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)

A sensitive nitrocellulose filter assay that measures the retention of 125I single-stranded calf thymus DNA has been used to detect and purify DNA-binding proteins that retain a biological function from Rauscher murine leukemia virus. By consecutive purification on oligo (dT)- cellulose and DEAE-Bio-Gel columns and centrifugation in 10 to 30% glycerol gradients, RNA-dependent DNA polymerase has been separated from a second virion DNA-binding protein. The binding of this protein to DNA was strongly affected by NaCl concentration but showed little change in activity over a wide range of temperature or pH. After glycerol gradient purification, polyacrylamide gel electrophoresis of this protein showed one major band with a molecular weight of approximately 9,800. This protein binds about as well as to single-stranded Escherichia coli or calf thymus DNA or 70S type C viral RNA. The binding to 125I single-stranded calf thymus DNA is very efficiently inhibited by unlabeled single-stranded DNA from either E. coli or calf thymus and by 70S murine or feline viral RNA. Much larger amounts of double-stranded DNA are required to produce an equivalent percentage of inhibition. This protein, therefore, shows preferential binding to single-stranded DNA or viral RNA.
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PMID:Low-molecular- weight Rauscher leukemia virus protein with preferential binding for single-stranded RNA and DNA. 5 75

The pattern of transcription has been examined for a cluster of genes encoding polypeptides some or all of which are assembled into a cross-linked component of the Bacillus subtilis spore coat. Three promoters, designated PVWX, PX and PYZ, were indicated by reverse transcriptase mapping. On the basis of Northern hybridization, it appeared that the cotV, W and X genes were transcribed as a polycistronic mRNA from PVWX as well as a monocistronic cotX mRNA from Px. The cotY and cotZ genes are cotranscribed from the PYZ promoter with a smaller cotY mRNA resulting from premature termination or RNA processing. All four transcripts were synthesized late during sporulation and were not produced in mutants lacking sigma K, which directs RNA polymerase to transcribe genes in the mother-cell compartment of sporulating cells. The DNA-binding protein GerE, which affects transcription of many genes in the mother cell during the late stages of sporulation, was also shown to be involved. There was essentially no cotX mRNA in a gerE mutant and the amounts of cotVWX, cotYZ and cotY mRNAs were somewhat reduced. In vitro run-off transcription studies with sigma K RNA polymerase and GerE confirmed the presence of the three promoters, and directly showed that GerE was necessary for transcription from PX as well as enhanced transcription from the PVWX and PYZ promoters. The DNase I footprints of GerE for all three promoters were immediately upstream of the -35 regions. These GerE binding sites were compared to those in other GerE-responsive promoters and a larger consensus sequence for GerE binding was recognized. This complex transcriptional pattern of the cotVWXYZ cluster is probably necessary to ensure that an optimal amount of each protein is made for the assembly of the spore coat.
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PMID:Regulation of the transcription of a cluster of Bacillus subtilis spore coat genes. 751 71

During processive DNA synthesis in vitro, the human immunoefficiency virus, type 1 (HIV-1) reverse transcriptase encounters template nucleotide positions at which continued synthesis is difficult. At these positions, the enzyme has a relatively high probability of dissociating from the template, and product molecules of corresponding length accumulate as the incubation proceeds. These positions, which are known as termination sites, could be associated with template secondary structures in some cases, but many termination sites appear to be template sequence-related rather than secondary structure-related. Mechanisms producing these blocks in processive DNA synthesis are not well understood. In this study, to examine further the effects of template sequence on termination, we engineered selected single-base changes in the M13mp2 template, and we found that such changes can influence termination. Several general trends emerged from the study. First, strong termination sites rarely correspond to dATP as the "incoming" substrate opposite template T. Second, the sequence of the template-primer stem is more important for termination than the sequence of the single-stranded template ahead of the primer. Thus, we note the phenomenon of action at a distance: changing sequence at one nucleotide position in the template-primer stem alters termination at other positions, a few nucleotides distant at the primer 3' end. A and C as template bases in the template-primer stem have opposite effects. A is the strongest terminator residue, and C is the weakest terminator residue, followed by G. Since termination sites are produced by reverse transcriptase dissociation from the template-primer, the results suggest that the HIV-1 reverse transcriptase has properties reminiscent of a sequence-specific double-stranded DNA-binding protein in that its binding mechanism can distinguish both base residues and positions in the double-stranded DNA template-primer stem.
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PMID:Mechanism of HIV-1 reverse transcriptase. Termination of processive synthesis on a natural DNA template is influenced by the sequence of the template-primer stem. 768 74

