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 tenascin-R (TN-R) gene encodes a multidomain extracellular matrix glycoprotein belonging to the tenascin family. It is detectable mainly in oligodendrocytes and neuronal subpopulations of the central nervous system. In this report, we describe the structure of the 5'-region of the mouse TN-R gene and characterise the activity of its promoter. By in silico cloning and genome walking, we have deduced the organisation of the gene and identified the promoter sequence by 5'-RACE technology. TN-R transcripts in adult mouse brain contain non-coding exons 1 and 2 as demonstrated by the reverse transcriptase-polymerase chain reaction. The promoter displays its activity in cultured cells of neural origin, but not in a fibroblast-like cell line or an undifferentiated teratocarcimoma cell line. As for the human and rat genes, the elements required for the full and cell type-specific activity of the promoter are contained in exon 1 and 167 bp upstream of this exon. The mouse TN-R promoter sequence is similar to that of rat and human in that it displays similarly unusual features: it lacks any classical TATA-box or CAAT-box, GC-rich regions or initiator elements. The promoter contains consensus sequences for binding of a variety of transcription factors, notably p53/p73 and glucocorticoid receptors.
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PMID:Structure of the murine tenascin-R gene and functional characterisation of the promoter. 1292 10

Hemoglobin II from the clam Lucina pectinata is an oxygen-reactive protein with a unique structural organization in the heme pocket involving residues Gln65 (E7), Tyr30 (B10), Phe44 (CD1), and Phe69 (E11). We employed the reverse transcriptase-polymerase chain reaction (RT-PCR) and methods to synthesize various cDNA(HbII). An initial 300-bp cDNA clone was amplified from total RNA by RT-PCR using degenerate oligonucleotides. Gene-specific primers derived from the HbII-partial cDNA sequence were used to obtain the 5' and 3' ends of the cDNA by RACE. The length of the HbII cDNA, estimated from overlapping clones, was approximately 2114 bases. Northern blot analysis revealed that the mRNA size of HbII agrees with the estimated size using cDNA data. The coding region of the full-length HbII cDNA codes for 151 amino acids. The calculated molecular weight of HbII, including the heme group and acetylated N-terminal residue, is 17,654.07 Da.
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PMID:The cDNA-derived amino acid sequence of hemoglobin II from Lucina pectinata. 1471 36

Human hepatic stimulator substance (hHSS) is a newly identified growth-promoting factor in liver. HSS is capable of stimulating the hepatic regeneration in hepaectomized rats and promoting the growth of hepatic tumor cells. To understand and elucidate the regulation of hHSS on hepatocyte growth at genetic level, the 4,890 bp of 5'-flanking region of hHSS gene have been isolated and sequenced. On basis of comparison of the 5'-flanking sequences with GenBank, hHSS gene was localized on human chromosome 16. Transcriptional start site was identified by reverse transcriptase-polymerase chain reaction (RT-PCR) and 5' rapid amplification of cDNA end (5'RACE). The initiation nucleotide was defined as 'C', which located at 254 bp upstream of the reported sequence of exon I.
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PMID:[Cloning and structural analysis of 5'-flanking sequence of human hepatic stimulator substance (hHSS) gene]. 1498 37

The expression of cytochrome P450scc, encoded by the CYP11A gene, was investigated in the rat kidney from birth to adulthood. In the male and female rat kidneys, the corresponding mRNA was detected by semi-quantitative reverse transcriptase and polymerase chain reaction (RT-PCR) analysis with specific primers, resulting in higher levels of expression during the first 15 days from birth. RT-PCR and sequence analysis showed that the P450scc mRNA coding region was the same for both kidney and testis, whereas 5'-RACE analysis (rapid amplification of cDNA ends) demonstrated that the renal transcription utilizes a distal transcription start site (TSS) located 76 b upstream of that used in ovarian and testicular P450scc mRNA expression, which is placed 43 b upstream of the first ATG. The 5'-UTR sequence of renal P450scc cDNA exactly matched the contiguous upstream untranslated region of the gene, suggesting that alternative splicing was not involved in the synthesis of this transcript. Northern hybridization detected a specific transcript only in the newborn male, but not in adult rat kidney, confirming the higher levels of expression in the first days of the rat's life. Positive immunodetections of cytochrome P450scc were found in renal cortical distal tubules and the results were confirmed by Western blotting analysis. As demonstrated by semi-quantitative RT-PCR, the male kidney also expresses the messengers corresponding to the steroidogenic acute regulatory (StAR) and steroidogenic factor 1 (SF-1) proteins, which are normally required for steroidogenesis in steroidogenic tissues, such as gonads and adrenal cortex. These studies suggest that the rat kidney has the capability for local steroid hormone production, although the physiological significance of the pregnenolone eventually produced remains to be established.
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PMID:Expression of cytochrome P450scc mRNA and protein in the rat kidney from birth to adulthood. 1502 86

