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)

We have used analysis of ethidium-bromide-stained reverse transcriptase-polymerase chain reaction (RT-PCR) products to assess the effects of X-chromosome inactivation during spermatogenesis in the mouse. RT-PCR was performed on total RNA from eight different spermatogenic cell types, including premeiotic spermatogonia, meiotic spermatocytes, and postmeiotic spermatids, to detect transcripts from five different X-linked structural genes (Pgk-1, Zfx, Pdha-1, Hprt, and Phka) and two autosomal genes (Pgk-2 and beta-actin). Relative intensities of ethidium-bromide-stained RT-PCR products representing transcripts from each gene in each cell type were analyzed by densitometry using the Image program (version 1.4, NIH), and normalized against beta-actin values. These results suggest a coordinate inactivation of the X-linked loci at the onset of meiosis, followed by variable rates of decline of corresponding transcript levels reflecting differential mRNA stabilities and/or leaky expression after inactivation. Technically, these results indicate that analysis of ethidium-bromide-stained RT-PCR products can be used to provide a "semiquantitative" indication of relative levels of specific transcripts in a developing cell lineage without using radioactive probes to quantitate these products.
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PMID:Semiquantitative analysis of X-linked gene expression during spermatogenesis in the mouse: ethidium-bromide staining of RT-PCR products. 128 26

The biological activity of insulin-like growth factor-I (IGF-I) is mediated by a transmembrane glycoprotein (type-1 IGF receptor or IGF-I receptor) that shows considerable sequence homology with the insulin receptor. In order to detect the expression of this gene in chicken liver tissue, a plasmid was constructed containing a fragment of chicken IGF-I receptor cDNA. The cDNA fragment corresponded to nucleotides 326-599 of the human IGF-I receptor cDNA and showed 86.1 and 69.3% homology at the nucleotide level and 96.7 and 80.2% homology at the amino acid level with the human IGF-I receptor and insulin receptor respectively. The construct was used to generate an antisense RNA probe for the detection of IGF-I receptor mRNA transcripts in 1- and 4-week-old chick liver tissue. IGF-I receptor gene expression was initially detected by the reverse transcriptase polymerase chain reaction using synthetic chicken IGF-I receptor oligonucleotides. Amplified fragments of the correct size were detected in both RNA samples. Northern blots were also used to detect IGF-I receptor mRNA transcripts in the liver RNA samples. The results indicated that the amount of receptor mRNA decreased significantly between 1 and 4 weeks after hatch. In contrast, chicken beta-actin gene expression remained constant over this period. A major IGF-I receptor RNA transcript (11 kb) was observed in blots from 1-week-old livers, less abundant transcripts were also observed ranging in size from 8 to 9 kb.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The expression of a putative insulin-like growth factor-I receptor gene in the liver of the developing chick. 132 34

Poly (A) RNA was isolated from microdissected guinea pig organ of Corti and converted into cDNA with RNase H- murine leukemia virus reverse transcriptase. After size fractionation, the cDNA was directionally ligated into the vector pSPORT 1 and the plasmids were transformed into DH10B E. coli via electroporation. The library was found to have 3.35 x 10(6) independent colonies with ten percent of the colonies lacking an insert. After checking 33 randomly selected colonies for inserts, the average insert size was 1218 base pairs, ranging from 3300 base pairs to 400 base pairs. The library was screened with a beta-actin oligonucleotide probe and 1.4% of the colonies contained an insert hybridizing to the probe.
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PMID:Construction of a cDNA library from microdissected guinea pig organ of Corti. 135 71

A new method for estrogen receptor (ER) mRNA was performed on 33 human breast tumors, using a reverse transcriptase-polymerase chain reaction (RT-PCR) assay by the method of Fuqua et al. In a preliminary experiment using the MCF-7 breast tumor cell line, ER/beta-actin ratio was almost same. ER protein was estimated by a dextran coated charcoal (DCC) assay and by an ER-immunocytochemical (ER-ICA) assay using a specific monoclonal antibody. We found RT-PCR assay correlates with ER-ICA assay (r = 0.664, p less than 0.01), whereas no significant correlation was seen between RT-PCR assay and DCC assay. These results suggests that RT-PCR assay is suitable for detection of ER from small amounts of tissue.
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PMID:[Detection of estrogen receptor (ER) mRNA by use of reverse transcriptase-polymerase chain reaction (RT-PCR) assay; comparison with dextran coated charcoal (DCC) assay and immunocytochemical assay]. 137 12

