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)

Myelin basic proteins (MBPs) are a family of alternatively spliced isoforms present in myelin sheaths of most vertebrates. A reverse transcriptase-polymerase chain reaction (RT-PCR) approach was used to clone MBP isoforms in species representing two superorders of elasmobranchs: Squalus acanthias, representing Squalomorph sharks, and Raja erinacia, representing Batoidea rays. Two products were generated from each species. The larger product encoded a 155 amino acid protein, the same size as MBPs from two Galeomorph sharks, Heterodontus francisci and Carcharhinus obscurus, which, based upon alignment with other vertebrate MBPs, contained six of the seven MBP exons; only exon II was absent. The smaller product encoded a 141 amino acid protein that lacked exon II and exon V. There were 26 and 30 nucleotide differences between Squalus and Heterodontus, and Raja and Heterodontus, respectively. Sequences from Squalus and Raja were far more similar, having only five nucleotide differences. Both isoforms of elasmobranch MBP contain 18.5% basic (lysine plus arginine) amino acids, compared with 17.5% in mammalian MBPs comprised of the corresponding exons. Northern blot analysis of whole brain total RNA revealed a single band of 2.5 kb in Squalus, and three bands of 1.2, 1.4, and 2.3 kb in Raja. The finding that MBPs of a Squalomorph shark and a Batoidea ray are closer to one another than either is to the Galeomorph sharks suggests that MBP sequence information may prove useful in classifying modern day Chondrichthytes.
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PMID:Molecular cloning of the myelin basic proteins in the shark, Squalus acanthias, and the ray, Raja erinacia. 769 75

Molecular genetic studies have revealed that the human hepatitis B viral (HBV) Pol protein, a polypeptide of about 94 kDa, contains four domains. These are the 5'-terminal protein, spacer, RNA reverse transcriptase/DNA polymerase, and RNase H, respectively, from the amino (N) to carboxy (C) terminus. No evidence indicates as yet the involvement of a specific protease in cleaving the Pol protein or the presence of protease-cutting sites in the Pol protein. An in vitro-translated Pol protein was shown to be cleaved by purified thrombin but not in the presence of its inhibitor, hirudin. Two thrombin-cutting sites, spanning 194 amino acids, were then deduced by thrombin digestion of Pol protein with various lengths of C-terminal deletion. These two putative cutting sites, one located in the spacer region and the other in the beginning of the polymerase region, were found to be conserved at similar positions in the Pol protein of all hepadnaviruses. By using a novel method called the LacZ localization assay (LLA), it was demonstrated that a tripartite fusion protein containing the nucleus localization sequence (NLS) of SV40 large T Ag, the putative thrombin cutting sequence (Ile-Arg-Ile-Pro-Arg320-Thr) of HBV Pol protein and the full length beta-galactosidase of E. coli, exhibited a lower percentage (approximately 53%) of targeting into the nucleus of transfected hepatoma cells when compared with a similar tripartite protein containing a single mutation (Arg320 residue into Trp320) of HBV Pol protein (approximately 78%) or with a bipartite protein of SV40 NLS-beta-galactosidase (approximately 90%). These results indicate that the putative thrombin-cutting site in the spacer region of HBV Pol protein could be cleaved by a cellular protease resulting in the separation of NLS sequence from the beta-galactosidase and rendering a lower frequency of X-gal staining in the nucleus.
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PMID:Demonstration of the presence of protease-cutting site in the spacer of hepatitis B viral Pol protein. 773 Apr 38

Complementary DNA fragments encoding the prepro-salmon gonadotropin-releasing hormone ([Trp7, Leu8]GnRH, sGnRH) of the red seabream Pagrus major were amplified from mRNA of the olfactory bulbs using a reverse transcriptase-polymerase chain reaction (RT-PCR), and the full-length cDNA was cloned from a cDNA library using the PCR-amplified cDNA as a probe. The cDNA consisted of 442 bp, including an open reading frame of 270 bp which encoded the prepro-sGnRH (90 amino acid residues). The prepro-sGnRH had the same architecture as that reported in other species. It was composed of a signal peptide, sGnRH and a GnRH-associated peptide (GAP), which was connected to sGnRH by a Gly-Lys-Arg sequence. The prepro-sGnRH of the red seabream had 90% amino acid identity to the prepro-sGnRH from an African cichlid Haplochromis burtoni which belongs to the same suborder as the red seabream; however, identity was lower to the prepro-sGnRH from Atlantic salmon Salmo salar (74%) and masu salmon Oncorhynchus masou (70%). The GnRH peptide itself and the Gly-Lys-Arg sequence in the prepro-GnRH are highly conserved among vertebrates. The red seabream GAP also shows significant amino acid identity to the GAPs of the African cichlid (89%), Atlantic salmon (74%), and masu salmon (67%), but exhibits no significant identity to chicken or mammalian GAP.
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PMID:Molecular cloning of a cDNA encoding the prepro-salmon gonadotropin-releasing hormone of the red seabream. 785 23

