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)

Luteinizing hormone/chorionic gonadotropin (LH/CG) receptor complementary DNA (cDNA) isoforms were amplified using pseudopregnant rat ovarian total RNA as a template and the primers reaching over the coding regions at both ends in a reverse transcriptase-polymerase chain reaction (RT-PCR). Agarose gel electrophoresis of the PCR products revealed three bands corresponding to about 2.1, 2.0 and 1.8 kilobases (kb). Subcloning of pooled PCR products into EcoRI site of pUCBM20 resulted in 167 clones, from which five different restriction patterns were obtained by digestion with EcoRI and HaeIII. One clone of each was further characterized. It could be predicted from the nucleotide sequences that the clone rLHR2100 encoded a full-length receptor (a 674 amino acid mature protein), the clone rLHR2075 lacked part of exon IX (nucleotides 693-717) and encoded a truncated 225 amino acid mature protein, the clone rLHR1950 lacked exons III and IV (nucleotides 246-395) and encoded a nearly full-length protein (a 624 amino acid mature protein), and the clones rLHR1834 and rLHR1759 lacked the same part of exon XI (nucleotides 960-1225), with exon V (nucleotides 396-470) also absent in the latter, the deletion in exon XI leading both these clones to premature termination. The clone rLHR1834 encoded a 316 amino acid mature protein and rLHR1759 a 291 amino acid mature protein, respectively. The sequence data suggest that all of these isoforms contain the putative signal sequence and are derived from a single copy gene via alternative splicing. These results point further to the fact that the expression of the 90 kDa LH/CG receptor is regulated via an extensive alternative splicing of the receptor gene primary transcript.
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PMID:Expression of the LH/CG receptor gene in rat ovarian tissue is regulated by an extensive alternative splicing of the primary transcript. 135 63

The majority of infants born to HIV-positive mothers are not infected in utero, suggesting that the pregnancy factors produced by fetal trophoblasts may provide protection against HIV-1 infection. Except for steroid female hormones, little is known of other pregnancy factors that may regulate either resistance or susceptibility to HIV-1. Human chorionic gonadotropin (hCG)--the major glycoprotein produced by the placental trophoblast throughout the pregnancy--was tested on reverse transcriptase activity in HIV-infected ACH-2 lymphocytes and U1 monocytes. The results suggest that low non-cytotoxic doses of hCG (0.01-1.0 IU range) may inhibit viral replication in maternal blood cells.
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PMID:Effect of human chorionic gonadotropin (hCG) on reverse transcriptase activity in HIV-1 infected lymphocytes and monocytes. 138 34

Detection of gonadotropin releasing hormone (GnRH) mRNA in rat ovary was carried out, and cloning of LH, chorionic gonadotropin (CG) receptor in human ovary was attempted by use of polymerase chain reaction (PCR). Firstly, in an attempt to detect the expression of GnRH or related gene in rat ovaries at the RNA level, GnRH message was amplified. Total RNA from rat ovaries was converted to cDNA using reverse transcriptase and amplified in PCR using a pair of specific primers complementary to the rat GnRH cDNA. The DNA products were subcloned into plasmid vectors and their sequence determined. 1. In the rat ovary, a prominent PCR product of 462 bp was identified as a fragment of prothymosin alpha cDNA previously found in the spleen. 2. In contrast, RT-PCR amplification of hypothalamus and granulosa cell messages indicated the presence of a 244 bp product with identical sequence to GnRH. To confirm the presence of GnRH message, a second set of GnRH primers was used. PCR amplification of cDNA from hypothalamus, granulosa cells and whole ovary yielded a product identical with the authentic GnRH cDNA sequence. These data demonstrated the presence of mRNA for GmRH and prothymosin alpha in the rat ovary. Secondly, a part of the human LH, CG receptor was obtained from human granulosa cells by selective amplification with PCR of DNA segments presenting possible sequence similarity with genes for the porcine or rat LH, CG receptor. Total RNA from human granulosa cells was converted to cDNA and amplified in PCR using degenerate oligonucleotide primers corresponding to possible conserved regions in extracellular segments of the porcine or rat LH, CG receptor.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Detection of gonadotropin releasing hormone (GnRH) messenger RNA in the ovary and cloning of LH, chorionic gonadotropin (CG) receptor]. 178 65

