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)

Tissue distribution and potential alternative splicing of insulin-like growth factor I (IGF-I) messenger RNA were studied using reverse transcriptase-polymerase chain reaction (RT-PCR) on RNA from several tissues at various stages of the life cycle of coho salmon (Oncorhynchus kisutch). DNA sequence analysis of RT-PCR products revealed three IGF-I mRNA transcripts, designated Ea-1, Ea-2, and Ea-3, which code for three distinct prohormones, IGF-IA-1, IGF-IA-2, and IGF-IA-3, respectively. The E-domain of proIGF-IA-1 is 35 amino acids long and shares 77% sequence identity with the E-domain of human proIGF-IA, which is also 35 amino acids long. The proIGF-IA-2 and proIGF-IA-3 E-domains are homologous to the proIGF-IA-1 E-domain but contain 27 and 39 amino acid inserts, respectively, between Lys86 and Glu87. In the human IGF-I gene Lys86 is coded by exon 4 and Glu87 is coded by exon 6. This suggests that Ea-2 and Ea-3 transcripts may be the result of alternative splicing during pre-mRNA processing. All three transcripts were readily detectable using a solution hybridization/RNase protection assay. Furthermore, RT-PCR and DNA sequencing analysis indicate the presence of three IGF-I prohormones in another member of the Salmonidae family, the Atlantic salmon (Salmo salar). An analysis of IGF-I and -II E-domains from several vertebrates suggests that certain chemical and physical properties of the molecule are well conserved despite wide variations in primary structure. Ea-1, Ea-2, and Ea-3 transcripts were found in whole embryos, and liver, muscle, and brain of juvenile and adult salmon. At least one IGF-I transcript was found in heart, kidney, testes, ovary, adipose tissue, and spleen of juvenile salmon. These results indicate that IGF-I is expressed during embryonic development of fish, and that most tissues are capable of IGF-I mRNA production. These data also indicate that pre-mRNA transcripts can be alternatively spliced to yield at least three prohormones.
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PMID:Nucleotide sequence and tissue distribution of three insulin-like growth factor I prohormones in salmon. 140 98

In species such as the pig and human, gonadal steroidogenesis is believed to be dependent upon the availability of low density lipoprotein (LDL) cholesterol. However, before ovulation, Graafian follicles are impermeant to lipoproteins in the LDL class. Thus, de novo cholesterol biosynthesis via the rate-determining enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is likely to provide a significant mechanism for generating sterol substrate for steroidogenesis by granulosa cells before follicular rupture. As serum-free monolayer culture of (swine) granulosa cells offers an in vitro model of hormonally responsive HMG-CoA reductase, we generated a (porcine) complementary DNA and homologous complementary RNA to investigate by sensitive and specific ribonuclease protection assay the hormonal regulation of HMG-CoA reductase gene expression in ovarian cells from immature Graafian follicles. Using reverse transcriptase-polymerase chain reaction, we cloned and sequenced a 238-base pair complementary DNA from porcine luteal tissue that encodes the catalytic region of HMG-CoA reductase. GenBank analysis of the DNA sequence homology between the pig and other species showed the greatest concordance with human (88%) and hamster (90%). Solution hybridization/ribonuclease protection analysis of total RNA isolated from serum-free monolayer cultures of porcine granulosa cells revealed that insulin (3 micrograms/ml) increased HMG-CoA messenger RNA (mRNA) concentrations corrected for constitutive 18S ribosomal RNA expression in a time-dependent fashion, with significant effects observed at 12 h and a 6-fold increase by 48 h. Recombinant human insulin-like growth factor I (IGF-I) peptide was able to mimic the action of insulin alone. Neither FSH (100 ng/ml) nor 8-bromo-cAMP (1 mM) had observable effects on HMG-CoA message accumulation at any time point studied. However, the combined action of either FSH and insulin or 8-bromo-cAMP and insulin resulted in synergistic increases in reductase mRNA by 31- and 17-fold, respectively. To assess the possible feedback effects of sterol on HMG-CoA gene expression, granulosa cells were treated with LDL. At physiological concentrations, LDL suppressed basal expression of HMG-CoA mRNA to levels below the control value. In addition, LDL inhibited insulin-stimulated HMG-CoA mRNA accumulation by 84% as well as the synergistic effects of insulin and FSH (by 94%) and of insulin and 8-bromo-cAMP (by 93%). We conclude that insulin alone or in combination with FSH or cAMP augments the accumulation of HMG-CoA reductase mRNA in ovarian (granulosa) cells.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Regulation of porcine granulosa cell 3-hydroxy-3-methylglutaryl coenzyme A reductase by insulin and insulin-like growth factor I: synergism with follicle-stimulating hormone or protein kinase A agonist. 758 48