The Mycobacterium tuberculosis genome encodes two ferric uptake regulator homologues, furA and furB, the function of which is under investigation. Using Mycobacterium smegmatis as a model system, we investigated the transcriptional pattern of Rv(Ms)2358-furB genes. Transcripts covering the two genes could be identified by northern blotting and by reverse transcriptase PCR. The transcriptional start point was mapped at one base upstream of the Ms2358 start codon by the RACE technique. By cloning M. smegmatis and M. tuberculosis DNA regions upstream of a reporter gene, we demonstrated the presence of one promoter, located immediately upstream of the Rv(Ms)2358 gene. Promoter induction was tested on several cultures grown under different conditions of pH and temperature, and in the presence of different concentrations of metallic ions. The promoter was found to be specifically induced by zinc. The recombinant M. tuberculosis FurB protein typically contained two zinc ions per protein monomer. Complete removal of zinc could not be obtained, even with strong denaturation treatment. Our data are in favour of the hypothesis that Rv2358 and FurB are transcriptional regulators involved in zinc homeostasis.
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PMID:The Mycobacterium tuberculosis Rv2358-furB operon is induced by zinc. 1505 32

An investigation was initiated to explore previously published results indicating that approximately 80 bp of the 5'-end of the iduronate sulfatase (IDS) cDNA sequence (Accession No L07291) are 100% homologous with the 3'-UTR of isoform I of the sodium hydrogen exchanger (Acc. No. U51112). 5'-RACE carried out on IDS mRNA demonstrated the apparent homology to be a cloning artifact. A sequence comparison of the IDS 5'-RACE product with a mouse BAC clone covering the region, and with various IDS ESTs, suggested that the region is highly susceptible to cloning artifacts, a common one of which is template switching by reverse transcriptase. The nucleotide sequence flanking the translation start site is unusual in containing two inverted repeats composed of the complementary trinucleotide microsatellites, (GCG)9 and (CGC)6. These likely form a highly stable stem of 20-21 nt, through which reverse transcription is compromised. Such a stem could be involved in the regulation of IDS expression by directly affecting translation, message turnover, or serving as a substrate for siRNA production. Though such mRNA features are relatively rare, they may be more abundant but overlooked due to difficulties in their reverse transcription.
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PMID:Inverted Gcg/CGC trinucleotide microsatellites in the 5'-region of Mus IDS mRNA: recurrent induction of aberrant reverse transcripts. 1529 86

A large number of plant retrotransposons have been characterized, but only three families ( Tnt1, Tto1 and Tos17) have been demonstrated to be transpositionally competent. We have used a novel approach to identify an active member of the Ty1- copia retrotransposon family with estimated 400 copies in the sweetpotato genome. Ty1- copia reverse transcriptase (RTase) sequences from the sweetpotato genome were analyzed, and a group of retrotransposon copies that probably arose by recent transposition events was identified and analyzed further. Transcripts containing long terminal repeats (LTRs) of this group were amplified from callus cDNA by the 3'RACE technique. Patterns of sequence-specific amplification polymorphism (S-SAP) of the LTR sequences in genomic DNA were compared between a normal plant and callus lines derived from it. In this way, a callus-specific S-SAP product was identified, which apparently resulted from the insertion of the retrotransposon detected by 3'RACE during cell culture. We conclude that our approach provides an effective way to identify active elements among the members of high-copy-number retrotransposon families.
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PMID:Isolation of an active element from a high-copy-number family of retrotransposons in the sweetpotato genome. 1548 Jul 92

The nucleotide sequence of the Clostridium cellulovorans xynB gene, which encodes the XynB xylanase, consists of 1,821 bp and encodes a protein of 607 amino acids with a molecular weight of 65,976. XynB contains a typical N-terminal signal peptide of 29 amino acid residues, followed by a 147-amino-acid sequence that is homologous to the family 4-9 (subfamily 9 in family 4) carbohydrate-binding domain. Downstream of this domain is a family 10 catalytic domain of glycosyl hydrolase. The C terminus separated from the catalytic domain by a short linker sequence contains a dockerin domain responsible for cellulosome assembly. The XynB sequence from mass spectrometry and N-terminal amino acid sequence analyses agreed with that deduced from the nucleotide sequence. XynB was highly active toward xylan, but not active toward carboxymethyl cellulose. The enzyme was optimally active at 40 degrees C and pH 5.0. Northern hybridizations revealed that xynB is transcribed as a monocistronic 1.9-kb mRNA. RNA ligase-mediated rapid amplification of 5' cDNA ends by PCR (RLM-5'RACE PCR) analysis of C. cellulovorans RNA identified a single transcriptional start site of xynB located 47 bp upstream from the first nucleotide of the translation initiation codon. Alignment of the xynB promoter region provided evidence for highly conserved sequences that exhibited strong similarity to the sigmaA consensus promoter sequences of gram-positive bacteria. Expression of xynB mRNA increased from early to middle exponential phase and decreased during the early stationary phase when the cells were grown on cellobiose. No alternative promoter was observed by RLM-5'RACE PCR and reverse transcriptase PCR analyses during expression. The analysis of the products from xylan hydrolysis by thin-layer chromatography indicated its endoxylanase activity. The results suggest that XynB is a consistent and major cellulosomal enzyme during growth on cellulose or xylan.
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PMID:Isolation and expression of the xynB gene and its product, XynB, a consistent component of the Clostridium cellulovorans cellulosome. 1557 84