A one-step (all reactants added simultaneously) reverse transcription and polymerase chain reaction (RT-PCR) procedure for amplification of full-length open reading frames (ORFs) of relatively rare transcripts was developed. It was applied for cloning rat luteinizing hormone/chorionic gonadotropin receptor cDNA isoforms larger than two kb. In the procedure developed, manual work is minimized, thus large numbers of samples can be handled, since after denaturation of template RNA and the primers and addition of other reagents, no further manual steps are needed. No inhibitory effect of avian myeloblastosis virus (AMV) reverse transcriptase (RT) on Thermus aquaticus (Taq) DNA Polymerase was found. This was because, under the conditions described, Taq DNA Polymerase effectively amplified picogram amounts of plasmid DNA or template reverse transcribed from nanograms of total ovarian RNA in the presence of AMV-RT. Even a large excess of AMV-RT did not inhibit Taq DNA Polymerase. Thus, our coupled one-step RT-PCR procedure amplifies fast and reproducibly full-length ORFs from nanogram amounts of total RNA.
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PMID:A coupled one-step reverse transcription PCR procedure for generation of full-length open reading frames. 751 6

Human chorionic gonadotropin (hCG)--a pregnancy-associated immunomodulating hormone--has been recently shown in vitro to suppress reverse transcriptase activity in chronically HIV-infected lymphocytes and monocytes and to block viral transmission resulting from cell-cell contact between virus-carrying lymphocytes and placental trophoblasts. In further pursuit of the query into the mechanism of action, purified alpha and beta subunits of hCG were tested for the inhibition of p24 gag protein synthesis in virus-producing ACH-2 lymphocytes and U1 monocytes. Unlike the alpha subunit, beta-hCG displayed a distinct U-shaped dose response, characteristic of the effect of dimer hCG. Maximum inhibition of viral expression has been achieved at 10-100 ng/ml, the concentration corresponding to blood levels of beta-hCG in pregnant women. The doses that were several logs higher of normal levels seemed to increase viral production in monocytes. The data presented supports our original observations regarding the effect of intact hCG on HIV replication. While the mechanism of action remains to be established, the results suggest that the virus-interfering activity of hCG is determined by hormone-specific beta chain but not by the alpha subunit--shared with the family of glycoprotein hormones from the pituitary--follicle-stimulating hormone, luteinizing hormone and thyrotropin.
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PMID:Anti-HIV effect of beta subunit of human chorionic gonadotropin (beta hCG) in vitro. 753 8

To obtain insight into the secretion pattern of human chorionic gonadotropin (hCG) and its free subunits, hCG alpha and hCG beta, in vivo, we analyzed hydrocele fluids of 13 patients with testicular cancer and correlated the respective values to those of cubital vein and testicular vein serum. As a control population, patients with nonmalignant hydroceles (n = 11) were studied. Analyses were performed with a set of highly sensitive and specific time-resolved fluoroimmunoassays based on our own panel of monoclonal antibodies. In the collective of testicular cancer patients, increased hydrocele levels of either hCG or free hCG alpha or free hCG beta were observed in 77, 54, and 92% of cases; the corresponding percentages for cubital vein serum were 62, 23, and 31%. The cubital vein ratio of hCG:hCG alpha (546:1) and hCG:hCG beta (51:1) decreased to 64:1 and to 7:1 in the hydrocele fluids. Surprisingly, hydrocele fluids of five patients with pure seminoma, who were negative for the three markers in the periphery, revealed an elevation of free hCG beta in all cases, while hCG alpha and holo-hCG were elevated twice. Final proof that hCG beta and hCG alpha are indeed produced by these previously termed "marker negative" seminomas has been achieved by reverse transcriptase-polymerase chain reaction with primers specific for the alpha-subunit and the four most abundantly transcribed hCG beta genes 3, 5, 7, and 8. From these data, we conclude that: (alpha) seminomatous and nonseminomatous testicular cancers, irrespective of histology, secrete hCG and its free subunits; (b) the amount of free subunits being secreted in vivo by these tumors has been underestimated; and (c) the classification in marker-positive and marker-negative testicular cancer should be reconsidered.
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PMID:Human chorionic gonadotropin (hCG) and its free subunits in hydrocele fluids and neoplastic tissue of testicular cancer patients: insights into the in vivo hCG-secretion pattern. 792 24