We have identified four transcription initiation sites in the salmon insulin-like growth factor I (IGF-I) gene. Use of the most upstream transcription start site generates a minor mRNA species with a 5' untranslated region (UTR) of approximately 450-550 nucleotides, whereas transcription starting at the downstream initiation sites results in more abundant IGF-I mRNAs with 5'-UTRs approximately 250, 245, and 165 nucleotides in length. No consensus TATA box-like elements are present immediately upstream of the most upstream start site identified, nor is this region particularly GC-rich. Transient expression assays, however, demonstrated orientation-dependent promoter activity in a 386-nucleotide-long fragment containing the major downstream transcription start sites. Additionally, reverse transcriptase polymerase chain reaction (RT-PCR) analyses demonstrated tissue and developmental stage-specific use of the various transcription start sites identified.
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PMID:Characterization of a salmon insulin-like growth factor I promoter. 794 38

A reverse transcriptase/PCR assay was employed to amplify an internal portion of insulin-like growth factor I (IGF I) and IGF II cDNA from the RNA of juvenile rainbow trout (Oncorhynchus mykiss) in an effort to detect analogues to the multiple size forms of IGF mRNA known to exist in mammals. After cloning and sequencing the PCR products, 4 variant size forms of trout IGF I mRNA and 1 form of IGF II mRNA were evident. The full cDNA nucleotide sequence of 1 form of IGF I mRNA and IGF II mRNA have been previously reported; the full cDNA nucleotide sequence of a second form of IGF I mRNA and PCR-derived nucleotide sequences of the 2 additional IGF I mRNA forms are reported herein. We define the 4 forms of IGF I mRNA (IGF IEa-1, Ea-2, Ea-3, and Ea-4) as having E-domain peptide lengths of 35, 47, 62, and 74 predicted amino acid residues, respectively. RNAse protection assays were used to quantify the mRNA levels of each of the 5 IGFs in 8 tissues of both juvenile and adult trout, as well as in testes and ovaries of adult trout. The IGF mRNA levels, normalized with 18S rRNA levels, suggest (1) at least 1 form of IGF I and IGF II mRNA are present at both developmental stages in all tissues examined; (2) all 5 IGF mRNA forms are present at their highest levels in the liver (p < 0.05); and (3) adults have significantly higher IGF mRNA levels than juveniles in the liver (p = 0.0047).
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PMID:Age-related and tissue-specific levels of five forms of insulin-like growth factor mRNA in a teleost. 819 70

