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)

A specific 'nested' reverse transcriptase/polymerase chain reaction (RT/PCR) procedure was used to characterize the expression patterns of PML-RAR-alpha chimeric mRNAs in 32 patients with acute promyelocytic leukemia (APL). The sensitivity of the technique was such that the fusion gene transcript could be detected from as little as 2.5 pg of total leukemic cell RNA against a background of 1 microgram of cellular RNA lacking the PML-RAR-alpha fusion gene transcript(s). In 19 cases the PML-RAR-alpha isoform referred to here as long was identified. A short isoform, which in comparison with the long form lacks three PML exons, was detected in 11 other cases. A third PML-RAR-alpha mRNA isoform, in which the most 3' PML exon present in the long-type isoform was truncated in its sequences lying immediately upstream of RAR-alpha B region, was found and characterized in a single patient. In one APL patient with a variant translocation t(11;17), the PCR product corresponding to PML-RAR-alpha chimeric mRNAs could not be amplified despite the presence of RAR-alpha gene rearrangement. Genomic and PCR analysis showed that the different PML-RAR-alpha isoforms found in APL patients arise as a result of distinct translocation breakpoints. In each case the exons encoding the B-F regions of RAR-alpha are expressed and are spliced downstream from variable PML gene exons. The 'nested' RT/PCR analysis of the PML-RAR-alpha fusion gene proved to be a rapid and sensitive tool for the diagnosis of the APL and for monitoring the residual APL chimeric mRNA expression during complete remission.
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PMID:Occurrence of distinct PML-RAR-alpha fusion gene isoforms in patients with acute promyelocytic leukemia detected by reverse transcriptase/polymerase chain reaction. 137 19

Radiation-induced gene expression was examined in rat astrocyte cultures using differential display of mRNA via reverse transcriptase-polymerase chain reaction. A 0.3-kb cDNA that was consistently observed in irradiated cultures but not in unirradiated cultures was cloned and sequenced. It was found to be identical to Ptk-3, a receptor tyrosine kinase gene identified recently. The protein encoded by Ptk-3 is a member of a novel class of receptor tyrosine kinases whose extracellular domain contains regions of homology with coagulation factors V and VIII and complement component C1. Northern blot analysis revealed that the expression of Ptk-3 was increased in rat astrocytes by 0.5 h after exposure to 10 Gy and remained at the same elevated level for at least 24 h. The maximum increase occurred after 5 Gy. Cloning studies indicated the presence of at least two Ptk-3 mRNA transcripts, which are probably the result of an alternative splicing mechanism. The short isoform lacks a 37-amino acid sequence in the glycine/proline-rich juxtamembrane region. The splicing pattern of the Ptk-3 gene was not altered by radiation. However, the ratios of the longer to the shorter mRNA transcripts differed between adult cortex, neonatal cortex and in vitro astrocyte cultures.
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PMID:Radiation induction of the receptor tyrosine kinase gene Ptk-3 in normal rat astrocytes. 759 35

The insulin receptor possesses tyrosine kinase activity which is thought to mediate the biological effects of insulin upon target cells. pp120 is a liver-specific glycoprotein of apparent molecular size of 120 kDa that is phosphorylated on tyrosine residues by the receptors for insulin, insulin-like growth factor-I, and epidermal growth factor. Previously, we demonstrated that pp120 is identical to a liver-specific ecto-ATPase. In the present study, we have cloned the rat gene encoding pp120/ecto-ATPase. The gene is contained within approximately 15 kilobases of genomic DNA, and consists of nine exons interrupted by eight introns. Using the reverse transcriptase/polymerase chain reaction, we isolated cDNA clones complementary to rat liver mRNA encoding pp120/ecto-ATPase. Sequence analysis indicated the presence of two populations of cDNA's that differ by the presence or absence of a 53-base pair (bp) fragment encoding the juxta-membrane region of the cytoplasmic domain. By cloning the corresponding region of the ecto-ATPase gene, we demonstrated that the 53-bp represents exon 7 of the gene. This 53-bp exon undergoes alternative splicing, thereby giving rise to two mRNA variants. Deletion of this 53-bp cassette exon introduces a frameshift, and results in a premature chain termination codon that truncates the cytoplasmic domain. The truncated cytoplasmic domain contains 10 rather than 71 amino acid residues. Because the short isoform of ecto-ATPase lacks the putative sites for tyrosine- and serine-specific phosphorylation, this alternative splicing may have a major effect upon the physiological function of the enzyme.
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PMID:pp120/ecto-ATPase, an endogenous substrate of the insulin receptor tyrosine kinase, is expressed as two variably spliced isoforms. 838 Apr 6