The Variable Coding Sequence (VCS) multigene family of Rattus norvegicus, is composed of at least 10 members, and shows extensive evolutionary divergence in the protein-coding region. Three members of the VCSA subclass, have been characterized: one of them, the VCSA1 gene mainly expressed in the submandibular gland (SMG) encodes the prohormone-like protein, SMR1-VA1. As VCSA-related genes have not been detected in Mus musculus, the VCSA genes subclass is presumed to have recently emerged. To study the evolution of this subclass, we have looked for VCSA genes in a closely related species, Rattus rattus. By Northern analysis, we demonstrate that VCS-related mRNAs are present in the SMG, and that the level of VCSA mRNA accumulation is approximately equal in both sexes. By contrast, in R. norvegicus, males accumulate about 3,000 times more VCSA1 mRNA than females. Using total SMG mRNA, an almost full-length cDNA, homologous to the cDNA of the R. norvegicus VCSA1 gene, was cloned by reverse transcriptase polymerase chain reaction (RT-PCR). The putative corresponding SMR1-VA1 protein is 146 amino acids long and presents the features characteristic of a secreted protein, with a potential signal peptide of 22 amino acids in the amino-terminal portion. The presence of potential processing multibasic sites suggests that small peptides could be generated (particularly a hexapeptide: Arg-Gln-His-Asn-Leu-Arg), as in the case of the SMR1-VA1 protein of R. norvegicus. From Southern blot analysis there appears that species-species modifications of VCSA gene copy number have occurred; R. rattus contains a greater VCSA1 copy number than R. norvegicus (two or three and one, respectively).
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PMID:Recent evolution of genes encoding the prohormone-like protein SMR1 in the rat submandibular gland. 786 31

The mechanism for myometrial quiescence during pregnancy is unknown. cGMP plays an integral role in the relaxation of smooth muscle, and nitric oxide (NO) is the most important endogenous activator of soluble guanylate cyclase. The purpose of this study was to determine the effect of gestational age on myometrial cGMP and NO synthase (NOS) activity in the guinea pig. Myometrial cGMP content (measured by RIA) rose slowly until 0.49 (fraction of pregnancy completed) gestation before abruptly increasing to 200 times the non-pregnant control value. It then declined precipitously after 0.87 gestation. Of the known isoenzymes of NOS, the messenger RNAs coding for both endothelial and neuronal NOS could be amplified from the myometrium of pregnant and nonpregnant animals using reverse transcriptase-polymerase chain reaction, but inducible NOS messenger RNA was not found. Myometrial calcium-dependent NOS activity (measured by the conversion of L-[U-14C]arginine to [U-14C]citrulline) declined slowly with advancing gestation (r2 = 0.096; slope = -0.34; P = 0.01), but never differed significantly from the activity in nonpregnant animals [31.1 +/- 11 (term pregnancy) vs. 56.9 +/- 16 (nonpregnant) pmol/min.g; P = NS]. Calcium-independent activity declined shortly after conception, and then rose toward the nonpregnant level (r2 = 0.19; slope = 0.45; P = 0.0009). However, at no time was it significantly different from that in the nonpregnant animal. Pregnancy had no effect on myometrial L-arginine and L-citrulline content. The administration of L-nitro-arginine methyl ester (200 mg/kg) to inhibit NOS dramatically increased blood pressure and reduced fetal renal NOS activity, but had no effect on the myometrial cGMP content. Estradiol (500 micrograms/kg for 5 days) modestly increased cGMP, but in contrast to many tissues in which estradiol increases NOS, it had no effect on myometrial NOS activity. We conclude that pregnancy dramatically increases cGMP by a mechanism independent of NOS. The stimulus remains to be identified. The temporal change in cGMP concentration is consistent with the hypothesis that cGMP mediates myometrial quiescence during pregnancy.
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PMID:Pregnancy increases guanosine 3',5'-monophosphate in the myometrium independent of nitric oxide synthesis. 798 34

