Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.48 (transcriptase)
 9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

cDNA of pea seed-borne mosaic potyvirus (PSbMV) RNA was synthesized and cloned in E. coli. Four overlapping clones that cover the complete PSbMV genome, except the extreme 5' terminus, were identified by restriction enzyme mapping, hybridization analysis, and partial sequencing. Overlapping cDNA clones covering 1386 nucleotides of the 3' terminus were sequenced. The nucleotide sequence contains one open reading frame (ORF), followed by an untranslated region of 163 nucleotides and a poly(A)-tract. The deduced amino acid sequence was found to include the C-terminus of the predicted RNA-dependent RNA polymerase and the coat protein. A glutamine-alanine dipeptide was identified as a putative 49-kD proteinase cleavage site at the N-terminus of the coat protein.
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PMID:Nucleotide sequence of pea seed-borne mosaic potyvirus coat protein gene. 179 54

1. Aminoacyl-transfer-RNA synthetase activity in extracts prepared from tobacco leaf was increased 3-5-fold when sodium thioglycollate (30mm) and magnesium chloride (16mm) were included in the extraction medium. Omitting sucrose (0.45m) from the extraction medium did not alter the activity. 2. Activity was a linear function of enzyme concentration up to 1 disk (30mg. fresh wt.)/ml. and was not affected by dialysis at any concentration. 3. Activity increased about 13-fold above control values when a mixture of 21 amino acids and amides (1mm) was added to the reaction mixture. 4. Under the conditions used in the standard assay for aminoacyl-transfer-RNA synthetase activity K(m) (ATP) was 0.65mm and K(m) (l-amino acids) was 70mum. 5. Activity above the control value was found with all amino acids and amides tested except alanine, arginine, glutamic acid, glutamine and hydroxyproline. Activity was highest with leucine, isoleucine, valine, cysteine and histidine. Total activity with a mixture of 21 amino acids and amides was 20% lower than the total activity of the enzymes assayed separately.
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PMID:The extraction and assay of aminoacyl-transfer-ribonucleic acid synthetases of tobacco leaf. 422 1

The poliovirus RNA-dependent RNA polymerase, 3Dpol, is known to share a region of sequence homology with all RNA polymerases centered at the GDD amino acid motif. The two aspartic acids have been postulated to be involved in the catalytic activity and metal ion coordination of the enzyme. To test this hypothesis, we have utilized oligonucleotide site-directed mutagenesis to generate defined mutations in the aspartic acids of the GDD motif of the 3Dpol gene. The codon for the first aspartate (3D-D-328 [D refers to the single amino acid change, and the number refers to its position in the polymerase]) was changed to that for glutamic acid, histidine, asparagine, or glutamine; the codons for both aspartic acids were simultaneously changed to those for glutamic acids; and the codon for the second aspartic acid (3D-D-329) was changed to that for glutamic acid or asparagine. The mutant enzymes were expressed in Escherichia coli, and the in vitro poly(U) polymerase activity was characterized. All of the mutant 3Dpol enzymes were enzymatically inactive in vitro when tested over a range of Mg2+ concentrations. However, when Mn2+ was substituted for Mg2+ in the in vitro assays, the mutant that substituted the second aspartic acid for asparagine (3D-N-329) was active. To further substantiate this finding, a series of different transition metal ions were substituted for Mg2+ in the poly(U) polymerase assay. The wild-type enzyme was active with all metals except Ca2+, while the 3D-N-329 mutant was active only when FeC6H7O5 was used in the reaction. To determine the effects of the mutations on poliovirus replication, the mutant 3Dpol genes were subcloned into an infectious cDNA of poliovirus. The cDNAs containing the mutant 3Dpol genes did not produce infectious virus when transfected into tissue culture cells under standard conditions. Because of the activity of the 3D-N-329 mutant in the presence of Fe2+ and Mn2+, transfections were also performed in the presence of the different metal ions. Surprisingly, the transfection of the cDNA containing the 3D-N-329 mutation resulted in the production of virus at a low frequency in the presence of FeSO4 or CoCl2. The virus derived from transfection in the presence of FeSO4 grew slowly, while the viruses recovered from transfection in CoCl2 grew at a rate which was similar to that of the wild-type poliovirus.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Mutation of the aspartic acid residues of the GDD sequence motif of poliovirus RNA-dependent RNA polymerase results in enzymes with altered metal ion requirements for activity. 785 86