We used a catecholaminergic neuron-like cell line (CATH.a cells) as a model system to investigate the likelihood that pituitary adenylate cyclase-activating polypeptide (PACAP) may participate in the regulation of specific gene expression in catecholaminergic neurons. Analysis by reverse transcriptase-PCR amplification revealed the presence in these cells of type I PACAP receptors, with a short isoform, together with a heavier so-called Hop splice variant. PACAP38 and PACAP27 enhanced, in a dose-dependent manner, both cyclic AMP formation and phosphoinositide breakdown, with EC50 values of, respectively, 0.6 x 10(-10) and 2 x 10(-9) M. These peptides, in addition, also elevated [Ca2+]i by mobilizing intracellular calcium pools. Vasoactive intestinal peptide (VIP) was approximately 1,000-fold less potent in stimulating cyclic AMP (with EC50 = 2 x 10(-7) M) and failed to change the turnover of phosphoinositides and to alter [Ca2+]i. Both forms of PACAP, as well as forskolin, stimulated transcriptional induction of tyrosine hydroxylase (TH) and c-fos promoters fused to a chloramphenicol acetyltransferase (CAT) reporter gene in transiently transfected cells (p < 0.01 vs. controls). Induction of CAT activity linked to both TH and c-fos promoters was obliterated upon coexpression of a dominant inhibitory mutant (Mt-RAB) of cyclic AMP-dependent protein kinase. We conclude that CATH.a cells do express functional PACAP type I receptors, the activation of which impinges on TH and c-fos transcription according to a process that is primarily dependent on the cyclic AMP-PKA pathway.
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PMID:Pituitary adenylate cyclase-activating polypeptide triggers dual transduction signaling in CATH.a cells and transcriptionally activates tyrosine hydroxylase and c-fos expression. 908 43

Leptin is the protein product of the recently cloned obesity gene. Leptin receptor mRNA is found in a number of central and peripheral locations. The hypothalamus is a presumed site of action. However, little is known about the specific locations of the receptor in peripheral organs. Epinephrine has potent anorectic effects and can cause weight loss by a variety of mechanisms. Excretion of epinephrine is reduced in the ob/ob mouse, which lacks leptin, suggesting an effect by leptin on the adrenal medulla. In the current study, the presence of the leptin receptor was identified on epinephrine-secreting cells in the adrenal medulla. Immunohistochemical studies found dense leptin receptor-like immunoreactivity in the adrenal medulla with no labeling in the adrenal cortex. Double immunofluorescent labeling confirmed that the leptin receptor was present on cells that were phenylethanolamine N-methyltransferase-like immunoreactive and therefore were epinephrine-secreting cells. Leptin receptor mRNA in the adrenal medulla was detected by reverse transcriptase-polymerase chain reaction, with the majority of the mRNA coding for the short isoform (Ob-Ra) of the receptor. Finally, autoradiography was performed using 125I-labeled leptin; specific binding was found in the adrenal medulla, with no specific binding in the adrenal cortex. These results suggest that leptin may have a direct effect on epinephrine-secreting cells in the adrenal medulla. Epinephrine may play a role in mediating the effects of leptin to reduce body weight.
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PMID:Leptin receptors in the adrenal medulla of the rat. 927

Primers specific to the long isoform of leptin receptor (OB-Rb) mRNA were used in reverse transcriptase-linked polymerase chain reactions (RT-PCR) to investigate the expression of this receptor in the hypothalamus and cerebellum of human and rat. For both regions, we observed RT-PCR cDNAs as well as restriction enzyme cleavage fragments of expected sizes. Additionally, in situ hybridization of human cerebellum using two independent [35S]oligonucleotide probes complementary to the OB-Rb mRNA sequence revealed a prominent hybridization signal within the granular layer. Overall, our findings demonstrate the expression of OB-Rb mRNA in the cerebellum and suggest that in such a location, leptin receptors may mediate a function presumably not linked to body weight homeostasis.
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PMID:Expression of leptin receptor mRNA (long form splice variant) in the human cerebellum. 933 26

Leptin and its structural gene, Ob, are exclusively expressed in adipose tissue. Leptin is secreted into the blood and is responsible for fat mass regulation via leptin receptors in the hypothalamus. This has been considered the major role of leptin, but leptin receptor isoforms are expressed not only in the brain but also in most other tissues in humans and rodents: heart, placenta, lung, liver, muscle, kidney, pancreas, spleen, thymus, prostate, testes, ovary, small intestine, and colon. This implicates leptin regulation in other systems apart from fat mass regulation, and leptin action has been demonstrated in human fetal development and reproductive development, liver metabolism, hematopoiesis, and insulin secretion. Four splice variants of the leptin receptor have been identified in humans: the long isoform huOb-R and the shorter isoforms B219.1 to B219.3. It is known that the long isoform has full intracellular signaling capacity, and is responsible for anorectic action in the hypothalamus. The roles of the other isoforms are yet to be elucidated. Here, we report the identification by reverse transcriptase-polymerase chain reaction (RT-PCR) of three leptin receptor isoforms coexpressed in human visceral adipose tissue: the long isoform huOb-R and the short isoforms huB219.1 and huB219.3. The possible roles of these isoforms are discussed.
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PMID:Leptin receptor isoforms expressed in human adipose tissue. 966 33

