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 complementary DNA (cDNA) sequence of epidermal growth factor (EGF) indicates that its 3' untranslated region (3' UTR) is 745 bases long, with polyadenylation occurring at residue 4749. However, when we used reverse transcriptase-polymerase chain reaction (RT-PCR) with an anchored 3' primer [3'rapid amplification of cDNA ends (RACE)] to amplify the 3' ends of cDNA, we actually detected two major products [800 and 600 base pairs (bp)] and a minor product (400 bp) in the thyroid or submaxillary glands (SMGs) of male mice. Analysis of genomic DNA with a battery of primer pairs gave only the predicted PCR products from the 3' UTR, demonstrating the lack of introns in this region of genomic DNA and eliminating alternate splicing as the explanation of the transcript diversity we detected. We confirmed that two potential polyadenylation sites proximal to residue 4749 are used in vivo by hybridizing the same 3' RACE products with probes specific for the 5' end of the 3' UTR, and also for poly-A tails. To assess the distribution of poly-A tail lengths on transcripts using the terminal polyadenylation site (4749), we used several different approaches to analyze 3' RACE products. Solution hybridization with 3' UTR probes revealed a striking difference between transcripts in SMG and thyroid: SMG contained two large 3' RACE populations (approximately 770 and 870 bp), whereas thyroid only contained one (approximately 770 bp). EGF transcript heterogeneity due to different poly-A tail lengths was confirmed using an upstream primer 400 bases closer to the 3' end of the 3' UTR, and TaqI digestion. Again we found two major populations in SMG (approximately 380 and 480 bp), but only one (380 bp) in thyroid, which upon TaqI digestion showed tissue-specific heterogeneity only in the 3' fragment. T4 treatment of male mice (0.25 microgram T4/gm ip) increased the intensity of both populations in SMG and the smaller population in thyroid within 24 h. However, after a week of T4 injections, only the intensity of the population with the longer poly-A tails in the SMG remained elevated, a finding consistent with tissue-specific enhanced stability of transcripts due to polyadenylation. Finally, to resolve poly-A tail lengths more precisely, we used an upstream primer that was specific for the 3' end of murine 3' UTR. This approach revealed that the thyroid contains three major populations of EGF transcripts, with poly-A tail lengths of approximately 20, 50, and 70 A's. After T3 treatment for 24 h, the intensity of transcripts containing 20 A's increased 52% (P < 0.02) and those with 50 A's increased 130% (P < 0.01), whereas there was no change in transcripts with tails > or = 70 A's. On the other hand, there were no distinct bands in SMG samples, but rather a heterogeneous distribution of poly-A tail lengths from approximately 20-120 A's that showed an overall increase of approximately 60% in response to T3.
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PMID:Assessment of transcript polyadenylation by 3' RACE: the response of epidermal growth factor messenger ribonucleic acid to thyroid hormone in the thyroid and submaxillary glands. 758 22

The secondary structure in mRNA is essential for many processes, but it can present a technical problem in making full-length cDNA with reverse transcriptases. Furthermore, different reverse transcriptases have differing abilities to transcribe through regions with secondary structure, which can alter the products obtained by reverse-transcribing RNA and then PCR-amplifying the product (RT-PCR). We have been interested in studying the posttranscriptional regulation of epidermal growth factor by RT-PCR and have tested the ability of several reverse transcriptases to reverse transcribe the 3'-untranslated region (3'UTR), a region that contains substantial secondary structure. When low levels of either total RNA or poly(A)+ mRNA were used, we found avian myeloblastosis virus reverse transcriptase (AMV-RT) to be the most robust of all the enzymes tested. Furthermore, contrary to reports that AMV-RT is inhibited by tRNA--which should make it less effective than Moloney murine leukemia virus reverse transcriptase (MMLV-RT) at reverse-transcribing total RNA--adding tRNA to poly(A)+ RNA actually increased the amount of specific RT-PCR product obtained with AMV-RT while it decreased the amount of product and enhanced mispriming with MMLV-RT. We found that pre-incubation of the oligo(dT) primer with total RNA at elevated temperature prior to reverse transcription improved the efficiency of both native and modified MMLV-RTs. These findings support the concept that secondary structures in RNA differentially affect the abilities of different reverse transcriptases to detect transcript diversity and raise the possibility that such structures could affect quantitation using RT-PCR with internal mRNA standards.
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PMID:Secondary structure in the 3' UTR of EGF and the choice of reverse transcriptases affect the detection of message diversity by RT-PCR. 858 21