To identify a gene responsible for multiple endocrine neoplasia type 1 (MEN1), we attempted to isolate potentially transcribable fragments from cosmid clones derived from a region on chromosome 11q13 where genetic linkage studies and analyses of loss of heterozygosity in MEN1-associated tumors have localized the MEN1 gene. By an exon-amplification method, we recovered three exon-like sequences from one of these clones, cCI11-367, and using these sequences as probes we were able to isolate new clones from cerebrum, cerebellum, and fetal-liver cDNA libraries. Sequence analysis of these cDNA clones revealed that the transcribed gene, designated ZFM1, encodes a novel 623-amino-acid protein containing domains with interesting structural properties including a nuclear transport domain, a metal binding motif, and glutamine- and proline-rich regions. Analysis by the reverse-transcriptase polymerase chain reaction (RT-PCR) indicated that this gene is expressed in various tissues including endocrine organs such as thyroid gland, pancreas, adrenal gland, and ovary. These data suggest that ZFM1 might be a candidate for mutations that cause MEN1.
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PMID:Isolation and characterization of a novel gene encoding nuclear protein at a locus (D11S636) tightly linked to multiple endocrine neoplasia type 1 (MEN1). 791 30

cDNA of tulip breaking virus-tulip (TBV-tulip) RNA was synthesized and cloned in E. coli. One clone that contains a 4.5 kb insert was identified by restriction enzyme analysis, dot immunobinding assay (DIBA), and partial sequencing. Then 1479 nucleotides of the 3'-terminus of the clone were sequenced and revealed that the sequence contains one open reading frame (ORF), followed by an untranslated region of 255 nucleotides and a poly(A) tract. The deduced amino acid sequence was found to include the C terminus of the predicted RNA-dependent RNA polymerase and the coat protein. A glutamine-alanine dipeptide was identified as a putative NIa protease cleavage site at the N terminus of the coat protein.
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PMID:Nucleotide sequence of the coat protein coding region of tulip breaking virus. 807 37

Hepatitis A virus (HAV) cDNAs encoding the P3 region proteins were expressed in vivo and in vitro to characterize the HAV 3D protein and to identify the cleavage site between 3C and 3D. Protein coding sequences were placed under control of a T7 promoter and an EMCV translational initiation signal. T7 RNA polymerase was provided by simultaneous infection of transfected BS-C-1 cells with a recombinant vaccinia virus vTF7-3 (T. R. Fuerst et al., Proc. Natl. Acad. Sci. USA 83, 8122-8126, 1986). Efficient synthesis and processing of P3 proteins occurred to yield 3CD (78 kDa), 3D (54 kDa), 3ABC (33 kDa), 3BC (25 kDa), and 3C (23 kDa). Similar products were produced by translation of T7 transcripts in a rabbit reticulocyte lysate in vitro. The 3C/D cleavage site was mapped by comparing the mobility of 3D in SDS-PAGE with 3D proteins engineered to begin at each of the two proposed cleavage sites; in addition, direct N-terminal sequencing of radiolabeled 3D protein from translation in vitro was performed. The results showed that 3D was formed by cleavage at the glutamine-arginine (Q/R) pair at position 1738 and 1739 of the HAV polyprotein. HAV 3D protein produced by autocatalytic cleavage of P3 precursor proteins in BS-C-1 cells is virtually completely insoluble and sediments after low-speed centrifugation. This is in contrast to the poliovirus 3D protein, produced from a similar construct, a significant portion of which remains soluble. Extracts containing the poliovirus 3D protein manifested high levels of RNA-dependent RNA polymerase activity, whereas those containing the HAV 3D protein showed no detectable activity by the same assay.
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PMID:Expression of hepatitis A virus precursor protein P3 in vivo and in vitro: polyprotein processing of the 3CD cleavage site. 829 Dec 34

In this study, we isolated two cDNA molecules encoding putative glutamate receptor subunits, fGluR1 alpha and fGluR1 beta, from an Oreochromis sp. brain cDNA library by hybridizing with the glutamate receptor cDNA, fGluR2 beta, of the same fish. The deduced amino acid sequence of the fGluR1 alpha consists of 908 residues with an 18-residue signal peptide and displays a sequence identity of 74% to the amino acid sequence of rat GluR1 subunit. Northern blotting indicates that the expression level of fGluR1 alpha in telencephalon is higher than that in optic tectum and cerebellum in adult fish brain. Reverse-transcriptase polymerase chain reaction and genomic analyses reveal the presence of variants created by alternative splicing at the flip-flop module and the carboxyl terminus of fGluR1 alpha transcripts. The amino acid sequence of fGluR1 alpha is unique in that it contains a glutamine-rich sequence inserted at the loop 1 (L1) between transmembrane domains 1 and 2. A second incomplete cDNA clone, designated fGluR1 beta, coding for a polypeptide showing sequence identity to the rat GluR1 and fGluR1 alpha was isolated from the same library. Insertion of a serine- and glutamine-rich sequence at the L1 was also detected in the translated sequence of fGluR1 beta.
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PMID:Molecular analysis of cDNA molecules encoding glutamate receptor subunits, fGluR1 alpha and fGluR1 beta, of Oreochromis sp. 883 30

