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 backbone dynamics of Escherichia coli ribonuclease HI (RNase HI) in the picosecond to nanosecond time scale were characterized by a combination of measurements of 15N-NMR relaxation (T1, T2, and NOE), analyzed by a model-free approach, and molecular dynamics (MD) simulation in water. The MD simulations in water were carried out with long-range Coulomb interactions to avoid the artificial fluctuation caused by the cutoff approximation. The model-free analysis of the 15N-NMR relaxation indicated that RNase HI has a rotational correlation time of 10.9 ns at 27 degrees C. The generalized order parameter (S2) for the internal motions varied from 0.15 to 1.0, with an average value of 0.85, which is much larger than that of the RNase H domain of HIV-1 reverse transcriptase (0.78). Large internal motions (small order parameters) were observed in the N-terminal region (Leu2-Lys3), the loop between beta-strands A and B (Cys13-Gly15), the turn between alpha-helix I and beta-strand D (Glu61, His62), the loop between beta-strand D and alpha-helix II (Asp70-Tyr71), the loop between alpha-helices III and IV (Ala93-Lys96), the loop between beta-strand E and alpha-helix V (Gly123-His127), and the C-terminal region (Gln152-Val155). The effective correlation time observed in these regions varied from 0.45 ns (Glu61, Lys96) to 2.2 ns (Leu14). The order parameters calculated from the MD agreed well with those from the NMR experiment, with a few exceptions. The distributions of most of the backbone N-H vectors obtained by MD are approximately consistent with the diffusion-in-a-cone model. These distributions, however, were elliptic, with a long axis perpendicular to the plane defined by the N-H and N-C alpha vectors. Distributions supporting the axial fluctuation model or the jump-between-two-cones model were also observed in the MD simulation.
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PMID:Characterization of the internal motions of Escherichia coli ribonuclease HI by a combination of 15N-NMR relaxation analysis and molecular dynamics simulation: examination of dynamic models. 775 90

To study the differential expression of the murine VLA-4 (alpha 4 beta 1) integrin, the 5'-flanking region of the gene for the alpha subunit (alpha 4m) was isolated and a cDNA for alpha 4m was obtained with reverse transcriptase polymerase chain reaction (RT-PCR). The cDNA sequence contained a difference in the signal peptide region compared to the previously described cDNA (Neuhaus et al., 1991). As a consequence, another start codon is predicted, resulting in a decrease in size of the signal peptide. This was confirmed by genomic sequencing. The promoter region was delimited by ribonuclease protection assay (RPA) and transfection experiments fusing 5'-upstream fragments to the luciferase gene. A fragment extending from -936 to +221 was capable of controlling the expected cell-type-specific expression. Sequence comparison of the mouse alpha 4m promoter region with the human alpha 4h promoter revealed little homology. Like most integrin subunits, alpha 4m lacks TATA anc CCAAT boxes. Putative recognition sites for DNA-binding nuclear factors (AP1, AP2, Sp1, and PU1) were identified. The characterization of the promoter region and further identification of the transcription regulatory elements should provide insight in the regulation of alpha 4m integrin gene expression.
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PMID:Cloning and characterization of the promoter region of the murine alpha-4 integrin subunit. 777 55

Peptide YY (PYY) is a 36-amino-acid peptide known to inhibit pancreatic and gastrointestinal secretion. Immediately following small bowel resection, intestinal PYY mRNA and plasma PYY levels rise. The purpose of this study was to determine whether PYY expression changes in the pancreas during the adaptive period after extensive small bowel resection. Female Sprague-Dawley rats (250 g) underwent 70% small intestinal resection or transection alone as control. Animals were sacrificed at 6 hr, 24 hr, 1 week, or 2 weeks following operation (N = 5/time group). Pancreatic tissue was harvested and RNA was isolated by the guanididium-thiocyanate method. PYY mRNA was analyzed by reverse transcriptase PCR, standardized to glyceraldehyde-3-phosphate dehydrogenase, and semiquantitated by Southern blotting and 32P cpm. Ribonuclease protection assay was used to confirm PCR results. PYY mRNA expression was increased 9 1/2-fold beginning 6 hr after resection compared to transection (P < 0.05). PYY mRNA levels remain elevated, 2 1/4-fold greater than control after 2 weeks (P < 0.05) as analyzed by reverse transcriptase PCR and ribonuclease protection assay. Quantitation by ribonuclease protection assay reveals a gradual elevation of PYY mRNA levels in transected animals compared to a nonoperated rat starting at 1 and 2 weeks. Pancreatic PYY mRNA levels increase rapidly after extensive intestinal resection and remain elevated 2 weeks postoperatively. These results confirm for the first time that the increase in PYY seen after extensive intestinal resection also occurs in extraintestinal sites. In the pancreas, elevated PYY levels may inhibit exocrine secretion, reducing luminal volume, and thereby facilitating intestinal adaptation.
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PMID:Pancreatic peptide YY mRNA levels increase during adaptation after small intestinal resection. 783 Apr 8

