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 sequence of bovine parathyroid hormone mRNA has been determined by sequence analysis of near full-length cloned DNA complementary to the mRNA. Restriction fragments hybridized to the mRNA and extended toward the 5' terminus with reverse transcriptase were analyzed to derive the sequence not present in cDNA. The reverse transcripts were heterogeneous in length with three major stopping points within 8 nucleotides of each other and a minor stop about 30 bases further toward the 5' terminus of the mRNA. The sequence of the gene corresponding to the minor reverse transcript begins with the sequence 5' XXXATATATAAAA which contains the consensus sequence for a TATA box, a putative eukaryotic promoter sequence. Assuming that the major reverse transcriptase stop nearest the 5' terminus of the mRNA, which is 24 bases downstream from the TATA box, represents the beginning of bovine PTH mRNA, the mRNA contains 672 nucleotides, 100 in the 5' noncoding region, 348 in the coding region and 224 in the 3' noncoding region. Bovine PTH mRNA contains 38% G and C bases. The 3' noncoding region is particularly rich in A and U bases with the last 100 nucleotides of the molecules containing 46% U and 32% A. As with other mRNAs, the sequences CG and UAG occur much less than expected. The 5' noncoding region does not contain an AUG before the initiator codon and contains two potential regions that could base-pair with sequences near the 3' terminus of 18S ribosomal RNA. The sequence AAUAAA is present 14 nucleotides from the polyadenylic acid at the 3' terminus. Bovine PTH mRNA exhibits extensive homology with human PTH mRNA.
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PMID:Nucleotide sequence of bovine parathyroid hormone messenger RNA. 618 74

DNA complementary (cDNA) to a partially purified preparation of bovine parathyroid hormone mRNA was synthesized using avian myeloblastosis viral reverse transcriptase. The PTH cDNA contained about 750 bases and was greater than 95% sensitive to digestion by S1 nuclease. Analysis of the mRNA preparation by excess RNA hybridization to the PTH cDNA revealed one rapidly hybridizing component consisting of 50% of the PTH cDNA. Sequential incubation of the PTH mRNA with reverse transcriptase and E. coli DNA polymerase I produced near full length double-stranded PTH cDNA. Of the 22 restriction endonucleases tested, double-stranded PTH cDNA could be cleaved with Alu I, Mbo II, Sau 3A, Sst I, and Taq I. The restriction fragments corresponding to the 5' terminus of the sense strand were identified for the last three enzymes by comparing the size of fragments obtained from PTH cDNA before and after cleavage of the hairpin loop connecting the two strands by S1 nuclease. The restriction map of the cDNA was used to detect clones of bacteria containing recombinant plasmids with near full length PTH cDNA inserts.
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PMID:Synthesis, restriction analysis, and molecular cloning of near full length DNA complementary to bovine parathyroid hormone mRNA. 625 49

Pseudohypoparathyroidism type Ib (PHP-Ib) is thought to be caused by a PTH/PTH-related peptide (PTHrP) receptor defect. To search for receptor mutations in genomic DNA from 17 PHP-Ib patients, three recently isolated human genomic DNA clones were further characterized by restriction enzyme mapping and nucleotide sequencing across intron/exon borders. Regions including all 14 coding exons and their splice junctions were amplified by polymerase chain reaction, and the products were analyzed by either temperature gradient gel electrophoresis or direct nucleotide sequencing. Silent polymorphisms were identified in exons G (1 of 17), M4 (1 of 17), and M7 (15 of 17). Two base changes were found in introns, 1 at the splice-donor site of the intron between exons E2 and E3 (1 of 17) and the other between exons G and M1 (2 of 17). Total ribonucleic acid from COS-7 cells expressing minigenes with or without the base change between exons E2 and E3 showed no difference by either Northern blot analysis or reverse transcriptase-polymerase chain reaction. Radioligand binding was indistinguishable for both transiently expressed constructs. A missense mutation (E546 to K546) in the receptor's cytoplasmic tail (3 of 17) was also found in 1 of 60 healthy individuals, and PTH/PTHrP receptors with this mutation were functionally indistinguishable from wild-type receptors. PHP-Ib thus appears to be rarely, if ever, caused by mutations in the coding exons of the PTH/PTHrP receptor gene.
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PMID:Pseudohypoparathyroidism type Ib is not caused by mutations in the coding exons of the human parathyroid hormone (PTH)/PTH-related peptide receptor gene. 774 8