To assess whether RAS oncogenes may affect the expression of cytokines in tumor cells, the presence of interleukins (IL) 1 alpha, 1 beta, 4, 6, 7, and 8, tumor necrosis factor (TNF) alpha and interferon gamma mRNA has been analyzed by reverse transcriptase-polymerase chain reaction in 19 melanoma clones derived from the metastatic cell line 665/2 and previously characterized for RAS mutation and expression. Five of these clones and the parental cell line showed a mutation at codon 61 of N-RAS that resulted in Gln-->Arg substitution (N-RAS/61+), while in the remaining 14, only the wild-type allele for N-RAS was present (N-RAS/61-). With the exception of interferon gamma and IL-4, all the cytokines tested were expressed by the parental 665/2 cell line, whereas IL-1 alpha, IL-6, and TNF-alpha were coordinately transcribed only in the subset of the clones bearing the mutated N-RAS gene. The other cytokine genes studied (IL-1 beta, IL-4, IL-7, and IL-8) displayed a variable degree of expression, and such an heterogeneity was not correlated to the N-RAS phenotype of the clones. The association between N-RAS oncogene and IL-1 alpha, IL-6, and TNF-alpha expression was also found in a 665/2 subline (665/2/5) in which loss of mutated N-RAS genes simultaneously occurred with the loss of IL-1 alpha, IL-6, and TNF-alpha expression. Direct evidence that N-RAS oncogene could influence the pattern of cytokine expression was provided by the coordinate induction of IL-1 alpha, IL-6, and TNF-alpha messenger RNA achieved in N-RAS/61+ transfectants of the N-RAS wild-type melanoma clone 2/21. Furthermore, IL-1 alpha, IL-6, and TNF-alpha could be detected by enzyme-linked immunosorbent assay in the culture medium obtained from N-RAS/61+ melanoma clones as well as from positive transfectants, indicating that lymphokine mRNA expression triggered by the activated N-RAS oncogene lead to a secreted protein. In an N-RAS/61+ melanoma clone, by adding specific antibodies against each cytokine, it was found that soluble IL-1 alpha exerted a positive control on IL-6 mRNA and a negative one on its own expression. In addition, IL-1 alpha and IL-6 were negatively regulated by soluble IL-6 and TNF-alpha.
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PMID:Expression of interleukin 1 alpha, interleukin 6, and tumor necrosis factor alpha genes in human melanoma clones is associated with that of mutated N-RAS oncogene. 806 79

We have analysed by heteroduplex formation (HF), single stranded conformational polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), and nucleotide sequencing the cDNAs of the Ahrb-1 and Ahrd allelic forms of the aromatic hydrocarbon receptor (AhR) present in inbred strains of mice. The Ahrb-1 allele, found in the C57BL and C57BR strains, encodes a 95 kDa receptor with an affinity for ligand 15-20 times higher than the affinity of the 104 kDa receptor encoded by the Ahrd allele, found in the DBA/2 strain. Five overlapping fragments of the AhR coding sequence were obtained from liver RNA by reverse transcriptase synthesis of a cDNA first strand, followed by polymerase chain reaction amplification of these cDNA sequences (RT-PCR). Analysis by HF and SSCP revealed the presence of sequence differences in three of the five fragments. When the complete nucleotide sequence of the coding regions was determined by PCR sequencing, we found a total of ten nucleotide differences between the two alleles, nine of which localized to the three fragments where differences were detected by HF and SSCP. Five of the differences are silent. Of the other five, one changes the opal termination codon in Ahrb-1 to the codon for Arg in Ahrd, extending translation of the mRNA by 43 amino acids and accounting for the larger size of the AhR peptide in DBA/2 mice. One of the four remaining differences causes the replacement of a leucine residue in Ahrb-1 by a proline residue in Ahrd, and breaks a potential alpha-helix near the AhR Q-rich region; it is likely that structural changes associated with this amino acid change are responsible for the differences in agonist affinity observed between the Ah receptors of these two strains of mice.
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PMID:Ten nucleotide differences, five of which cause amino acid changes, are associated with the Ah receptor locus polymorphism of C57BL/6 and DBA/2 mice. 814 72