GnRH regulates gonadotropin biosynthesis and release in the anterior pituitary via specific receptors. Although extrapituitary expression and action of GnRH have been shown in some species, in the human it is not clear whether GnRH has a peripheral action. In this study we sought to determine whether the human ovary expresses GnRH receptor (GnRHR) messenger ribonucleic acid (mRNA). Ovarian tissues from 11 women (32-61 yr old) and granulosa-lutein (GL) cells purified from follicular aspirates of 51 women undergoing oocyte retrieval for in vitro fertilization were analyzed by ribonuclease protection assay and reverse transcriptase-polymerase chain reaction (RT-PCR). Human pituitaries, lymphocytes, and placenta were also studied. Measurable levels of GnRHR mRNA were found by ribonuclease protection assay in 2 of 10 ovaries, in 2 of 4 GL cells preparations from women whose ovarian hyperstimulation involved a GnRH agonist, in GL cells from 3 women whose ovarian hyperstimulation involved a GnRH antagonist, and in human pituitaries. Relative to the total amount of RNA analyzed, the level of GnRHR mRNA was about 200-fold lower in the ovary than in the pituitary. A sequence of 314 basepairs of GnRHR mRNA was amplified by RT-PCR in the pituitary, in 9 of 10 ovaries, and in 4 of 5 GL cell preparations. No message could be amplified in human lymphocytes, and placental specimens showed a weak signal. The relative GnRHR mRNA levels in GL cells from 13 women analyzed by quantitative RT-PCR showed a wide range of individual differences. These results suggest that GnRHR mRNA is expressed in GL cells and the human ovary across different functional stages, implying that multiple ovarian compartments may express GnRH receptors. The administration of GnRH analogs may have a further direct action on the human ovary.
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PMID:Gonadotropin-releasing hormone receptor gene expression in human ovary and granulosa-lutein cells. 785 1

The conformation of the last 201 nucleotides located at the 3'-end of brome mosaic virus (BMV) RNAs was investigated in solution using different chemical and enzymatic probes. Bases were probed with dimethylsulfate (which methylates N-1 positions of A, N-3 positions of C and N-7 positions of G), a carbodiimide (which modifies N-1 positions of G and N-3 positions of U) and diethylpyrocarbonate (which modifies N-7 positions of A). Ribonucleases T1, U2 and S1 were used to map unpaired nucleotides and ribonuclease V1 to monitor paired bases or stacked nucleotides. Cleavage or modification sites were detected by gel electrophoresis either indirectly by analyzing DNA sequence patterns generated by primer extension with reverse transcriptase of the modified RNAs or by direct identification within the statistical cleavage patterns of the RNA. On the basis of these biochemical results, an atomic model was built by computer modeling and its stereochemistry refined. The deduced secondary structure of the RNA confirms data previously proposed by others but contains additional base-pairs (A27-U32, A28-G31, G41-A134, G64-C68, U80-A99, G81-A98, G88-U91, G100-U126, U104-U125, G162-G166 and A172-A191), one new tertiary long-range interaction (U103-U164) and a small triple helical conformation with (G41-A134)-A18 and (C42-G133)-A17 interactions. The new secondary structure also indicates the existence of a second pseudoknot involving pairing between residues A181 to A184 and residues U197 to U194, outside the domain conferring tyrosylation ability to BMV RNA. The main outcome from the model stems from its intricate folding, which allows a new assignment for the domains mimicking the anticodon- and D-loop regions of tRNA. Interestingly, the stem and loop region found structurally to be analogous to the anticodon arm of tRNA(Tyr) does not contain the tyrosine anticodon involved in the aminoacylation process. The structural analogies with canonical tRNA(Tyr) illustrate the functional mimicry existing between the BMV RNA structure and canonical tRNA(Tyr) that allows for their efficient aminoacylation by tyrosyl-tRNA synthetase. This structural model rationalizes mutagenic and footprinting data that have established the importance of specific regions of the viral RNA for recognition by its replicase, (ATP,CTP):tRNA nucleotidyl-transferase and yeast tyrosyl-tRNA synthetase. The new fold has biological implications that can be used as a predictive tool for elaborating new experiments.
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PMID:Solution structure of the 3'-end of brome mosaic virus genomic RNAs. Conformational mimicry with canonical tRNAs. 828 79