The prophenoloxidase (proPO) system forms the basis of the non-specific defence system in crustaceans. The aim of this study was to develop an RT-PCR procedure to determine proPO gene expression. We used several degenerate primers designed from the conserved regions in amino acid sequences of proPOs from other species to clone the possible cDNA(s) from the haemocytes of the prawn Macrobrachium rosenbergii. One DNA fragment, 2016 bp long, was cloned by a combination of reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (3'/5'-RACE). This fragment could encode a putative polypeptide with 671 amino acids. Further analysis showed that this putative polypeptide contained six histidine residues and a thiol ester-like motif (GCGWPRHM) just like the structural features of proPOs from the shrimp Penaeus monodon. A partial fragment of the sequence with 934 bp containing three histidine residues and the thiol ester-like motif was used as a target to monitor the activation of the proPO gene by the semi-quantitative RT-PCR analysis. The result showed that the highest level of proPO mRNA was detected at 1h after in vivo injection with 5 microg of CpG oligodeoxynucleotide 2006 per prawn; in the same experiment, the highest PO activity was detected at 6 h after injection. In the control, a continuous and slow elevation of PO activity was observed during the experimental period, but such elevation of proPO mRNA was not observed. From our previous study and the time course of gene expression of proPO and enzymatic activity of PO in this study, it can be concluded that the enhancement effect was through its transcriptional level, its translational level and then its post-translational level sequentially. These results suggest that, both for reliability, sensitivity and for the timing of sampling, the change in proPO mRNA is more useful than the PO activity in monitoring the activation of the proPO non-specific defence system of prawns after treatment with stimulants.
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PMID:Cloning the prophenoloxidase cDNA and monitoring the expression of proPO mRNA in prawns (Macrobrachium rosenbergii) stimulated in vivo by CpG oligodeoxynucleotides. 1596 18

PLC (phospholipase C) isoenzymes catalyse the conversion of PtdIns(4,5)P2 into the Ca2+-mobilizing second messenger, Ins(1,4,5)P3, and the protein kinase C-activating second messenger, diacylglycerol. With the goal of identifying additional mammalian PLC isoenzymes, we screened the NCBI non-redundant database using a BLAST algorithm for novel sequences with homology with the conserved PLC catalytic core. Two unique sequences corresponding to two unknown PLC isoenzymes were identified, and one of these, designated PLC-eta2, was cloned and characterized. Most of the coding sequence of PLC-eta2 was constructed from two ESTs (expressed sequence tags), which included an overlapping sequence that was confirmed by multiple ESTs and mRNAs. 5'-RACE (rapid amplification of cDNA ends) also identified an upstream exon not deduced from available EST or mRNA sequences. Sequence analysis of PLC-eta2 revealed the canonical domains of a PLC isoenzyme with an additional long C-terminus that contains a class II PDZ-binding motif. Genomic analyses indicated that PLC-eta2 is encoded by 23 exons. RT-PCR (reverse transcriptase-PCR) analyses illustrated expression of PLC-eta2 in human retina and kidney, as well as in mouse brain, eye and lung. RT-PCR with exon-specific primers also revealed tissue-specific expression of four splice variants in mouse that represent alternative use of sequences in exons 21, 22 and 23. PLC-eta2-specific antisera recognized one of these splice variants as an approx. 155 kDa species when expressed in COS-7 cells; PLC-eta2 natively expressed in 1321N1 human astrocytoma cells also migrated as an approx. 155 kDa species. PLC activity was observed in vitro and in vivo for three different constructs of PLC-eta2, each containing possible alternatively spliced first exons. Co-expression of PLC-eta2 with Gbeta1gamma2 dimers of heterotrimeric G-proteins resulted in marked stimulation of inositol lipid hydrolysis. Thus PLC-eta2 may in part function downstream of G-protein-coupled receptors.
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PMID:Molecular cloning and characterization of PLC-eta2. 1623 48


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