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)

Sequential reverse transcriptase, DNA polymerase, and S1 nuclease reactions can be employed to synthesize double-stranded DNA representing messenger RNA. Using reverse transcriptase products made from partially purified lysozyme, ovomucoid, and ovalbumin messengers from hen oviduct, we have characterized the Escherichia coli DNA polymerase I reaction. We have optimized for a high yield of full length second strands under conditions which require only a small amount of mRNA. The effects of several parameters (time, enzyme levels, salt concentration, monovalent cation, and temperature) on the length of products synthesized by DNA polymerase I have been investigated. Each has a significant influence on the proportion of products which are full length. Under our conditions the three reactions are efficient in synthesizing full length duplex DNA from partially purified mRNA fractions or from total poly(A)-containing RNA.
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PMID:Synthesis of double-stranded DNA complementary to lysozyme, ovomucoid, and ovalbumin mRNAs. Optimization for full length second strand synthesis by Escherichia coli DNA polymerase I. 7 87

Bacillus subtilis W23 was infected with a clear-plaque variant of SP-10 phage, namely, SP-10c. Exogenous thymidine was not incorporated into phage DNA (even in the presence of deoxyadenosine), nor was there any transfer of thymidine nucleotides from bacterial to viral DNA. The lytic program was unaffected by concentrations of 5-fluorodeoxyuridine sufficient to reduce bacterial DNA synthesis by greater than 95%. Although these data are consistent with the interpretation that thymidine nucleotides are excluded from phage DNA, formic acid digests of SP-10c DNA contained what appeared to be the four conventional bases; however, adenine and thymine were not recovered in equimolar yields. DNA-RNA hybridization and hybridization competition experiments were done. Synthesis of host RNA started to wane moments postinfection and stopped completely by 36 min. SP-10c coded for discrete classes of early and late RNA. The possibility of discrete subclasses of early RNA exists. Replication of the bacterial genome appeared to terminate 12 min postinfection. Degradation of the host DNA to acid-soluble material started at 36 min and, by the end of the latent period, greater than 90% of the host chromosome was hydrolyzed. Four apparent phage-coded enzymes have been identified. A di- and triphosphatase degraded dUTP, dUDP, dTTP, and dTDP (and, to a lesser extent, dCDP and d CTP) to the corresponding monophosphates; the enzyme had no apparent activity on dATP and dGTP. SP10c also coded for a DNA-dependent DNA polymerase, lysozyme, and a nuclease that degrades native bacterial DNA. Judging from the dependence of enzyme synthesis on the time of addition of rifampin (an inhibitor of the initiation of RNA synthesis), messengers for the di- and triphosphatase, as well as the nuclease, are transcribed from promoters that start to function 6 min postinfection. Promoters for polymerase and lysozyme did not become functional until 8 and 16 min postinfection, respectively.
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PMID:SP-10 bacteriophage-specific nucleic acid and enzyme synthesis in Bacillus subtilis W23. 13 89

The effect of several enzymes of the DNA metabolism of Escherichia coli on the biological activity of native and single-stranded T7 DNA was studied by transfection of lysozyme-EDTA spheroplasts prepared from various E. coli mutants. It is shown that the presence of the recBC DNase in the recipient cells decreases the infectivity of native and denatured DNA by about 100- and 10-fold, respectively. Lack of exonuclease I did not stimulate transfection by single-stranded DNA. Separated light (l) and heavy (r) strands of T7 DNA are fully infective, with a linear dependence on DNA concentrations, whereas heat-denatured DNA shows a two-hit kinetics. Single-stranded DNA was observed to depend on a functional DNA polymerase III for infectivity in polAB cells, whereas transfection with native T7 DNA was independent of the host DNA polymerases. The results are discussed with respect to the mode of T7 DNA replication.
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PMID:In vivo effects of recBC DNase, exonuclease I, and DNA polymerases of Escherichia coli on the infectivity of native and single-stranded DNA of bacteriophage T7. 32 5