The presence of reverse transcriptase (RT) activity in a DNA-binding protein complex of the goat bone marrow has been reported earlier from our laboratory. Here we report a procedure for the purification of the enzyme with RT activity from bovine bone marrow and show that the basic function is associated with a approximately 66-kDa protein. This enzyme can use RT specific homopolymers as template and short oligonucleotides as primers, while displaying a Mg(2+)-ion requirement. Eukaryotic RTs have been shown to have endogenous RNAs associated with the enzymes. Evidence is presented here to show that some endogenous RNAs are associated with the RT activity in bovine bone marrow. Even though the enzyme activity appears to be associated with a approximately 66-kDa protein, the results indicate that for a full expression of its activity, the enzyme needs to interact with a 55-kDa protein that co-purifies with the enzyme during ion-exchange chromatography.
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PMID:Reverse transcriptase activity in bovine bone marrow: purification of a 66-kDa enzyme. 1089 2

The AML1 gene encodes a DNA-binding protein that contains the runt domain and is the most frequent target of translocations associated with human leukemias. Here, point mutations of the AML1 gene, V105ter (single-letter amino acid code) and R139G, (single-letter amino acid codes) were identified in 2 cases of myelodysplastic syndrome (MDS) by means of the reverse transcriptase-polymerase chain reaction single-strand conformation polymorphism method. Both mutations are present in the region encoding the runt domain of AML1 and cause loss of the DNA-binding ability of the resultant products. Of these mutants, V105ter has also lost the ability to heterodimerize with polyomavirus enhancer binding protein 2/core binding factor beta (PEBP2beta/CBFbeta). On the other hand, the R139G mutant acts as a dominant negative inhibitor by competing with wild-type AML1 for interaction with PEBP2beta/CBFbeta. This study is the first report that describes mutations of AML1 in patients with MDS and the mechanism whereby the mutant acts as a dominant negative inhibitor of wild-type AML1.
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PMID:Mutations of the AML1 gene in myelodysplastic syndrome and their functional implications in leukemogenesis. 1104 97

In this work, we characterize genes in Mycobacterium tuberculosis that are regulated by IdeR (iron-dependent regulator), an iron-responsive DNA-binding protein of the DtxR family that has been shown to regulate iron acquisition in Mycobacterium smegmatis. To identify some of the genes that constitute the IdeR regulon, we searched the M. tuberculosis genome for promoter regions containing the consensus IdeR/DxR binding sequence. Genes preceded by IdeR boxes included a set encoding proteins necessary for iron acquisition, such as the biosynthesis of siderophores (mbtA, mbtB, mbtI), aromatic amino acids (pheA, hisE, hisB-like) and others annotated to be involved in the synthesis of iron-storage proteins (bfrA, bfrB). Some putative IdeR-regulated genes identified in this search encoded proteins predicted to be engaged in the biosynthesis of lipopolysaccharide (LPS)-like molecules (rv3402c), lipids (acpP) and peptidoglycan (murB). We analysed four promoter regions containing putative IdeR boxes, mbtA-mbtB, mbI, rv3402c and bfrA-bfd, for interaction with IdeR and for iron-dependent expression. Gel retardation experiments and DNase footprinting analyses with purified IdeR showed that IdeR binds to these IdeR boxes in vitro. Analysis of the promoters by primer extension indicated that the IdeR boxes are located near the -10 position of each promoter, suggesting that IdeR acts as a transcriptional repressor by blocking RNA polymerase binding. Using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) coupled to molecular beacons, we showed that mRNA levels of mbtA, mbtB, mbtI, rv3402c and bfd are induced 14- to 49-fold in cultures of M. tuberculosis starved for iron, whereas mRNA levels of bfrA decreased about threefold. We present evidence that IdeR not only acts as a transcriptional repressor but also functions as an activator of bfrA. Three of the IdeR- and iron-repressed genes, mbtB, mbtI and rv3402c, were induced during M. tuberculosis infection of human THP-1 macrophages.
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PMID:The Mycobacterium tuberculosis IdeR is a dual functional regulator that controls transcription of genes involved in iron acquisition, iron storage and survival in macrophages. 1172 47

Telomere maintenance is essential for the continuous growth of tumor cells. In most human tumors telomeres are maintained by telomerase, a specialized reverse transcriptase. Tankyrase 1, a human telomeric poly(ADP-ribose) polymerase (PARP), positively regulates telomere length through its interaction with TRF1, a telomeric DNA-binding protein. Tankyrase 1 ADP-ribosylates TRF1, inhibiting its binding to telomeric DNA. Overexpression of tankyrase 1 in the nucleus promotes telomere elongation, suggesting that tankyrase 1 regulates access of telomerase to the telomeric complex. The recent identification of a closely related homolog of tankyrase 1, tankyrase 2, opens the possibility for a second PARP at telomeres. We therefore sought to establish the role of tankyrase 1 at telomeres and to determine if tankyrase 2 might have a telomeric function. We show that endogenous tankyrase 1 is a component of the human telomeric complex. We demonstrate that telomere elongation by tankyrase 1 requires the catalytic activity of the PARP domain and does not occur in telomerase-negative primary human cells. To investigate a potential role for tankyrase 2 at telomeres, recombinant tankyrase 2 was subjected to an in vitro PARP assay. Tankyrase 2 poly(ADP-ribosyl)ated itself and TRF1. Overexpression of tankyrase 2 in the nucleus released endogenous TRF1 from telomeres. These findings establish tankyrase 2 as a bona fide PARP, with itself and TRF1 as acceptors of ADP-ribosylation, and suggest the possibility of a role for tankyrase 2 at telomeres.
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PMID:Role for the related poly(ADP-Ribose) polymerases tankyrase 1 and 2 at human telomeres. 1173 45