A second prostaglandin G/H synthase (PGHS-2), encoded by a gene separate from that for the original PGHS (PGHS-1), has recently been identified. We have shown that PGHS-2 is expressed in cultured mouse calvariae and have compared regulation of PGHS-2 and PGHS-1 messenger RNA (mRNA) levels. PGHS-2 mRNA was not detectable in freshly isolated bones, but was induced during culture and further stimulated by interleukin-1 (IL-1) and PTH. Both factors also increased PGHS-2 protein levels. Changes in medium prostaglandin E2 (PGE2) production correlated with increases in PGHS-2 mRNA levels. However, with IL-1, PGE2 production was increased more than PGHS-2 mRNA levels (treated/control ratio, 3.4 and 1.5, respectively), whereas with PTH there was a closer correspondence (2.0 and 2.1). Cortisol reduced PTH-stimulated PGE2 production (treated/control ratio decreased from 3.1 to 0.2) more than PGHS-2 mRNA levels (2.8 to 0.8). In the presence of exogenous arachidonic acid, changes in PGHS-2 mRNA levels with IL-1, PTH, and cortisol correlated closely with changes in PGE2 production. PGE2 itself increased PGHS-2 mRNA, and nonsteroidal antiinflammatory drugs decreased PGHS-2 mRNA levels by 80%. In contrast, PGHS-1 mRNA was expressed constitutively and was not affected by IL-1, PTH, or cortisol when measured by competitive reverse transcriptase-polymerase chain reaction. We conclude that regulation of PGE2 production is predominantly through PGHS-2, rather than PGHS-1; that IL-1 and cortisol may also regulate arachidonic acid release; and that PGE2 may amplify its own production through stimulation of PGHS-2.
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PMID:Regulation of the two prostaglandin G/H synthases by parathyroid hormone, interleukin-1, cortisol, and prostaglandin E2 in cultured neonatal mouse calvariae. 807 Mar 58

PTH-related peptide (PTHrP), the factor mediating the syndrome of humoral hypercalcemia of malignancy, is also expressed in smooth muscle cells (SMC) of the urinary bladder and uterus in response to mechanical distention and fetal occupancy, respectively. Vascular SMC also produce PTHrP, and its expression is induced by serum and vasoconstrictors, such as angiotensin-II. To determine whether mechanical distension affected vascular PTHrP gene expression, the abdominal aorta of adult male rats was balloon-distended, and aortae were collected at various times after the intervention. PTHrP mRNA was determined by competitive reverse transcriptase-polymerase chain reaction, using sequential dilutions of a cloned internally truncated PTHrP RNA fragment as standard. The molar concentration of PTHrP mRNA was obtained by extrapolating at a standard/wild-type band intensity ratio of 1:1. Aortic PTHrP mRNA was induced from a basal level of 19, to 22, 46, 36, 13, 12, 22, and 20 attamoles/mg total RNA 1, 2, 12, 24, and 48 h and 7 and 48 days after balloon distension, respectively. To determine whether mechanical events directly regulate vascular PTHrP gene expression, primary rat aortic SMC were plated and placed on a rocking device at 20 oscillations/min to create a gentle flowing motion of the culture medium. Rocking induced PTHrP mRNA of SMC exposed to either serum-free medium or 10% serum by 2.5-and 4.0-fold at 4 h, and 2.9- and 3.7-fold at 24 h, respectively. These effects were oscillation rate dependent, potentiated by angiotensin-II, and specific, as similar changes were not observed in alpha-actin mRNA content. Flow motion-induced PTHrP mRNA at 24 h was partially decreased by 10(-6) M colchicine (which inhibits microtubule assembly), but not by cytochalasin-E (which disrupts actin polymerization). As PTHrP is a known vasorelaxant, we propose that mechanical events induce the release of PTHrP by SMC, possibly to serve as a compliance factor or an agent for vascular remodeling.
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PMID:Mechanical stimuli induce vascular parathyroid hormone-related protein gene expression in vivo and in vitro. 815 26