High-resolution microscopy in conjunction with colloidal gold-labeled insulin-like growth factor I (IGF-I) has been used to provide evidence that the IGF-I receptor is first detected in 8-cell-stage mouse embryos, confirming the results of previous reverse transcriptase polymerase chain reaction (RT-PCR) studies. Specificity for the IGF-I receptor was demonstrated by displacement with unlabeled IGF-I and dual-labeling experiments with colloidal gold-labeled or unlabeled insulin. Labeled IGF-I ligand is internalized by means of receptor-mediated endocytosis following its concentration in coated pits, and it can be visualized within cytoplasmic organelles. Immunocytochemical analyses at the blastocyst stage, using gold-labeled antibodies to the receptor, confirmed the expression of IGF-I receptors on all cells of the embryo. Similar studies with antibodies directed against the ligand demonstrated that IGF-I internalized by the embryo in vivo is maternally derived. Approximately 40% of blastocysts showed apical plasma membrane binding of gold-labeled ligand ("responders"), while approximately 60% did not demonstrate binding ("nonresponders"); however, both classes of embryo expressed receptors on basolateral membranes of trophectoderm cells and on the surface of inner masses. Functional studies show that incubating embryos in physiological levels of IGF-I (40 ng/ml) results in increased numbers of cells in the inner cell mass (p < 0.05), but not the trophectoderm, as compared to controls.
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PMID:Mouse preimplantation embryos exhibit receptor-mediated binding and transcytosis of maternal insulin-like growth factor I. 835 75

Osteocytes have been proposed to be the cells primarily responsible for sensing the effects of mechanical loading in bone. Osteocytes respond to loading in vivo, and have been shown to express osteotropic agents and their receptors, and cell/matrix adhesion molecules in vitro, but the functional significance of such findings is not clear. One obstacle to increased understanding of the role of osteocytes in the regulation of bone mass is that the cells are not easily accessible for study. In situ studies are difficult, and although it is possible to extract and culture osteocytes from neonatal bones, the responses of such cells might be very different from those in older bones in situ. We have developed a technique to investigate osteocyte gene expression in vivo, using the reverse transcriptase linked polymerase chain reaction (PCR), and have shown that they express mRNA for beta-actin (beta-ACT), osteocalcin (OC), connexin-43 (Cx43), insulin-like growth factor I (IGF-I), c-fos and c-jun, but not tumor necrosis factor alpha (TNF-alpha) or tartrate-resistant acid phosphatase (TRAP). The principle behind the method is that after removal of the periosteum, tangential cryostat sections of a tubular bone contain RNA only from osteocytes and a very small number of endothelial cells as long as the marrow cavity is not broached. Using this method, we have investigated gene expression in cells from rat ulnar cortical bone under forming and resorbing bone surfaces. In addition, we have investigated the effect on gene expression of mechanical loading which, if repeated daily, initiates new bone formation on quiescent or resorbing surfaces. Although the expression of the genes we have studied in osteocytes is different from those expressed by the periosteal surfaces overlying the cortex, we have not detected loading-related changes in osteocyte gene expression in any cortical bones. This may be because of the extreme sensitivity of the PCR technique which can only resolve large differences in expression. The use of quantitative methods in the future may allow demonstration of regulated gene expression in osteocytes.
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PMID:Constitutive in vivo mRNA expression by osteocytes of beta-actin, osteocalcin, connexin-43, IGF-I, c-fos and c-jun, but not TNF-alpha nor tartrate-resistant acid phosphatase. 885 45

A cDNA for equine insulin-like growth factor I (IGF I) has been isolated by reverse transcriptase-polymerase chain reaction and subsequently sequenced. The sequenced fragment contained 465 bp including the coding regions for the signal peptide, the entire mature protein, and 4 amino acids into the E-peptide. Like its human counterpart, the mature equine IGF I peptide contains 70 amino acids and was 100% homologous between horse and man. The 49-amino-acid signal peptide had the threonine in position 26 of the human signal peptide substituted by isoleucine. The nucleotide homology across the entire clone was 96.3% between horse and man and 91.6% between horse and rat. The isolated cDNA hybridized to the same transcripts in fetal and adult tissues.
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PMID:Cloning and sequencing of an equine insulin-like growth factor I cDNA and its expression in fetal and adult tissues. 886 Mar 3