Initiation of new DNA synthesis was observed in B. subtilis cells upon gamma-ray irradiation followed by toluene treatment and incubation in the presence of the four deoxynucleotide triphosphates and Mg2+. This DNA synthesis took place in the absence of ATP and was refractory to 6-(p-hydroxyphenylazo)-uracil which is a specific inhibitor for the type III polymerase of Bacillus subtilis. This repair-type DNA synthesis was greatly reduced in mutant cells deficient in DNA polymerase I. Restoration of transforming activity of cellular DNA was found to occur in parellel with the above repair type DNA synthesis. A protein factor which enhances the priming activity of gamma-irradiated DNA for DNA polymerase I was detected in DNA-free extracts prepared from B. subtilis cells by means of lysis with a buffer containing lysozyme, Brij-58 and EDTA.
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PMID:Studies on DNA repair in Bacillus subtilis. I. A cellular factor acting on gamma-irradiated DNA and promoting its priming activity for DNA polymerase I. 80 54

Plasmid DNA released from bacteria by boiling in the presence of lysozyme and Triton x-100 and without further purification can be sequenced by the dideoxy method using T7 DNA polymerase, when conditions during alkali denaturation and subsequent ethanol precipitation are adjusted to remove contaminants. The samples remain in the same microcentrifuge tubes from the harvesting of the bacteria until the splitting of the sample into four aliquots for the termination reactions. Less background label is observed with end-labelled primers (radioactivity or fluorescence), but even when radioactive nucleotides are incorporated during the sequencing reactions, 250 bases or more can be read from template prepared from 1.5 ml bacterial culture. The DNA can also be cut by restriction enzymes; the purification procedure described thus provides the rapid preparation of plasmids for a variety of purposes.
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PMID:Rapid and simple preparation of plasmids suitable for dideoxy DNA sequencing and other purposes. 180 39

In our attempts to establish a cell-free DNA replication system for the yeast Saccharomyces cerevisiae, we have observed that recombinant DNA plasmids purified from Escherichia coli by a common procedure (lysozyme-detergent lysis and equilibrium banding in cesium chloride ethidium bromide gradients) often serve as templates for DNA synthesis by elongation enzymes. The templates could be elongated equally well by enzymes present in the yeast cell-free extracts, by the large proteolytic fragment of E. coli DNA polymerase I or by T4 DNA polymerase. The template activity of the purified plasmids was dependent on the presence of heterologous DNA segments in the bacterial vectors. The template activity could be diminished by treatment with alkali. We propose that the ability of recombinant plasmids isolated from bacterial hosts to serve as elongation templates may lead to erroneous conclusions when these plasmids are used as templates for in vitro replication or transcription reactions.
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PMID:DNA synthesis in yeast cell-free extracts dependent on recombinant DNA plasmids purified from Escherichia coli. 388 51

Previous work from this laboratory has shown that the cytosine-containing T4 deoxyribonucleic acid (DNA) made by deoxycytidine triphosphatase (dCTPase) amber mutants is extensively degraded, and that nucleases controlled by genes 46 and 47 participate in this process. In this paper, we examine other consequences of a defective dCTPase. Included are studies of DNA synthesis and phage production, and of the control of both early and late protein synthesis after infection of Escherichia coli B with various T4 mutants defective in genes 56 (dCTPase), 42 (dCMP hydroxymethylase), 1 (deoxynucleotide kinase), 43 (DNA polymerase), 30 (polynucleotide ligase), 46 and 47 (DNA breakdown) or e(lysozyme). By varying the temperature of infection with a temperature-sensitive dCTPase mutant, we have been able to control intracellular dCTPase activity, and thus vary the cytosine content of the phage DNA. We have produced and characterized viable T4 phage in which cytosine replaces 20% of the 5-hydroxymethylcytosine (HMC) in the DNA. We present evidence which suggests that intact, cytosine-containing T4 DNA is much less efficient than is normal T4 DNA in directing the synthesis of tail-fiber antigen. Lysozyme production is much less affected by progressively decreasing dCTPase activity; however, complete substitution of cytosine is correlated with a depression of lysozyme synthesis greater than expected from the defective synthesis of DNA. Low but significant lysozyme synthesis is observed late after infection of E. coli B with T4 amber mutants defective in a number of genes controlling DNA synthesis. The "20% cytosine" T4 phage, once produced, can initiate an apparently normal infection at permissive temperatures; the synthesis of early enzymes, DNA, and phage does not appear to be impaired. Two roles for HMC in T4 DNA have been indicated previously: (i) involvement in host-controlled restriction of the phage, in which glucosylation of the hydroxymethyl group plays a crucial role (16, 29, 53, 58), and (ii) protection of vegetative DNA against phage-controlled nucleases, a protection not dependent on glucosylation (41, 66, 67). A third role is suggested by our present results: transcription of at least some late genes can occur only from HMC-containing DNA and not from cytosine-containing DNA.
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PMID:Biological effects of substituting cytosine for 5-hydroxymethylcytosine in the deoxyribonucleic acid of bacteriophage T4. 430 78

