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:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A recombinant plasmid containing chick pro-alpha2 collagen gene sequences has been constructed and cloned in Escherichia coli. Using partially purified collagen mRNA as template, we synthesized double-stranded DNA by the successive action of reverse transcriptase (RNA-directed DNA nucleotidyltransferase) from avian myeloblastosis virus and the Klenow A fragment of E. coli DNA polymerase I. From this complex mixture of double-stranded DNAs, a specific 200-base-pair restriction fragment was generated by cleavage with the restriction endonucleases BamHI and EcoRI. These enzymes also make unique cuts in the plasmid vector pBR322. The restriction fragment was inserted into pBR322 via these BamHI and EcoRI sites and cloned in E. coli chi1776. The cloned recombinant plasmid was shown to contain pro-alpha2 collagen DNA by its specific hybridization to chick pro-alpha2 collagen mRNA, as assayed in an in vitro translation system. Thus, a clone containing pro-alpha2 collagen DNA was constructed without first obtaining highly purified collagen mRNA.
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PMID:Construction of a recombinant bacterial plasmid containing a chick pro-alpha2 collagen gene sequence. 36 5

Following pneumonectomy in animals, the contralateral lung increases in volume, weight, collagen content, protein, and cell number, reaching levels approximate to those of both lungs of control animals. The volume and weight response in quicker and more complete in young animals compared to old animals. The increase in the amount of DNA was found to be greater in young rats compared to old ones. However, little is known about the effects of pneumonectomy in immature animals, in which combined effects of normal and the compensatory lung growth may be expected. The present studies were aimed at elucidating early changes in terms of morphology and biochemistry in the contralateral lung following pneumonectomy in premature rats. Male Sprague-Dawley rats (2 week-old) were subdivided into 3 groups. Group P, underwent left pneumonectomy, group S was sham operated, which group C was matched by age, sex, and weight with group P. Morphological studies consisted of light microscopic morphometry and immunohistochemistry using anti-bromodeoxyuridine (BrdU) were performed. Biochemical studies included measurement of DNA polymerase activity, DNA and RNA content, collagen and elastin content. The wet lung weight in group P after one week reached approximately the same as that of bilateral lungs of groups S and C. The fixed lung volume of group P reached that of group S or group C at three weeks. The activity of DNA polymerase and BrdU positive alveoli were increased only during the early period following pneumonectomy. DNA content in group P reached the same range as group S and C at 4 weeks, suggesting the occurrence of cellular hyperplasia.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Early changes of postpneumonectomy lung growth in premature rats]. 171 Mar 1

The aggregation factor (AF) of the marine sponge Geodia cydonium recognizes the aggregation receptor (AR) which is inserted in the plasma membrane, under formation of species-specific aggregates. The specific cell-binding fragment of the AF was used to investigate for the first time the phosphoinositide metabolism in a lower avertebrate system. We found that after binding of the cell-binding fragment to the aggregation receptor a strong and rapid stimulation of the phosphate incorporation into phosphatidylinositol occurs followed by an increased turnover of phosphoinositides in the Geodia cells. The consequences of an increased degradation of phosphatidylinositol 4,5-bisphosphate into the two second messengers inositol-1,4,5-trisphosphate and diacylglycerol are 2-fold. First, after the addition of the extracellular stimulus the cytosolic Ca2+ concentration rises, resulting in a rapid increased Ca2+ efflux rate. The functional consequence of the increase of the extracellular Ca2+ level is an initiation of the aggregate formation that is mediated by the collagen assembly factor (= primary aggregation factor). Second, some experimental evidences are presented, showing that the other second messenger formed, diacylglycerol, causes a translocation of protein kinase C within the cell. Incubation of Geodia cells with the cell-binding fragment of the AF, or with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, resulted within 5 min after treatment in a 70% decrease in protein kinase C activity in the cytosolic fraction and in a 700% increase in enzyme activity in the membrane fraction. It is proposed that by membrane association protein kinase C becomes activated. As a result of this event a series of cellular proteins are phosphorylated, a process which ultimately leads to an unusually strong induction of DNA polymerase alpha activity. From these data we conclude that inositol trisphosphate and protein kinase C also play a fundamental role in cellular signal transduction in lower eukaryotes.
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PMID:Role of the aggregation factor in the regulation of phosphoinositide metabolism in sponges. Possible consequences on calcium efflux and on mitogenesis. 303 73