The reverse transcriptase-polymerase chain reaction (RT-PCR), in combination with 5' and 3' rapid amplification of cDNA ends (RACE), was used to clone a G protein-coupled receptor from turkey brain mRNA. This cDNA clone has an open reading frame of 1,311 base pairs encoding a 436-residue protein with seven transmembrane-spanning domains and exhibits high homology with previously cloned mammalian D2 dopamine receptors. Northern blot analysis of turkey brain mRNA detected an approximate 2.4-kb transcript. RT-PCR and subsequent nucleotide sequence analysis of turkey brain and peripheral tissue mRNA also demonstrated the presence of an alternatively spliced mRNA corresponding to the predicted D2 short isoform. RT-PCR experiments demonstrated a widespread distribution of alternatively spliced D2 dopamine receptor transcripts throughout the turkey brain and in select peripheral tissues as well. In situ hybridization experiments detected strong autoradiographic signals over much of the turkey telencephalon, diencephalon, mesencephalon, cerebellum, pituitary, and pineal gland. Dopamine has several important functions as a neurotransmitter and hormone in mammals and may have similar actions in avian species. The cloning and tissue distribution of the D2 receptor subtype should enable the investigation of any functional role dopamine and dopamine receptors exert on the physiology and behavior of birds.
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PMID:Molecular cloning and tissue distribution of an avian D2 dopamine receptor mRNA from the domestic turkey (Maleagris gallopavo). 1023 44

Leptin, the adipocyte-derived hormone, is secreted into the blood and regulates body weight via its receptors in the hypothalamus. Leptin receptors are also present in many peripheral tissues implicating leptin in the regulation of other body functions, including reproduction, liver and enteric metabolism, hematopoiesis, and immunity. Four splice variants of the leptin receptor have been identified in humans: the long isoform that has full intracellular signaling capacity and 3 shorter isoforms that differ in the length of their cytoplasmic tail. Here, we report the quantification by reverse transcriptase-polymerase chain reaction (RT-PCR) of the relative expression levels of the 2 major leptin receptor splice variants, the long (OB-RL) and the shortest membrane bound variant (OB-RS) in mononuclear cells from peripheral blood of 15 healthy human subjects (9 women and 6 men), with a body mass index (BMI) that ranged from 19.7 to 41.6. Both OB-RL and OB-RS were coexpressed in all mRNAs tested. However, the expression of the short form (OB-RS), was on average 8-fold higher than the expression of the long form (OB-RL) (120.8 +/- 12.9 v 14.6 +/- 3.0 relative intensity units, P < .001). The predominance of the short splice variant over the long one was apparent in all samples and ranged from 4- to 27-fold. There was no significant difference in the expression of either isoform between men and women. However, the relative expression of both OB-RS and OB-RL isoforms was significantly lower in the overweight (BMI > 26), compared with the lean subjects (BMI < 25) (78.8 +/- 9.1 and 6.2 +/- 1.1 v148.8 +/- 14.4 and 18.9 +/- 4.0 relative intensity units, respectively, P < .03) and was inversely correlated with the BMI and plasma leptin levels (P < .01). In conclusion, the expression of OB-RS and OB-RL leptin receptor isoforms appears to be reduced in human peripheral blood mononuclear cells from obese individuals, with OB-RS remaining the predominant leptin receptor isoform. This might have implications for the bioavailability and/or action of circulating leptin not only on these cells, but also on other target tissues.
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PMID:Expression of the long and short leptin receptor isoforms in peripheral blood mononuclear cells: implications for leptin's actions. 1114 13

The human insulin receptor (IR) exists in two isoforms (IR-A and IR-B). IR-A is a short isoform, generated by the skipping of exon 11, a small exon encoding for 12 amino acid residues at the carboxyl terminus of the IR alpha-subunit. Recently, we found that IR-A is the predominant isoform in fetal tissues and malignant cells and binds with a high affinity not only insulin but also insulin-like growth factor-II (IGF-II). To investigate whether the activation of IR-A by the two ligands differentially activate post-receptor molecular mechanisms, we studied gene expression in response to IR-A activation by either insulin or IGF-II, using microarray technology. To avoid the interfering effect of the IGF-IR, IGF-II binding to the IR-A was studied in IGF-IR-deficient murine fibroblasts (R- cells) transfected with the human IR-A cDNA (R-/IR-A cells). Gene expression was studied at 0.5, 3, and 8 h. We found that 214 transcripts were similarly regulated by insulin and IGF-II, whereas 45 genes were differentially transcribed. Eighteen of these differentially regulated genes were responsive to only one of the two ligands (12 to insulin and 6 to IGF-II). Twenty-seven transcripts were regulated by both insulin and IGF-II, but a significant difference between the two ligands was present at least in one time point. Interestingly, IGF-II was a more potent and/or persistent regulator than insulin for these genes. Results were validated by measuring the expression of 12 genes by quantitative real-time reverse transcriptase-PCR. In conclusion, we show that insulin and IGF-II, acting via the same receptor, may differentially affect gene expression in cells. These studies provide a molecular basis for understanding some of the biological differences between the two ligands and may help to clarify the biological role of IR-A in embryonic/fetal growth and the selective biological advantage that malignant cells producing IGF-II may acquire via IR-A overexpression.
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PMID:Differential gene expression induced by insulin and insulin-like growth factor-II through the insulin receptor isoform A. 1288 24


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