A target RNA/DNA-specific nuclease could be constructed if a specific RNA/DNA binding domain allowing target RNA/DNA recognition was fused to a (deoxy)ribonucleolytic domain allowing target RNA/ DNA cleavage. The design and construction of such a chimeric enzyme could be of value for both basic research involving structure-function relationships and applied research requiring inactivation of harmful RNA/DNA molecules of cellular or pathogenic origin. The feasibility of this designer nuclease approach for inactivating specific RNA/DNA molecules was assessed using human immunodeficiency virus type-1 (HIV-1) RNA as a model. Trans-activator of transcription (Tat) protein is one of the key regulatory proteins encoded by HIV-1. It binds to the trans-activation-responsive (TAR) RNA element located within the 5' non-coding region of HIV-1 RNAs. The TAR RNA binding domain of this protein was fused to the ribonuclease (RNase) H domain of HIV-1 reverse transcriptase (RT). RNase H by itself lacks an RNA binding domain. The chimeric Tat-RNase H protein was shown to specifically recognize and cleave HIV-1 TAR RNA in vitro. Cleavage was abolished by mutations in the Tat binding region within the TAR RNA, indicating that it is specific to HIV-1 TAR RNA.
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PMID:Fusion with an RNA binding domain to confer target RNA specificity to an RNase: design and engineering of Tat-RNase H that specifically recognizes and cleaves HIV-1 RNA in vitro. 865 73

Nitric oxide is reportedly involved in the regulation of several ovarian processes, yet the isoforms of nitric oxide synthase (NOS) expressed in the ovary are unknown. Our purpose was to identify and localize NOS isoenzymes in the rat ovary and to examine++ if mRNA expression of NOS isoenzymes change after gonadotropin stimulation. Using reverse transcriptase-PCR, we demonstrated that inducible (iNOS) and endothelial (eNOS), but not neuronal, NOS mRNAs are expressed in the ovary. In a gonadotropin-stimulated rat model, unstimulated ovaries had the highest levels of iNOS mRNA as quantified by ribonuclease protection assay. After gonadotropin injection, iNOS mRNA declined to undetectable levels in ovaries containing ovulatory follicles before increasing slighty in ovaries containing copora lutea. In situ hybridization studies localized iNOS to granulosa cells of secondary follicles and small antral follicles. Western blots of unstimulated ovaries demonstrated iNOS protein. In contrast to iNOS, eNOS mRNA levels, determined by quantitative PCR, increased after gonadotropin stimulation and peaked in ovaries containing ovulatory follicles before declining in the luteal phase. eNOS protein was localized to blood vessels in the ovary by immunohistochemistry. We conclude that two isoforms of NOS are expressed in the ovary and the mRNA levels for these isozymes are differentially regulated.
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PMID:Expression and localization of inducible and endothelial nitric oxide synthase in the rat ovary. Effects of gonadotropin stimulation in vivo. 867 39

Cyclin and cyclin-dependent kinase(cdk) complexes, and their inhibitors (CKIs) play important roles in growth regulation on the cells. p27/kip1 is a CKI associated with G1 arrest induced by cell to cell contact, transforming growth factor-beta and cyclic AMP. The abnormality of p27/Kip1 genes in human tumors usually appears as a steady level defect of expression, since mutations in them is rare. Thus it is important to estimate the expression level of this gene. To detect the change of p27/Kip1 mRNA level in blood cells, we developed the ribonuclease protection assay using nonradioactive riboprobe which was produced by reverse transcriptase-polymerase chain reaction (RT-PCR) with T7 promoter-added antisense primer and the in vitro transcription system. Our assay may be useful for clinical evaluation of the mRNA level.
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PMID:[Detection of p27/kip1 mRNA in blood cells by nonradioactive ribonuclease protection assay]. 867 70