Gap junctions (GJ) are aggregates of intercellular channels, composed of connexin (Cx) protein, between adjacent cells. The vertebrate ovarian follicle contains homocellular (granulosa cell-granulosa cell) and heterocellular (granulosa cell-oocyte) GJ. However, the function of GJ during final oocyte differentiation (maturation) is controversial. The objectives of this study are to reexamine the number and identity of Cx genes that are expressed in the Xenopus ovary, and to examine the potential role of GJ in oocyte maturation by determining the temporal association between changes in ovarian Cx mRNA content and the process of maturation. We used reverse transcriptase-polymerase chain reaction to amplify ovarian cDNA fragments using degenerate Cx primers. We amplified three Cx-like fragments: one was novel and two corresponded to known Cx of Xenopus ovaries (Cx38 and 43). The novel fragment was used to screen an ovarian cDNA library. One positive clone was identified and its nucleotide sequence determined. Its deduced amino acid sequence showed that it corresponded to a novel Cx, Cx41, belonging to the Group II class of Cx. Xenopus Cx41 showed the highest homology to rat Cx37 (65% identity, 80% similarity). Also, the last 10 C-terminal amino acids of Cx41 were identical to those of rat, mouse, and human Cx37. Cx41 transcripts were detected by riboprobe mapping in ovarian somatic cells, heart, leg muscle, liver and eye, but not in brain or in oocytes of any developmental stage. Full-grown follicles incubated in vitro with human chorionic gonadotropin became committed to mature within 1-4 hr, and physical signs of maturation (germinal vesicle breakdown) were seen at 4-5 hr. Significant reductions in the levels of Cx41 and 43, but not 38 transcripts were seen at 4 hr, after oocytes had committed to mature. Thus, if availability of Cx mRNA determines availability of Cx protein and GJ, our results would suggest that irreversible commitment to maturation occurred prior to major declines in follicular GJ during the periovulatory period. The present study is the first to report the presence of at least two hormone-responsive Cx gene transcripts (Cx41 and 43 in Xenopus) in ovaries of a single animal species.
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PMID:Molecular cloning, tissue distribution, and hormonal control in the ovary of Cx41 mRNA, a novel Xenopus connexin gene transcript. 856 53

Production of the placental hormone, chorionic gonadotropin (CG), increases dramatically as cytotrophoblasts fuse to form syncytiotrophoblasts. The CG alpha- and beta-promoters are both responsive to cAMP, although the kinetics of cAMP stimulation are different. In an effort to understand the mechanisms of coordinate induction of these genes, AP-2 binding sites were identified in the promoter regions of the alpha and CGbeta genes. AP-2 bound to the upstream regulatory element (-186 to -156 base pairs (bp)) in the alpha-promoter and to several different regions of the CGbeta promoter, including footprints 2 and 4B (FP2, -311 to -279 bp; FP4B, 221 to -200 bp). AP-2 antibodies induced supershifts of these complexes, confirming the identity of the protein-DNA complex. In JEG-3 cells, which contain abundant AP-2, mutations in these CGbeta AP-2 sites reduced basal activity and decreased cAMP stimulation. In AP-2-deficient Hep-G2 cells, co-transfection of AP-2 stimulated expression of the CGbeta promoter 10-20-fold, and the alpha-promoter was induced by 3-6-fold. Mutations that eliminate AP-2 binding to CGbeta FP4B reduced AP-2 stimulation by more than 80%, whereas mutations in FP2 reduced AP-2 stimulation by less than 50%. Analyses of AP-2 mutants revealed a requirement for the DNA binding/dimerization domain and the amino-terminal proline-rich and acid-rich transactivation domains for stimulation of the CGbeta promoter. Primary cultures of placental cytotrophoblasts were differentiated into syncytiotrophoblasts in vitro to examine AP-2 expression by reverse transcriptase-polymerase chain reaction. AP-2 mRNA levels increased by day 2 and continued to rise in parallel with a marked increase in alpha and CGbeta gene expression. We conclude that both the alpha and CGbeta promoters contain binding sites for AP-2 and suggest that this transcription factor provides a mechanism for coordinating the induction of these genes during placental cell differentiation.
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PMID:Regulation of the human chorionic gonadotropin alpha- and beta-subunit promoters by AP-2. 918 71