Although the differentiation of mature osteoblasts has been well studied, there is still a need for a convenient way to study preosteoblast differentiation. Our laboratory has recently described a method for isolating small numbers of authentic osteoblast precursor cells from human bone marrow (Rickard et al., J Bone Miner Res 11:312-324, 1996). Here we describe the conditional immortalization of these cells by retroviral transfection with the amphotrophic vector, pZipSV40tsa58, which encodes for a temperature-sensitive mutant form of the simian virus large T-antigen. At the permissive temperature of 34 degrees C, the cell lines proliferated, but differentiation was arrested, whereas at the restrictive temperature of 39.5 degrees C, proliferation was decreased and differentiation was induced. As assessed by semiquantitative reverse transcriptase PCR after 4 days of culture at 39.5 degrees C, the six cell lines expressed similar mRNA levels both constitutively and in response to dexamethasone (Dex) and 1alpha,25-dihydroxyvitamin D3 (1,25(OH2)D3) for osteoblast (alkaline phosphatase [ALP], type I collagen [Col I], osteocalcin [OC], and parathyroid hormone receptor [PTH-R] and adipocyte (lipoprotein lipase [LPL]) genes. In the presence of 10(-8) M Dex, gene expression for ALP, PTH-R, and LPL increased, but that for OC decreased. Stimulation with 10(-8) M 1,25(OH2)D3 increased gene expression for ALP, OC, and Col I. Changes in protein production for ALP, OC, and type I procollagen in response to Dex and 1,25(OH2)D3 were similar to changes in mRNA levels. When cultured at 39.5 degrees C with ascorbate and beta1-glycerolphosphate for 21 days, mineralization of matrix occurred, whereas culture with Dex plus 1,25(OH2)D3, or rabbit serum led to enhanced formation of cytoplasmic lipid droplets within 6 days. Thus, these cell lines are capable of bipotential differentiation and should serve as an excellent tool to study the molecular mechanisms that regulate and select for osteoblast and adipocyte differentiation in humans.
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PMID:Development and characterization of conditionally immortalized osteoblast precursor cell lines from human bone marrow stroma. 949 13

HEM-1 was isolated as a putative factor responsible for oncogenic osteomalacia by Kumar et al. (Proc Assoc Am Phys 107:296-305; 1995). The cDNA was identified on the basis of PTH-like immunoreactivity; however, no studies have been reported of the expression of HEM-1 mRNA in oncogenic osteomalacia tumors. In this study, expression of HEM-1 mRNA was investigated in two oncogenic osteomalacia tumors and in a series of normal tissues. An HEM-1 PCR product was amplified from a cDNA library from one of the tumors, with six base changes identified, as compared with the published sequence. No expression was detected, however, in the oncogenic osteomalacia tumors either by Northern blot analysis or by reverse transcriptase PCR. This indicates that, although a region of HEM-1 sequence is present in the tumor cell cDNA library, any HEM-1 expression must be at very low levels. It is unlikely, therefore, that the HEM-1 product is the active factor responsible for oncogenic osteomalacia. In the normal tissues examined, human placenta, fibroblasts, parathyroid gland, liver, fetal bone, and rat kidney cortex, HEM-1 mRNA was not detected, suggesting that it does not have a physiological role in these tissues.
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PMID:Tumor expression studies indicate that HEM-1 is unlikely to be the active factor in oncogenic osteomalacia. 985 64