In the absence of a growth factor or an appropriate extracellular matrix (ECM), cells are arrested in the G0/G1 phase. In this report, we demonstrate the evidence that TNF-alpha induced DNA synthesis of primary mouse hepatocytes in vitro by activating two distinct pathways. TNF-alpha induced drastic spreading of hepatocytes on hydrophobic plastic, while the adhesion was not influenced. The effect was time and dose dependent. The cell spreading was accompanied by the phosphorylation of paxillin, indicating the stimulation of focal adhesion molecules. TNF-alpha-induced spreading of hepatocytes was not transient, and kinetic analysis and morphologic observation suggest that the effect was different from epidermal growth factor- or hepatocyte growth factor-induced transient hepatocyte spreading. TNF-alpha-induced hepatocyte spreading was blocked by cytochalasin D, Arg-Gly-Asp peptides, cycloheximide, or anti-integrin beta1 Ab. Results of competitive PCR for ECM proteins demonstrated that TNF-alpha increased the expression of laminin alpha3 and gamma1 chains in hepatocytes. These data suggested that TNF-alpha induced cell anchorage for hepatocytes by up-regulating ECM production. More importantly, TNF-alpha, but neither epidermal growth factor nor hepatocyte growth factor, induced DNA synthesis following the spreading in primary hepatocytes on hydrophobic plastic, while mere cell spreading on collagen did not induce DNA synthesis. The DNA synthesis was blocked by the inhibition of either cell spreading or DNA polymerase, demonstrating that TNF-alpha induced DNA synthesis in primary hepatocytes by activating two distinct pathways, i.e., forming the scaffold and inducing growth signals. Taken together, TNF-alpha bifunctionally regulates the proliferation of primary hepatocytes, serving as both an ECM inducer and a growth factor.
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PMID:TNF-alpha bifunctionally induces proliferation in primary hepatocytes: role of cell anchorage and spreading. 936 9

The tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is present in tobacco smoke and is hepatocarcinogenic in rats. Its bioactivation in rat hepatocytes leads to methylation and pyridyloxobutylation of DNA. Rat hepatocytes were cultured in serum-free William medium E on collagen-coated dishes. We demonstrated that some enzymes of the base and/or excision-repair pathways were involved in repair of NNK-induced DNA damage, measured by [methyl-3H] thymidine incorporation. Unscheduled DNA synthesis (UDS) induced by N-methyl-N-nitrosourea (MNU), NNK, N'-nitrosonornicotine (NNN) and 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc) increased 2.9-, 2.8-, 1.5- and 3.5-fold, respectively, suggesting that methylated and/or pyridyloxobutylated-DNA by these four nitroso compounds is repaired by the excision pathway. Moreover, levels of NNK-induced UDS were dose (1-3 mM) and time (1-18 h) dependent. Enzymes involved in the excision repair pathways were selectively inhibited. Inhibitors of DNA topoisomerase I (camptothecin) and topoisomerase II (etoposide, nalidixic acid) did not decrease the induction of UDS, suggesting that topoisomerases are not involved in the repair of NNK-induced damage. While aphidicolin and arabinocytidine (DNA polymerase alpha, delta, epsilon inhibitors) totally inhibited NNK- and NNKOAc-induced UDS, dideoxythymidine (DNA polymerase beta inhibitor) inhibited NNK- and NNKOAc-induced UDS by 40 and 33%, respectively. We conclude that DNA polymerase alpha, delta or epsilon and to a lesser degree polymerase beta are involved in the repair of pyridyloxobutylated DNA. Previous studies showed that inhibition of poly(ADP-ribosyl) polymerase (PARP) by 3-aminobenzamide (3-ab) facilitated DNA ligation. Our results demonstrate that 3-ab increased NNK-induced UDS, but does not affect NNKOAc-induced UDS. These observations suggest that the ligation step is rate limiting in the repair of methylated DNA but not of pyridyloxobutylated DNA.
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PMID:Modulation of DNA repair by various inhibitors of DNA synthesis following 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induced DNA damage. 956 22