Tomato ringspot nepovirus (TomRSV) RNA-1 encodes a putative NTP-binding protein (NTB), a putative viral genome-linked protein (VPg), a putative RNA-dependent RNA polymerase (Pol) and a serine-like protease (Pro), which have been suggested to be involved in viral RNA replication. Proteolytic processing of protease precursors containing these proteins was studied in Escherichia coli and in vitro. The TomRSV protease could cleave the precursor proteins and release the predicted mature proteins or intermediate precursors. Although processing was detected at all three predicted cleavage sites (NTB-VPg, VPg-Pro and Pro-Pol), processing at the VPg-Pro cleavage site was inefficient, resulting in accumulation of the VPg-Pro intermediate precursor in E. coli and in vitro. In addition, the presence of the VPg sequence in the precursor resulted in increased cleavage at the Pro-Pol cleavage site in E. coli and in vitro. Direct N-terminal sequencing of the genomic RNA-linked VPg, of the mature protease purified from E. coli extracts and of radiolabelled mature polymerase purified from in vitro translation products revealed the sequences of the NTB-VPg, VPg-Pro and Pro-Pol dipeptide cleavage sites to be Q/S, Q/G and Q/S, respectively. In vitro processing at the NTB-VPg and Pro-Pol cleavage sites was not detected upon mutation or deletion of the conserved glutamine at the -1 position of the cleavage site. These results are discussed in light of the cleavage site specificity of the TomRSV protease.
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PMID:Proteolytic processing of tomato ringspot nepovirus 3C-like protease precursors: definition of the domains for the VPg, protease and putative RNA-dependent RNA polymerase. 1009 22

We had previously demonstrated that a cellular protein specifically interacts with the 3' end of poliovirus negative-strand RNA. We now report the identity of this protein as heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Formation of an RNP complex with poliovirus RNA was severely impaired by substitution of a lysine, highly conserved among vertebrates, with glutamine in the RNA recognition motif (RRM) of recombinant hnRNP C1, suggesting that the binding is mediated by the RRM in the protein. We have also shown that in a glutathione S-transferase (GST) pull-down assay, GST/hnRNP C1 binds to poliovirus polypeptide 3CD, a precursor to the viral RNA-dependent RNA polymerase, 3D(pol), as well as to P2 and P3, precursors to the nonstructural proteins. Truncation of the auxiliary domain in hnRNP C1 (C1DeltaC) diminished these protein-protein interactions. When GST/hnRNP C1DeltaC was added to in vitro replication reactions, a significant reduction in RNA synthesis was observed in contrast to reactions supplemented with wild-type fusion protein. Indirect functional depletion of hnRNP C from in vitro replication reactions, using poliovirus negative-strand cloverleaf RNA, led to a decrease in RNA synthesis. The addition of GST/hnRNP C1 to the reactions rescued RNA synthesis to near mock-depleted levels. Furthermore, we demonstrated that poliovirus positive-strand and negative-strand RNA present in cytoplasmic extracts prepared from infected HeLa cells coimmunoprecipitated with hnRNP C1/C2. Our findings suggest that hnRNP C1 has a role in positive-strand RNA synthesis in poliovirus-infected cells, possibly at the level of initiation.
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PMID:Functional interaction of heterogeneous nuclear ribonucleoprotein C with poliovirus RNA synthesis initiation complexes. 1573 Dec 20

The peptide transporter PEPT2 mediates transmembrane uptake of small peptides. So far, its expression has not been evidenced in the gastrointestinal tract. We have investigated peptide transport activity in the neuromuscular layers of the gastrointestinal tract by using the fluorescent tracer-dipeptide beta-Ala-Lys-Nepsilon-7-amino-4-methyl-coumarin-3-acetic acid (Ala-Lys-AMCA). Whole-mount preparations from mouse, rat, and guinea pig stomach and small and large intestine were incubated with Ala-Lys-AMCA in the presence or absence of the uptake-inhibitors L-histidine, D-phenylalanyl-L-alanine (D-Phe-Ala), glycyl-L-sarcosine (Gly-Sar), glycyl-L-glutamine (Gly-Gln), benzylpenicillin, and cefadroxil. Fluorescence microscopy revealed that Ala-Lys-AMCA specifically accumulated in both ganglionic layers of the enteric nervous system (ENS) in all regions and species studied. This could be inhibited by Gly-Sar, D-Phe-Ala, Gly-Gln, and cefadroxil, but not by free histidine and benzylpenicillin, indicating uptake via PEPT2. Accordingly, dipeptide uptake was completely abolished in PEPT2-deficient mice. Reverse transcriptase-polymerase chain reaction analysis detected a PEPT2-specific transcript in extracts from the ganglionic ENS layers of mouse small and large intestine, further proving that enteric dipeptide transport activity is specifically mediated via PEPT2. The cellular site of dipeptide uptake was immunohistochemically localized to enteric glial cells and tissue-resident macrophages. In addition, dipeptide uptake occurred in a neurochemically defined subset of neurons in the guinea pig ENS. Our results constitute the first functional evidence for dipeptide transport activity in the ENS. PEPT2-mediated dipeptide transport in enteric glia could contribute to the clearance of neuropeptides in the ENS. In addition, the fluorophore-coupled dipeptide uptake via PEPT2 is a novel vital marker for glial cells in the ENS.
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PMID:Functional expression of the peptide transporter PEPT2 in the mammalian enteric nervous system. 1604 13


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