A variant type of hyperphenylalaninemia is caused by a deficiency of tetrahydrobiopterin (BH4), the obligatory cofactor for phenylalanine hydroxylase. The most frequent form of this cofactor deficiency is due to lack of 6-pyruvoyl-tetrahydropterin synthase (PTPS) activity, the second enzyme in the biosynthetic pathway for BH4. The human liver cDNA for PTPS was previously isolated, and the recombinant protein was found to be active when expressed in Escherichia coli. We now have investigated two patients for their molecular nature of this autosomal recessive disorder. Both patients were diagnosed as PTPS deficient, one with the central and one with the peripheral form, on the basis of an elevated serum phenylalanine concentration concomitant with lowered levels of urinary biopterin and PTPS activity in erythrocytes. Molecular analysis was performed on the patients' cultured primary skin fibroblasts. PTPS activities were found in vitro to be reduced to background activity. Direct cDNA sequence analysis using reverse transcriptase-PCR technology showed for the patient with the central from a homozygous G-to-A transition at codon 25, causing the replacement of an arginine by glutamine (R25Q). Expression of this mutant allele in E. coli revealed 14% activity when compared with the wild-type enzyme. The patient with the peripheral form exhibited compound heterozygosity, having on one allele a C-to-T transition resulting in the substitution of arginine 16 for cysteine (R16C) in the enzyme and having on the second allele a 14-bp deletion (delta 14bp), leading to a frameshift at lysine 120 and a premature stop codon (K120-->Stop). Heterologous expression of the enzyme with the single-amino-acid exchange R16C revealed only 7% enzyme activity, whereas expression of the deletion allele delta 14bp exhibited no detectable activity. All three mutations, R25Q, R16C, and K120-->Stop, affect evolutionarily conserved residues in PTPS, result in reduced enzymatic activity when reconstituted in E. coli, and are thus believed to be the molecular cause for the BH4 deficiency. This is the first report describing mutations in PTPS that lead to BH4 deficiency.
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PMID:Hyperphenylalaninemia due to defects in tetrahydrobiopterin metabolism: molecular characterization of mutations in 6-pyruvoyl-tetrahydropterin synthase. 817 19

Translation of the yeast retrotransposon Ty1 TYA1(gag)-TYB1(pol) gene occurs by a +1 ribosomal frameshifting event at the sequence CUU AGG C. Because overexpression of a low abundance tRNA-Arg(CCU) encoded by the HSX1 gene resulted in a reduction in Ty1 frameshifting, it was suggested that a translational pause at the AGG-Arg codon is required for optimum frameshifting. The present work shows that the absence of tRNA-Arg(CCU) affects Ty1 transposition, translational frameshifting, and accumulation of mature TYB1 proteins. Transposition of genetically tagged Ty1 elements decreases at least 50-fold and translational frameshifting increases 3-17-fold in cells lacking tRNA-Arg(CCU). Accumulation of Ty1-integrase and Ty1-reverse transcriptase/ribonuclease H is defective in an hsx1 mutant. The defect in Ty1 transposition is complemented by the wild-type HSX1 gene or a mutant tRNA-Arg(UCU) gene containing a C for T substitution in the first position of the anticodon. Overexpression of TYA1 stimulates Ty1 transposition 50-fold above wild-type levels when the level of transposition is compared in isogenic hsx1 and HSX1 strains. Thus, the HSX1 gene determines the ratio of the TYA1 to TYA1-TYB1 precursors required for protein processing or stability, and keeps expression of TYB1 a rate-limiting step in the retrotransposition cycle.
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PMID:A rare tRNA-Arg(CCU) that regulates Ty1 element ribosomal frameshifting is essential for Ty1 retrotransposition in Saccharomyces cerevisiae. 824 96

Ten mutations were generated in the env gene of Moloney murine leukaemia virus DNA. The mutations were made by site-directed mutagenesis to alter basic amino acids (lysine or arginine) in the surface glycoprotein gp70. Mutants were investigated following transfection into NIH/3T3 cells. All 10 mutants released virion particles into the medium, suggesting that none of the mutations affected overall viral gene expression or virion budding. Two mutants were positive in XC plaque assay, reverse transcriptase assay and re-infection experiments, showing that these mutations occurred in parts of the molecule not essential for infection. Three mutants were negative in both the XC plaque assay and re-infection experiments, suggesting that they make non-infectious virus particles. The results indicate a defect in the early phase of infection, perhaps in receptor binding or in the fusion of virion and host membranes. The other mutations resulted in reduced infectivity of released virion particles.
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PMID:Mutational analysis of Moloney murine leukaemia virus surface protein gp70. 846 56


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