Arctic foxes (Alopex lagopus) were collected from Greenland and Svalbard (N = 319). Twenty-four were infected with Trichinella (7.5%). Molecular analysis (random-amplified polymorphic DNA polymerase chain reaction) confirmed that all animals were infected with Trichinella nativa. Motile larvae were found in muscle tissue from all foxes after carcasses had been frozen for 1 yr at -18 C. Infective larvae were found in 2 foxes after a total of 4 yr storage at -18 C, which is longer than any previous observations. Morphological examination of the cysts showed large nurse cells and significant deposition of collagen and connective tissue. It is suggested that, within the geographical distribution of T. nativa, the more freeze-resistant isolates are found at higher latitudes.
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PMID:Freeze tolerance, morphology, and RAPD-PCR identification of Trichinella nativa in naturally infected arctic foxes. 1020 84

It is essential to identify specific food components that inhibit PCR in order to increase the sensitivity of the PCR method for rapid detection of pathogens contaminating a food. We found that collagen, a major component of several foods, inhibited PCR. The inhibitory action of collagen on PCR could be partially reversed by adjusting the concentration of magnesium ion in the reaction mixture and by the use of various DNA extraction methods to remove the collagen from the DNA. Also, the source of thermostable DNA polymerase was affected by the presence of collagen. These results suggest the need to optimize the extraction and assay conditions for rapid detection of enterotoxigenic Clostridium perfringens by PCR with respect to the kind of food being analyzed.
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PMID:Inhibitory effects of collagen on the PCR for detection of Clostridium perfringens. 1069 95

Reinsertion of autogenous nucleus pulposus, an innovative method to delay further disc degeneration, has been proved with an experimental animal model. This study examined whether coculture of nucleus pulposus cells with annulus fibrosus cells (a) activates annulus fibrosus cells and (b) retards disc degeneration when reinserted into the disc in a rabbit model of disc degeneration. Coculture of the two cell types stimulated proliferation of each, as indicated by increased DNA synthesis measured by increases in DNA polymerase alpha expression and uptake of 5-bromo-2'deoxy-uridine assessed by an enzyme-linked immunosorbent assay. In a model of disc degeneration in rabbits, reinsertion of activated nucleus pulposus cells delayed the formation of clusters of chondrocyte-like cells, the destruction of disc architecture, and the elaboration of type-II collagen as measured immunohistochemically compared with no treatment. The direct reinsertion of activated nucleus pulposus cells into the disc offers a promising line of investigation for delaying intervertebral disc degeneration, although these results obtained with notochordal cells may not necessarily apply when mature central nucleus pulposus cells are used.
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PMID:Reinsertion of stimulated nucleus pulposus cells retards intervertebral disc degeneration: an in vitro and in vivo experimental study. 1119 61

Mycoplasma virus P1 is one of only four viruses isolated from the genus Mycoplasma. The host for P1, Mycoplasma pulmonis, possesses complex, phase-variable restriction and modification enzymes and the Vsa family of phase-variable surface proteins. The ability of P1 virus to infect host cells is influenced by these phase-variable systems, rendering P1 a valuable tool for assessing host properties. The double-stranded P1 DNA genome was sequenced (11,660 bp) and 11 ORFs were identified. The predicted P1 DNA polymerase is similar to that of phages that are known to have terminal protein (TP) attached to the 5' end of their genome, consistent with previous studies indicating that P1 DNA has covalently attached TP. Most of the other predicted P1 proteins have little sequence similarity to known proteins, and P1 virus is unrelated to the other mycoplasma virus, MAV1, for which the genome sequence is known. One of the predicted P1 proteins, the ORF 8 gene product, contains a repetitive collagen-like motif characteristic of some bacteriophage tail fiber proteins and is a candidate for interacting with the Vsa proteins.
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PMID:Complete nucleotide sequence of the mycoplasma virus P1 genome. 1132 26

White spot syndrome virus (WSSV) is at present a major scourge to worldwide shrimp cultivation. We have determined the entire sequence of the double-stranded, circular DNA genome of WSSV, which contains 292,967 nucleotides encompassing 184 major open reading frames (ORFs). Only 6% of the WSSV ORFs have putative homologues in databases, mainly representing genes encoding enzymes for nucleotide metabolism, DNA replication, and protein modification. The remaining ORFs are mostly unassigned, except for five, which encode structural virion proteins. Unique features of WSSV are the presence of a very long ORF of 18,234 nucleotides, with unknown function, a collagen-like ORF, and nine regions, dispersed along the genome, each containing a variable number of 250-bp tandem repeats. The collective information on WSSV and the phylogenetic analysis on the viral DNA polymerase suggest that WSSV differs profoundly from all presently known viruses and that it is a representative of a new virus family.
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PMID:The white spot syndrome virus DNA genome sequence. 1144 54


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