Information on over- and underexpressed genes in prostate cancer in comparison to adjacent normal tissue was sought by DNA microarray analysis. Approximately 12,600 mRNA sequences were analyzed from a total of 26 tissue samples (17 untreated prostate cancers, 9 normal adjacent to prostate cancer tissues) obtained by prostatectomy. Hierarchical clustering was performed. Expression levels of 63 genes were found significantly (at least 2.5-fold) increased, whereas expression of 153 genes was decreased (at least 2.5-fold) in prostate cancer versus adjacent normal tissue. In addition to previously described genes such as hepsin, overexpression of several genes was found that has not drawn attention before, such as the genes encoding the specific granule protein (SGP28), alpha-methyl-acyl-CoA racemase, low density lipoprotein (LDL)-phospholipase A2, and the anti-apoptotic gene PYCR1. The radiosensitivity gene ATDC and the genes encoding the DNA-binding protein inhibitor ID1 and the phospholipase inhibitor uteroglobin were significantly down-regulated in the cancer samples. DNA microarray data for eight genes were confirmed quantitatively in five normal and five cancer tissues by real-time reverse transcriptase-polymerase chain reaction with a high correlation between the two methods. Laser capture microdissection of epithelial and stromal compartments from cancer and histological normal specimens followed by an amplification protocol for low levels of RNA (<0.1 microg) allowed us to distinguish between gene expression profiles characteristic of epithelial cells and those typical of stroma. Most of the genes identified in the nonmicrodissected tumor material as up-regulated were indeed overexpressed in cancerous epithelium rather than in the stromal compartment. We conclude that development of prostate cancer is associated with down-regulation as well as up-regulation of genes that show complex differential regulation in epithelia and stroma. Some of the gene expression alterations identified in this study may prove useful in the development of novel diagnostic and therapeutic strategies.
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PMID:Decrease and gain of gene expression are equally discriminatory markers for prostate carcinoma: a gene expression analysis on total and microdissected prostate tissue. 1205 20

Plants respond to environmental stress with a number of physiological and developmental changes. Water deficit is one of the major factors limiting plant growth and development and crop productivity. One response of plants to water deficit is accumulation of abscisic acid (ABA). An increase of ABA is responsible for the induction of many genes, presumably some of which contribute to drought tolerance. Analysis of gene expression in barley seedling shoots by differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) led to the isolation of several drought-, cold- and ABA-induced partial cDNA fragments. Here we extensively characterize one of these cDNAs, designated DD6. First, a larger cDNA was extended from DD6 by 5'-RACE (rapid amplification of cDNA ends). Subsequently, the corresponding gene was isolated by screening a barley BAC library, and the sequences of the transcribed and flanking regions were determined. The deduced amino acid sequence has similarity to an Arabidopsis hypothetical protein and to a human and mouse DNA-binding protein. The corresponding gene, named Srg6 (stress-responsive gene), was mapped in a barley doubled haploid mapping population to chromosome 7H between markers ABC455 and salfp76, within a region that previously has been linked to osmotic adaptation in barley and other grass genomes.
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PMID:Identification and mapping of a putative stress response regulator gene in barley. 1213 5

The WT1 gene encoding a zinc finger DNA-binding protein was identified as a tumor suppressor gene being responsible for Wilms' tumor. Recently, aberrant expression of WT1 gene and an inverse correlation between its expression levels and prognosis have been demonstrated in acute myeloid leukemia (AML), suggesting it is a novel tumor marker for leukemic blast cells. To explore whether the WT1 may be used as a marker for detection of minimal residual disease (MRD) in acute leukemia, we examined the sensitivity of the nested reverse transcriptase-polymerase chain reaction (RT-PCR) by using WT1 gene primers in comparison with tumor-specific marker genes, such as PML/RARalpha gene in NB4 cells or bcr-abl gene in K562 cells. In all samples, the integrity of RNA was confirmed by amplification of the c-abl gene as an internal control. The limits in amount of leukemic cells detected by two-step RT-PCR with primers for WT1 or tumor specific fusion gene were 10(-4) and 10(-5) in NB4 cells and 10(-3) to 10(-4) and higher than 10(-6) for K562 cells, respectively. None was WT1 positive in peripheral blood mononuclear cells (MNC) from 29 blood donors, while bone marrow MNCs from eight of 21 cases (38.1%) of nonmalignant patient WT1 gene expression were found. Our results suggested that monitoring of WT1 expression makes it possible to rapidly assess the effectiveness of treatment and follow up MRD in AML cases regardless of the presence or absence of tumor-specific markers.
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PMID:[Expression of the Wilms' Tumor Gene WT1 and Detection of Minimal Residual Disease in Acute Leukemia] 1257 85


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