We previously showed that the processing of proparathyroid hormone (proPTH) to PTH was accomplished most efficiently by furin (17). Colocalization studies demonstrated that furin is expressed in the parathyroid, whereas proprotein convertase (PC)1 and PC2 are not. Since that time, another member of the PC family, called PC7, has been identified. Here we show, using coinfection studies, that PC7, as well as furin, can appropriately cleave PTH from proPTH. ProPTH and PTH were purified from cell extracts by reversed-phase HPLC and were identified by Western blot analysis and delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Colocalization studies, using Northern blot and reverse transcriptase-PCR analyses, showed that PC7 messenger RNA (mRNA) is expressed in the parathyroid gland. Therefore, PC7, like furin, has the potential to be involved in the physiological processing of proPTH to PTH. The two major regulators of parathyroid cell synthetic and secretory activity are the extracellular fluid calcium and 1,25-dihydroxyvitamin D [1,25(OH)2D] levels. We investigated whether either of these agents might modulate processing of proPTH to PTH by altering parathyroid convertase gene expression. In both in vitro and in vivo systems in which regulation of PTH mRNA levels were clearly apparent, there was no effect of either calcium or 1,25(OH)2D3 on parathyroid furin or PC7 mRNA levels. This is in contrast to the processing of proinsulin to insulin in the pancreatic beta-cell, which is up-regulated by glucose stimulation of PC1 and PC2 synthesis.
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PMID:Proparathyroid hormone processing by the proprotein convertase-7: comparison with furin and assessment of modulation of parathyroid convertase messenger ribonucleic acid levels by calcium and 1,25-dihydroxyvitamin D3. 1043 21

Skeletal tissue contains a network of nerve fibers expressing several neuropeptides, including vasoactive intestinal peptide (VIP) and the related peptide pituitary adenylate cyclase activating peptide (PACAP). These peptides have been demonstrated to regulate osteoclast formation and osteoclast activity. Using atomic force microscopy and by analysing changes of the intracellular calcium concentrations, we have recently demonstrated that multinucleated rat osteoclasts have cell membrane binding sites recognising VIP and PACAP. In the present study, we have further studied the expression of VIP receptor subtypes in mouse bone marrow cultures and isolated osteoclasts. A micromanipulation technique was used to isolate pure populations of osteoclasts formed in PTH-stimulated mouse bone marrow cultures. By reverse transcriptase polymerase chain reaction (RT-PCR), we studied the expression of mRNA for VIP-1, VIP-2, and PACAP receptors. The purity of the microisolated osteoclasts was determined by studying the expression of specific mRNA associated with the phenotypic trait of osteoclasts or osteoblasts/stromal cells. In this study, we show that mouse osteoclasts express VIP-1 and PACAP, but not VIP-2, receptor mRNA.
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PMID:Microisolated mouse osteoclasts express VIP-1 and PACAP receptors. 1091 50

Parathyroid hormone-related peptide (PTHrP) is released under ischaemic conditions and it improves contractile function of stunned myocardium. The actions of PTHrP are mediated primarily by the type 1 parathyroid hormone receptor (PTH.1R), while PTHrP and PTH.1R expression levels are increased in ventricular hypertrophy associated with experimental hyperthyroidism. Since chronic administration of thyroxine (T4) improves postischaemic recovery in isolated heart models subjected to ischaemia-reperfusion stress, we tested the hypothesis that experimentally induced hyperthyroidism is associated with elevated expression of PTHrP and PTH.1R in rat myocardium. Hyperthyroid and control male Wistar rats were subjected to ischaemia-reperfusion stress using the Langendorff technique, and the PTHrP and PTH.1R expression was assessed by relative quantitative reverse transcriptase-polymerase chain reaction, Western blot analysis and immunohistochemistry. In the Langendorff model, the recovery of left ventricular developed pressure at the end of the stablization period and 45 min into the reperfusion period was used to assess the cardioprotective actions of T4 administration. Our data show that hyperthyroid animals had increased tolerance to the ischaemia-reperfusion stress and that this was associated with an increase of PTHrP and PTH.1R expression levels compared with those of control animals. In the control animals, the expression of PTHrP was increased 45 min into the reperfusion phase, while the PTH.1R expression pattern was significantly and gradually decreased throughout the ischaemia and reperfusion phases. In the hyperthyroid animals, the PTHrP and PTH.1R expression pattern was significantly higher throughout the ischaemia and reperfusion phases compared with that of control hearts. Our data suggest that increasing levels of PTHrP and PTH.1R expression can mediate, at least in part, the T4 administration-induced cardioprotection in rat ventricular myocardium.
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PMID:Experimental hyperthyroidism increases expression of parathyroid hormone-related peptide and type-1 parathyroid hormone receptor in rat ventricular myocardium of the Langendorff ischaemia-reperfusion model. 1791 57


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