A pilot survey of hepatitis C virus (HCV) infection in Nigeria was carried out on healthy adult blood donors and children of preschool age. Sixteen of 200 (8%) donors were positive for antibodies using a second generation enzyme-linked immunosorbent assay (ELISA) but all of the children were negative. Supplementary testing of the ELISA-positives using a recombinant immunoblot assay (RIBA-2) confirmed the presence of antibody in four and two others were indeterminate. Four of the anti-HCV-positive sera and one found positive by ELISA but which was negative by RIBA-2 were found to be positive for HCV RNA using reverse transcriptase-polymerase chain reaction (RT-PCR) and primers specific for the 5' untranslated region (5'UTR) of the HCV genome. The NS5 and core regions also were amplified and the PCR products from all three regions were sequenced. Sequences from the 5'UTR could be divided into two groups: one group comprised three isolates with greater than 95% sequence identity with published sequences of genotype 1 and the other comprised two isolates with greater than 93% sequence identity with genotype 4. Analysis of three sequences amplified from the NS5 region confirmed this assignment to genotypes 1 and 4. Pairwise comparisons of the NS5 region sequences with representatives of 1a, 1b, 1c (for the first group) and 4a-4h (for the second group) show the first group to include subtypes classifiable as 1a and a novel sequence and the second group to include a novel sequence within genotype 4. Sequence analysis of the core region was consistent with this interpretation. These data confirm the presence of at least two major HCV genotypes in Nigeria (genotypes 1 and 4) and we report two novel sequences which have been designated provisionally as genotypes 1d and 4i.
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PMID:Genotypes of hepatitis C virus in Nigeria. 881 62

Pituitary adenylate cyclase-activating polypeptide (PACAP) elicits its diverse biological actions by interacting with both PACAP-selective type I PACAP receptors (PACAPRs) and type II PACAPRs that do not distinguish between PACAP and vasoactive intestinal polypeptide. Using long distance polymerase chain reaction, we amplified and characterized the entire coding region of the rat type I PACAPR (rPACAPR) gene, which spans 40 kilobases and contains 15 exons. Mapping of the exons and sequencing of all intron-exon boundaries revealed a structural organization of the rPACAPR gene that is very similar to those encoding other members of the calcitonin/secretin/parathyroid hormone receptor family. Southern blot analysis demonstrated a single copy of the rPACAPR gene. A combination of rapid amplification of cDNA ends and reverse transcriptase polymerase chain reaction revealed an unexpected diversity in the rPACAPR mRNA in the 5'-untranslated (5'-UTR) region. Four rPACAPR cDNAs were identified with 5'-UTR sequences that all diverged from the genomic sequence at a site 76 bp upstream of the ATG start codon, where a consensus 3' slice acceptor sequence was located. Sequence analysis of these amplified transcripts demonstrated that they arise by tissue-specific differential usage of four exons in the 5' noncoding region of the rPACAPR gene. This study is the first to elucidate the structural organization of a PACAPR gene and to demonstrate that alternative splicing generates rPACAPR transcripts with unique 5'-UTRs.
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PMID:Genomic organization of the rat pituitary adenylate cyclase-activating polypeptide receptor gene. Alternative splicing within the 5'-untranslated region. 911 82