Several immune mechanisms are likely to be responsible for renal allograft rejection. The relative importance of delayed-type hypersensitivity versus cytotoxic T lymphocytes is controversial. We analyzed human renal allografts biopsies for intragraft expression of IL-1 beta, IL-6, and TNF alpha genes--putative mediators of DTH--as well as IL-2, IL-2 receptor (R) beta, and a CTL-specific serine protease gene. Total RNA was extracted from tissue samples and the mRNA fraction was converted to cDNA using oligo dT and reverse transcriptase. Then cDNA was amplified by the polymerase chain reaction (PCR) for 35 cycles using specific oligonucleotide primers. Each PCR analysis included beta-actin oligonucleotide primers to coamplify this constitutively expressed gene as an internal control. A total of 24 core allograft biopsies were studied and classified into a 3 histological categories: acute cellular rejection, equivocal components of rejection, and no evidence of rejection. There was no statistically significant difference in beta-actin expression among these histologic categories (P greater than 0.08). Interestingly, in this sample size, no significant difference was found between rejecting and nonrejecting samples for transcripts of any of the cytokines or IL-2R beta mRNAs. Apparently, DTH-like mechanisms are present in all allografts. However, detection of CTL-specific serine protease gene expression was almost exclusive to rejecting samples (P less than 0.003). These findings suggest that activation of CTLs play an active, but hardly exclusive, role as effectors of graft dysfunction in the rejection process. While this study does not define the relative importance of the genes examined, it does suggest that evidence of CTL-specific serine protease expression may provide a means of monitoring for rejection episodes or as a diagnostic aid when conventional diagnostic criteria are not conclusive.
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PMID:The strong correlation of cytotoxic T lymphocyte-specific serine protease gene transcripts with renal allograft rejection. 173 89

Psoriatic skin lesions contain HLA-DR positive T lymphocytes, and other activation antigens, which suggest that the T cells may be producing lymphokines. Gamma interferon is produced by activated T cells, and its presence in psoriasis has been inferred by the lesional keratinocyte expression of 3 gamma interferon-inducible proteins i.e. HLA-DR, intercellular adhesion molecule-1, and gamma-IP-10. To determine whether gamma interferon is being produced directly in psoriatic lesions, punch biopsies of normal and diseased skin were separated into epidermal sheets and dermal fragments. Total cellular RNA was isolated from each epidermal and dermal compartment, and reverse transcribed followed by amplification of the resultant DNA by polymerase chain reaction. The amplification process involved the use of 5' and 3' primers for gamma interferon, and tumor necrosis factor-alpha, with beta-actin serving as a control. Gamma interferon mRNA, but not tumor necrosis factor alpha mRNA, was detectable in 4 of 5 psoriatic epidermal specimens. Neither mRNA was detectable in any normal skin dermal/epidermal specimens. Gamma interferon mRNA was also detectable in a single psoriatic dermal specimen. If reverse transcriptase was omitted, no polymerase chain reaction products were detected, indicating that the fragments detected were not derived from contaminating genomic DNA. These results indicate that gamma interferon mRNA can be extracted and successfully detected from human psoriatic lesional skin biopsies, using polymerase chain reaction technology. This molecular approach can easily be expanded to measure many other cytokines in both epidermal and dermal locations. The detection of gamma interferon in this clinical setting may be of particular pathophysiological significance because injection of gamma interferon has been reported to induce psoriatic lesions.
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PMID:Detection of interferon-gamma mRNA in psoriatic epidermis by polymerase chain reaction. 190 50