A number of cytokines have previously been localised within the developing and adult hair follicle, however, the role they play in producing a mature hair follicle remains unknown. In an attempt to identify dermal papilla specific cytokines and thus those that may have an important controlling role, cytokine gene expression profiles, obtained by reverse transcriptase-polymerase chain reaction (RT-PCR), were compared between whole anagen rat hair follicles, passage 2 dermal papillae (a cell type with hair inductive capacity), and footpad fibroblasts (a non-hair inducing cell type). Based on this qualitative data, we were unable to identify a dermal papilla specific gene. The analysis of the pattern and timing of cytokine gene expression during the hair cycle is likely to be more informative. A semi-quantitative RT-PCR technique was therefore developed for studying trends in the level of in vivo expression of the following cytokines and their receptors from early anagen to early catagen in the rat hair growth cycle: insulin-like growth factor I, transforming growth factor beta 1, tumour necrosis factor, and basic fibroblast growth factor. These genes were found to be differentially expressed and this was correlated with their possible functions in controlling the hair growth cycle, providing valuable insights into the role of cytokines in regulating the hair growth process.
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PMID:In vivo cytokine and receptor gene expression during the rat hair growth cycle. Analysis by semi-quantitative RT-PCR. 888 67

In mammals, one of the major actions of insulin-like growth factor I (IGF-I) is to increase skeletal growth by stimulating new cartilage formation. IGF-I stimulates chondrocytes in vitro to synthesize new cartilage matrix, measured by enhanced uptake of 35S-sulfate, but the addition of insulin does not produce a similar effect except when added at high concentrations. However, recent studies have shown that, in teleosts, both insulin and IGF-I are potent activators of 35S-sulfate uptake in gill cartilage. To further characterize the growth-promoting activities of these hormones in fish, we have used reverse transcriptase-linked PCR to analyze the expression of insulin receptor family genes in salmon gill cartilage. Partial cDNA sequences encoding the tyrosine kinase domains from six distinct members of the IR gene family were obtained, and sequence comparisons revealed that four of the cDNAs encoded amino acid sequences that were highly homologous to human IR whereas the encoded sequences from two of the cDNAs were more similar to the human type I IGF receptor (IGF-R). Furthermore, a comparative reverse transcriptase-linked PCR assay revealed that the four putative IR mRNAs expressed in toto in gill cartilage were 56% of that found in liver whereas the expressed amount of the two IGF-R mRNAs was 9-fold higher compared with liver. These results suggest that the chondrogenic actions of insulin and IGF-I in fish are mediated by the ligands binding to their cognate receptors. However, further studies will be required to characterize the binding properties and relative contribution of the individual IR and IGF-R genes.
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PMID:Expression of multiple insulin and insulin-like growth factor receptor genes in salmon gill cartilage. 935 69

Growth hormone (GH) is produced in progestin-induced hyperplastic ductular mammary epithelia in dogs. Progestins also induce the development of cystic endometrial hyperplasia (CEH) in this species. The study reported here investigated whether GH gene expression could also be demonstrated in progestin-induced hyperplastic epithelium in the canine uterus. Eight beagle bitches were treated with 10 mg medroxyprogesterone acetate (MPA) kg-1 body mass s.c. at intervals of 3 weeks, for a total of five times in four dogs (group I) and for a total of 13 times in the other four dogs (group II). Blood samples were collected twice during each 3 week period for measurement of plasma concentrations of GH, insulin-like growth factor I (IGF-I) and IGF-II. At the end of the series of injections uterine tissue was obtained by ovario-hysterectomy. Histological examination confirmed that CEH was present in all uteri after MPA treatment; the changes in the dogs of group I were less marked than those in group II. Immunohistochemical examination of the uterine tissues showed that immunoreactive(i) GH was present in a number of uteri with CEH. iGH was usually located in the cytoplasm of glandular epithelial cells. However, reverse transcriptase PCR using GH-specific primers failed to demonstrate mRNA encoding GH in the uterine tissue of all dogs. It is concluded that local production of GH is not involved in progestin-induced hyperplasia of uterine epithelial cells in dogs.
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PMID:Lack of association of progestin-induced cystic endometrial hyperplasia with GH gene expression in the canine uterus. 940 6


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