Serine proteases (granzymes) in killer lymphocytes are required for lymphocyte cytotoxic granules to lyse target cells. Herein we report the development of a 3-step PCR cloning technique to amplify novel granzyme genes and two new rat granzymes are described. Degenerate oligonucleotide primers were designed based on sequence motifs selectively expressed in granzymes. These motifs flank "delta" regions that are unique for each granzyme. Total RNA of RNK-16 cells or activated splenocytes was amplified by reverse transcriptase-PCR to obtain cDNA fragments of several new granzymes. Gene-specific primers based on these delta regions were then used for 3'-RACE to obtain clones with the 3' gene ends. Reverse (antisense) delta-based or active site serine primers were used with a granzyme 5'-UTR primer to obtain clones extending to the 5' ends. Using this technique, two new cDNAs, RNKP-4 and RNKP-7, which encode granzymes of 248 and 241 amino acids, respectively, were cloned from activated lymphocytes. RNKP-4 is likely the rat equivalent of mouse granzyme C. RNKP-7 is most closely related to granzymes F and G. Modeling of the predicted proteins suggests large/polar P1 (Gln/Asn) specificity for RNKP-4 and large/hydrophobic P1 (e.g., Phe) specificity for RNKP-7. These specific protease activities were found in cytotoxic RNK-16 lymphocyte granules indicating that the two new genes may be translated and stored as active granzymes.
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PMID:P-4 and RNKP-7, new granzyme-like serine proteases expressed in activated rat lymphocytes. 914 69

A sero-epidemiologic survey of 74 IVDUs and 86 age-matched controls was performed to investigate the prevalence, clinical significance, and genetic distribution of GB virus C (GBV-C)/hepatitis G virus (HGV) infection among intravenous drug users (IVDUs) in Japan. GBV-C/HGV RNA was detected by reverse transcriptase-hemi-nested polymerase chain reaction (RT-hemi-nested PCR) for the 5'-untranslated region (5'-UTR) in 43.2% (32/79) of the IVDUs compared with 1.2% (1/86) of the controls (P < 0.001). The duration of drug use did not correlate with the prevalence of GBV-C/HGV. Thirteen subjects positive for GBV-C/HGV RNA without HCV RNA had normal serum aminotransferase concentrations. Phylogenetic tree showed that the 32 GBV-C/HGV-positive isolates from IVDUs were classified within a new GBV-C/HGV group, which has been identified mainly in Asian subjects (type 3). The present study illustrated, (1) the high incidence of GBV-C/HGV infection among Japanese IVDUs, (2) GBV-C/HGV alone may not be an important pathogenic factor for hepatic injury, and (3) type 3 GBV-C/HGV was the most prevalent among IVDUs in Japan.
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PMID:GB virus C (GBV-C)/hepatitis G virus (HGV) infection among intravenous drug users in Japan. 921 90

Combined methodologies of histochemistry, immunohistochemistry, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), reverse transcriptase polymerase chain reaction (RT-PCR) and a histochemical method specific for myofibrillar ATPase (mATPase) of the type IIX myosin heavy chain (MyHC) isoform were used to study human and rat single fibres to examine the homology between type II MyHC isoform-based fibres of both species. We demonstrate that human type II fibres exhibit antigenic mATPase and 3'-untranslated region (3'-UTR) sequence determinants homologous to the IIA and IIX but not the IIB MyHC isoforms of the rat. Both immunolabelling with anti-MyHC monoclonal antibodies and the mATPase method used with frozen sections confirmed that all human type II fibres express type IIA and/or type IIX MyHC. Quantitative immunohistochemistry failed to recognize human fibres with antigenic characteristics corresponding to hybrid IIXB MyHC-based fibres. Ca2+-stimulated maximum myosin ATPase activity, determined by quantitative histochemistry, revealed that human IIX fibres (with an optical density or OD = 0.707) display enzyme activity which is comparable to that of the rat type IIX (OD = 0.687) but lower than that of the rat type IIB fibres (OD = 0.836). The results do not support the notion that MyHC IIB is expressed in human limb muscles, even in hybrid fibres. We conclude that human type II fibres have been misclassified in numerous previous publications and that this has important implications in attempts to compare the physiological characteristics of fibre types, particularly when animal models are used.
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PMID:Comparison of the molecular, antigenic and ATPase determinants of fast myosin heavy chains in rat and human: a single-fibre study. 935 15