Direct recognition of viral gene sequences can be used to detect human immunodeficiency virus (HIV-1) in clinical specimens. A modification of the polymerase chain reaction (PCR) for amplification of gene sequences was used for detection of HIV-1-specific RNA prepared from peripheral blood mononuclear cells (PBMC). The RNA served as a template for reverse transcriptase using primers derived from both the 3'ORF and the LTR regions of HIV-1, as well as from the control cellular sequences encoding beta-actin and T cell receptor. The resultant DNA was amplified with DNA polymerase. A transcriptional step using the bacteriophage T7 promoter recognition sequences, incorporated into the primers, was used to enhance the efficiency of the amplification process. This assay detects as few as 100 RNA copies of cloned HIV-1 genome. Starting with 1 microgram RNA isolated from PBMC, we were able to detect HIV-1 sequences in patients with symptomatic and asymptomatic HIV-1 infection. The inclusion of T cell-specific primers permitted simultaneous evaluation of an immunologic parameter. The PCR can be applied to RNA samples for detection of viral and cellular sequences and is a rapid and efficient means for detection of HIV-1 sequences as well as potentially informative cellular sequences.
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PMID:Confirmation of HIV infection using gene amplification. 252 May 45

We developed a reverse transcription-polymerase chain reaction (RT-PCR) method which permits the simultaneous amplification of several mRNAs, even though their relative levels may be very different. First-strand cDNAs were synthesized from total RNA by MMLV reverse transcriptase with oligo(dT)15 priming. Analysis was performed in the linear phase of PCR, allowing the detection of the products by polyacrylamide gel electrophoresis followed by ethidium bromide staining. In order to obtain a similar amplification of multiple targets in the same PCR system, it was necessary: (i) to adjust the relative concentration of each set of primers and (ii) to use high-stringency conditions (annealing temperature and addition of organic solvent). These conditions allowed the rapid quantitation of several mRNAs in multiple samples, minimizing experimental variations. The reliability of the method was established by measuring variations of c-Ki-Ras, ornithine decarboxylase, alpha-amylase, and beta-actin mRNA levels during the growth of pancreatic tumoral AR4-2J cells. Glyceraldehyde-6-phosphate dehydrogenase expression showed very small variations which confirm that it represents a reliable internal standard for studies of gene expression. Results from competitive PCR amplification of target cDNA and internal competitive template were in agreement with those of simultaneous amplification, validating the quantitative aspect of the method.
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PMID:Quantitation of changes in the expression of multiple genes by simultaneous polymerase chain reaction. 750 50

Rab proteins are ras-like low molecular mass GTP-binding proteins, which are postulated to act as specific regulators of membrane trafficking in exocytosis and endocytosis. We have previously shown that synthetic peptides, corresponding to the effector domain of Rab3 proteins, stimulate a complete exocytotic response in mast cells. We have used a PCR-cloning strategy to investigate the presence of mRNA encoding Rab3 in mast cells. RNA based PCR was then performed on mast cell RNA using degenerate oligonucleotide primers based on two conserved sequences among Rab3 proteins. However, no PCR products were obtained, even for proteins known to be expressed in high copy numbers in mast cells (beta-actin and Fc receptor). We have found that the problem resides in the presence of mast cell secretory granule derived heparin, that copurifies with the RNA; heparin has been shown to inhibit the activity of reverse transcriptase and Taq polymerase in PCR. After treating the RNA (obtained from about 500 mast cells) with heparinase, several PCR products of varying size were obtained using primers specific for Rab3 proteins. These products were cloned and sequenced. We have found clones containing sequences that had a 100% homology at the deduced amino acid level to a portion of Rab3B and Rab3D (amino acids 16 to 83).
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PMID:RT-PCR cloning of Rab3 isoforms expressed in peritoneal mast cells. 750 66

To assess regulation of constitutive prostaglandin G/H synthase (PGHS-1) by interleukin-1 (IL-1) in osteoblastic MC3T3-E1 cells, we compared analysis by competitive reverse transcriptase-polymerase chain reaction (RT-PCR) with Northern blot analysis. Using RT-PCR, IL-1 increased PGHS-1 mRNA levels by 1.84 +/- 0.10 or 2.07 +/- 0.17, depending on the method of calculation. Using Northern blot analysis, the effect of IL-1 on PGHS-1 mRNA levels was more variable, and the variability was increased by normalization of PGHS-1 mRNA levels to the housekeeping genes, beta-actin and glyceraldehyde phosphate dehydrogenase (GAPDH), because their mRNA levels were also regulated by IL-1. We conclude that competitive RT-PCR is a reproducible and accurate method for studying small changes in mRNA levels.
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PMID:Measurement of interleukin-1 stimulated constitutive prostaglandin G/H synthase (cyclooxygenase) mRNA levels in osteoblastic MC3T3-E1 cells using competitive reverse transcriptase polymerase chain reaction. 752 76


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