence acts as a leader and is essential for effective expression of glycosylated HERV-K Env protein. Although HERV-K sequences code for all necessary retroviral proteins, infectious particles could not yet be demonstrated. The putative implication of HERV sequences in pathophysiological processes, for example, testicular malignancies, remains to be elucidated.
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PMID:HERV-K: the biologically most active human endogenous retrovirus family. 879 33

The mammalian testis is innervated by extrinsic catecholaminergic nerves and responds to catecholamines with steroid secretion. Although the primate testis has also been shown to be innervated, potential differences in the density of this innervation between immature and sexually developed individuals have not been described. A recent study demonstrated that the primate ovary contains a network of neuron-like cells and that some of these cells are catecholaminergic. It is thus possible that the male gonad is also endowed with a similar intragonadal source of catecholamines. The present study addresses these two issues. Catecholaminergic nerves were identified as such by their content of immunoreactive tyrosine hydroxylase (TH; the rate-limiting step in catecholamine biosynthesis), and in some cases by glyoxylic acid histochemistry. Fibers containing TH were abundant in testes from juvenile animals (1-2 yr of postnatal life), but the density of this innervation was not maintained in adult animals, whose testis showed only a few TH-positive fibers scattered in the interstitial tissue. Testicular norepinephrine (NE) concentration was much lower in adult than in juvenile animals, suggesting that the marked increase in testicular weight that occurs with the attainment of sexual maturity is not accompanied by corresponding changes in NE content. At the ultrastructural level, testicular nerve fibers contained pleiomorphic, dense-core and clear vesicles, suggesting the presence of catecholamines and other neurotransmitters. In addition to this extrinsic catecholaminergic innervation, prepubertal testes, but not adult gonads, contained an intrinsic population of TH-immunopositive neuron-like elements, identified as cells by confocal scanning laser microscopy. To determine whether the prepubertal monkey testis indeed expresses the TH gene, testicular RNA was subjected to reverse transcriptase polymerase chain reaction to amplify the 5' end of TH mRNA, which encodes the regulatory domain of the enzyme. The cDNA that was obtained predicts an amino acid sequence similar, but not identical, to that encoded by the alternatively spliced type 1 TH mRNA form present in the adrenal gland. These results indicate 1) that the primate testis receives a dual catecholaminergic input, one provided by the extrinsic innervation and the other by neuron-like cells located within the gonad itself, and 2) that the influence exerted by both sources on testicular function may be more prominent during the prepubertal period than in adulthood. The presence in the testis of a TH mRNA variant encoding amino acid substitutions in its 5' end suggests that regulation of testicular TH enzyme activity may include a gonad-specific component.
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PMID:Testis of prepubertal rhesus monkeys receives a dual catecholaminergic input provided by the extrinsic innervation and an intragonadal source of catecholamines. 886 66

The neurotrophin tyrosine kinase receptors trkA, trkB, and trkC have been isolated and sequenced from several mammalian species. Their cognate ligands nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4), and neurotrophin-3 (NT-3) act as survival and trophic factors for neurons in the peripheral nervous system (PNS). In this study we have focused on the isolation and expression of the chicken trkA homologue. In addition to a near full-length cDNA sequence described, including an extracellular six amino-acid motif earlier found in neuronal TrkA in human and rat, a novel insert of 150 base pairs (bp) between subdomains IX and X in the otherwise well-conserved intracellular kinase domain is reported. Phylogenetic analysis showed the relationship between chicken trkA and the mammalian trkA receptors. Comparisons of the extracellular domains showed some amino-acid motifs of putative NGF binding function to be well conserved in chicken TrkA. The early expression of trkA mRNA, including the alternatively spliced insert form, was localized by in situ hybridization. As early as embryonal day 3 (E3), trkA mRNA is expressed in the condensing dorsal root ganglia, and at E4 distinct trkA mRNA expression appears in the primary sympathetic chain ganglia. Finally, using a reverse transcriptase-polymerase chain reaction (RT-PCR) approach, we found that among several tested growth factors only fibroblast growth factor-2 (FGF-2) upregulated trkA mRNA expression in E9 sympathetic ganglion explants. This upregulation of trkA was corroborated by subsequent NGF-stimulated fiber outgrowth.
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PMID:Molecular cloning of the chicken trkA and its expression in early peripheral ganglia. 889 7

The corpus luteum undergoes tremendous growth, development and regression each oestrous or menstrual cycle. These changes are reflected by equally impressive growth and regression of the luteal vasculature. We have previously shown that angiogenic factors from corpora lutea are primarily heparin binding and that one of these factors is similar to vascular endothelial growth factor (VEGF). In an effort to identify this factor, and to define its role in luteal vascular development, the cDNA for the coding region of ovine VEGF was sequenced and a sensitive RNase protection assay was developed to quantitate mRNA encoding VEGF in luteal tissues from ewes in the early (days 2-4), mid- (day 8) and late (days 14-15) stages of the oestrous cycle. In addition, an N-terminal peptide was synthesized from the translated ovine cDNA sequence for VEGF and an antiserum was raised against this peptide for use in western immunoblotting procedures. Nested reverse transcriptase (RT)-PCR of RNA from ovine corpora lutea resulted in three products that correspond in size to the alternatively spliced variants of VEGF (VEGF120, VEGF164, and VEGF188) predicted from other species. The RNase protection assay revealed that the proportion of mRNA encoding VEGF was 2- to 3-fold greater on days 2-4 than on day 8 or days 14-15. Densitometric analysis of gels from the RNase protection assay showed that VEGF120 represented approximately one third of the total mRNA encoding VEGF in the corpus luteum and that this proportion did not vary with stage of the oestrous cycle. SDS-PAGE and western immunoblot analysis of a homogenate from corpora lutea showed a single 18 kDa protein. These data demonstrate that VEGF is expressed in luteal tissue throughout the ovine oestrous cycle and that expression of mRNA encoding VEGF is upregulated during the period of rapid luteal development, when luteal vascular growth is at its maximum.
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PMID:Characterization and expression of vascular endothelial growth factor (VEGF) in the ovine corpus luteum. 895 42


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