CR1 elements are a family of retroposons. They are classified as long interspersed elements (LINEs) or non-long-terminal-repeat (non-LTR) retrotransposons, and they have been found in the genomes of many vertebrates. However, they have been only partially characterized, and only a 2-kb region of the 3' end of chicken CR1 has been sequenced. In the present study, we determined the entire consensus sequence of CR1 elements in the turtle genome, designated PsCR1. The first open reading frame (ORF1) of PsCR1 has two unusual arrangements of Cys residues. One of them includes a zinc finger motif, CX2CX14CX2C. The putative zinc finger has cysteine residues with identical spacing and a similar amino acid composition to those found in the species-specific transcription initiation factors SL1 and TIF-IB. The 5' untranslated region (5' UTR) of PsCR1 contains a sequence similar to part of the human L1 promoter, L1 site A, and several cis elements of the type found in eukaryotic genes. Within a region of about 500 bp, there are nine "E boxes," cis elements that are recognized by the basic helix-loop-helix (bHLH) family of proteins. This observation raises the possibility that cellular transcription factors that bind to these sequences might act in concert to regulate the expression of PsCR1. The extent of the sequence divergence of the 3' UTR of CR1 between species was found to be lower than the rate of nonsynonymous substitutions per site in ORF2, suggesting that a strict functional constraint must exist for this region. This result strongly suggests that the conserved 3'-end sequence of CR1 is the recognition site for the reverse transcriptase of CR1. A discussion is presented of a possible mechanism for the integration of CR1 elements and also of the intriguing possible recruitment of the reverse transcriptase for the retroposition of SINEs.
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PMID:Determination of the entire sequence of turtle CR1: the first open reading frame of the turtle CR1 element encodes a protein with a novel zinc finger motif. 940 32

CD151 (PETA-3/SFA-1) is a member of the Transmembrane 4 Superfamily (TM4SF) of cell-surface proteins and, like other TM4SF members CD9 and CD63, is expressed by platelets, megakaryocytes and endothelial cells. The precise function of CD151 is unknown however complexes containing CD151 and beta1 integrins have been isolated from a number of cell systems and studies using anti-CD151 monoclonal antibodies have suggested a role in transmembrane signalling and cell adhesion. To further investigate the function of CD151 we have determined the genomic organisation of mouse CD151 (Cd151). Cd151 spans 4 kb and contains six coding region exons. Using 5' RACE and reverse transcriptase-polymerase chain reaction (RT-PCR) we have identified three 5' UTR splice variants which arise through alternate splicing of three exons. Splice variants were detected in a number of mouse tissues by RT-PCR. Analysis of the Cd151 genomic structure reveals a high degree of structural conservation with other TM4SF molecules supporting the theory that family members have arisen from gene duplication of a common ancestral gene. Cd151 maps to chromosome 7, in close linkage to the p gene (OCA2 in humans), and helps define a boundary in the human/mouse homology relationships.
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PMID:Characterisation of the mouse homologue of CD151 (PETA-3/SFA-1); genomic structure, chromosomal localisation and identification of 2 novel splice forms. 960 68

The spectrum of protein tyrosine phosphatases (PTPs) expressed in bone marrow-derived murine macrophages (BMMs) was examined using reverse transcriptase-polymerase chain reaction. Ten different PTP cDNAs were isolated and in this study we focus on mDEP-1, a type III receptor PTP. Three mDEP-1 transcripts were expressed in primary macrophages and macrophage cell lines and were induced during macrophage differentiation of M1 myeloid leukemia cells. A variant mRNA was identified that encodes an alternate carboxyl-terminus and 3' UTR. The expression of mDEP-1 was down-regulated by CSF-1 (macrophage colony-stimulating factor) and up-regulated by bacterial lipopolysaccharide, an important physiological regulator of macrophage function that opposes CSF-1 action. Whole mount in situ hybridization, and immunolocalization of the protein, confirmed that mDEP-1 is expressed by a subset of embryonic macrophages in the liver and mesenchyme. mDEP-1 was also detected in the eye and peripheral nervous system of the developing embryo. Attempts to express mDEP-1 constitutively in the macrophage cell line RAW264 were unsuccessful, with results suggesting that the gene product inhibits cell proliferation.
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PMID:Murine DEP-1, a receptor protein tyrosine phosphatase, is expressed in macrophages and is regulated by CSF-1 and LPS. 982 76


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