The expression of insulin receptor substrate-I (IRS-I) mRNA was demonstrated in rat luteal cells by Northern blot analysis, in situ hybridization as well as by reverse transcriptase polymerase chain reaction. Western blot with a polyclonal anti IRS-I antibody showed the presence of a 183 kDa protein which corresponds to the size of IRS-I reported in other tissues. Further studies were performed to determine whether human chorionic gonadotropin (hCG) can interact with the insulin-like growth factor-I (IGF-I) signaling pathway to increase tyrosine phosphorylation of IRS-I. While hCG alone was ineffective in stimulating the phosphorylation of IRS-I, IGF-I mediated phosphorylation of IRS-I was increased by prior exposure to hCG. These results were further confirmed by the immunoprecipitation of IRS-I from the lysate of hCG- and IGF-I-treated luteal cell cultures followed by Western blotting with anti-phosphotyrosine antibody. Similarly, pretreatment with forskolin also increased IGF-I stimulated IRS-I phosphorylation. The increased tyrosine phosphorylation of IRS-I seen in response to IGF-I stimulation following treatment with either hCG or forskolin was not due to an increase in IRS-I content. Furthermore, IGF-I receptor tyrosine kinase activity was not affected by forskolin, suggesting that the increase in IRS-I tyrosine phosphorylation was not the result of an increase in its activity. Thus, we conclude that hCG/LH and IGF-I signaling pathways 'cross-talk' to increase the levels of IRS-I tyrosine phosphorylation. The observed increase in IRS-I tyrosine phosphorylation may be the result of an increase in the stability of the phosphorylated form of IRS-I.
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PMID:IRS-I expression on the luteinized rat ovary: IGF-I and cyclic AMP effects on IRS-I tyrosine phosphorylation. 924 69

The interleukin-1 (IL-1) system has been shown to play an important role in human and murine embryo implantation. Recent studies have documented immunohistochemical evidence of interleukin-1 beta (IL--1 beta), interleukin-1 receptor antagonist (IL-1ra) and interleukin-1 receptor type I (IL-1R tI) in human preimplantation embryos and protein levels of interleukin-1 alpha (IL-1 alpha), IL-1 beta and IL1ra in human preimplantation embryo culture fluid have been correlated with successful implantation and pregnancy. Our aim in this study was to detect IL-1 beta, Il-1ra and Il-1R tI mRNA in single preimplantation mouse embryos and to describe the frequency of positive mRNA-expression at different developmental stages. B6C3F1-mice, 12 weeks old were pregnant mare serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG)-stimulated and mated. Animals were sacrificed at day 0.5, and zygotes were flushed from the tubes and cultured in HAMs-F10 medium. 2-cell- (2C-), 8-cell- (8C-), morula- (M-), early blastocyst- (EB-) and hatching blastocyst- (HB-) stage embryos were examined by one round of reverse transcriptase (RT) followed by two rounds of polymerase chain reaction (PCR) carried out on individual mouse embryos for beta-actin (internal standard), IL-1 beta, IL-1ra and IL-1R tI-mRNAs. The frequencies of positive mRNA-expressions were as follows (2C/8C/M/EB/HB); beta-actin: 91/96/100/100/98%; IL-1b: 0/0/2.5/6.25/19; IL-1ra; 0/5/30/41/74% and IL-1R tI: 0/0/10/20/25%. The incidence of IL-1ra mRNA expression increased with developmental stage. IL-1ra mRNA seems to be expressed in a very high percentage (74%) of embryos near the time of implantation, whereas the percentage of IL-1 beta-mRNA positive embryos is surprisingly low (19%).
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PMID:Different pattern of interleukin-1 beta-(IL-1 beta), interleukin-1 receptor antagonist- (IL-1ra) and interleukin-1 receptor type I- (IL-1R tI) mRNA-expression in single preimplantation mouse embryos at various developmental stages. 929 78


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