Using an antibody that recognizes the products of all known members of the fos family of immediate early genes, it was demonstrated that destruction of the nigrostriatal pathway by 6-hydroxydopamine (6-OHDA) lesions of the medial forebrain bundle produces a prolonged (>3 months) elevation of Fos-like immunoreactivity in the striatum. Using retrograde tract tracing techniques, we have previously shown that this increase in Fos-like immunoreactivity is located predominantly in striatal neurons that project to the globus pallidus. In the present study, Western blots were performed on nuclear extracts from the intact and denervated striatum of 6-OHDA-lesioned rats to determine the nature of Fos-immunoreactive protein(s) responsible for this increase. Approximately 6 weeks after the 6-OHDA lesion, expression of two Fos-related antigens with apparent molecular masses of 43 and 45 kDa was enhanced in the denervated striatum. Chronic haloperidol administration also selectively elevated expression of these Fos-related antigens, suggesting that their induction after dopaminergic denervation is mediated by reduced activation of D2-like dopamine receptors. Western blot immunostaining using an antibody which recognizes the N-terminus of FosB indicated that the 43 and 45 kDa Fos-related antigens induced by dopaminergic denervation and chronic haloperidol administration may be related to a truncated form of FosB known as deltaFosB. Consistent with this proposal, retrograde tracing experiments confirmed that deltaFosB-like immunoreactivity in the deafferented striatum was located predominantly in striatopallidal neurons. Gel shift experiments demonstrated that elevated AP-1 binding activity in denervated striata contained FosB-like protein(s), suggesting that enhanced deltaFosB levels may mediate some of the effects of prolonged dopamine depletion on AP-1-regulated genes in striatopallidal neurons. In contrast, chronic administration of the D1-like receptor agonist CY 208243 to 6-OHDA-lesioned rats dramatically enhanced deltaFosB-like immunoreactivity in striatal neurons projecting to the substantia nigra. Western blot immunostaining revealed that deltaFosB and, to a lesser extent, FosB are elevated by chronic D1-like agonist administration. Both the quantitative reverse transcriptase-polymerase chain reaction and the ribonuclease protection assay demonstrated that deltafosB mRNA levels were substantially enhanced in the denervated striatum by chronic D1-like agonist administration. Lastly, we examined the effects of chronic administration ofD1-like and D2-like dopamine receptor agonists on striatal deltaFosB expression in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) primate model of Parkinson's disease. In monkeys rendered Parkinsonian by MPTP, there was a modest increase in deltaFosB-like protein(s), while the development of dyskinesia produced by chronic D1-like agonist administration was accompanied by large increases in DeltaFosB-like protein(s). In contrast, administration of the long-acting D2-like agonist cabergoline, which alleviated Parkinsonian symptoms without producing dyskinesia reduced deltaFosB levels to near normal. Taken together, these results demonstrate that chronic alterations in dopaminergic neurotransmission produce a persistent elevation of deltaFosB-like protein(s) in both the rodent and primate striatum.
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PMID:Chronic alterations in dopaminergic neurotransmission produce a persistent elevation of deltaFosB-like protein(s) in both the rodent and primate striatum. 871 7

Stem cell factor (SCF) is a growth factor known to have profound effects on the proliferation, migration, differentiation, and survival of numerous cell types, including those of the ovary. The objectives of the present study were to identify and characterize expression of this growth factor in the ovine corpus luteum (CL). A 952-bp cDNA was amplified from Day 3 (Day 0 = estrus) ovine luteal total cellular (tc) RNA by reverse transcriptase-polymerase chain reaction and determined to encode SCF. Northern analysis of Day 10 luteal poly(A)+ RNA indicated one major transcript of approximately 6.5 kb. SCF mRNA was localized within Day 3 and Day 10 CL by in situ hybridization and was expressed throughout luteal tissue on both days examined. To asses expression throughout the luteal phase, SCF mRNA was quantified by ribonuclease protection assay in tcRNA collected on Day 3, 7, 10, 13, and 16; values did not differ across days (p > 0.10). Similarly, SCF mRNA was quantified in tcRNA isolated from pools of Day 10 large and small steroidogenic cells (n = 4 and 3, respectively); levels did not differ (p > 0.10) between cell types. In addition, SCF protein was detected in CL on Days 3 and 10, and was expressed in a cell-specific manner in cells with morphological characteristics of large and small luteal cells. These data indicate that SCF may be involved in communication among steroidogenic cells and/or between steroidogenic and nonsteroidogenic cells of the CL.
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PMID:Characterization of ovine stem cell factor messenger ribonucleic acid and protein in the corpus luteum throughout the luteal phase. 872 15


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