During nonpermissive infection by a T7 amber mutant in gene 1 (phage RNA polymerase-deficient), synthesis of the products of the phage genes 3 (endonuclease), 3, 5 (lysozyme), 5 (DNA polymerase), and 17 (serum blocking power) was shown to occur at about half the rate as during wild-type infection. This relatively high rate of expression of "late" genes (transcribed normally by the phage RNA polymerase) seems to be a general feature of all T7 mutants in gene 1 from our collection. In contrast, T3 gene 1 mutants and a T7 gene 1 mutant from another collection showed late protein synthesis at very reduced rates. Synthesis of the gene 3 endonuclease by T7 gene 1 mutants was very sensitive to the addition of rifampin 2 min after infection, conditions under which there was very little inhibition during wild-type infection. This supports the notion that late gene expression during nonpermissive infection by gene 1 mutants is dependent on the transcription of the T7 genome by the host RNA polymerase. In contrast to T3 gene 1 mutants, the T7 gene 1 mutants of our collection directed the synthesis of phage DNA during nonpermissive infection. This DNA accumulated as a material sedimenting faster than mature T7 DNA.
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PMID:Synthesis of bacteriophage-coded gene products during infection of Escherichia coli with amber mutants of T3 and T7 defective in gene 1. 457 63

Synthesis of many T7 proteins is prevented in F' episome-containing cells. In order to quantitate the degree of inhibition, we measured the activity of several T7 proteins in extracts prepared from T7-infected F(-) and F' cells and cells containing F factors mutant in phage inhibition [F'(PIF(-)2A) and F'(PIF(-)2A,2B)]. In addition, we were able to assign specific T7 proteins to the three translational units previously defined by polyacrylamide gel analysis of T7 proteins made in F(-) and episome-containing cells. After T7 infection, the presence of the wild-type F' (PIF(+)) episome led to greater than 90% inhibition of T7 DNA polymerase (product of gene 5), T7 lysozyme (gene 3.5), and gene 10 capsid protein synthesis. Nearly normal amounts of T7 RNA polymerase (gene 1) were made in these cells. T7 infection of cells containing the mutant F' (PIF(-)2A) episome led to normal synthesis of T7 RNA polymerase and T7 DNA polymerase; T7 lysozyme was synthesized at 30% of the maximal level in these cells; T7 gene 10 capsid protein synthesis was inhibited by 90%, and T7 DNA synthesis was arrested in these cells. T7 infection of cells containing the mutant F' (PIF(-)2A,2B) episome led to synthesis of normal levels of the enzymes assayed.
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PMID:T7 protein synthesis in F' episome-containing cells: assignment of specific proteins to three translational groups. 458 55

These data demonstrate that actinomycin D inhibits only 75-80% of DNA synthesis in cells of E. coli treated by lysozyme and ethyl enediaminetetraacetate. The residual synthesis is not the result of untemplated polymerization of dNTP. The DNA synthesis in spheroplasts does not correlate with replication of chromosomal DNA of E. coli catalyzed by DNA polymerase III sensitive to sulfhydryl blocking agents. N-ethylmaleimide does not inhibit this synthesis. No ATP stimulation of DNA synthesis is observed. The enzyme(s) responsible for DNA synthesis on endogenous template is (are) concentrated in interphase (D-fraction) as revealed by high speed centrifugation of spheroplasts lysate and they are absent in the chromosomal DNA fraction. dTTP 4-(N-2-chloroethyl-N-mehylamino) benzylamide suppresses completely the insensitive to actinomycin D action DNA synthesis and practically does not act on the sensitive one. It is suggested, that the DNA synthesis stable to the action of the antibiotic is catalyzed by RNA dependent DNA polymerase.
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PMID:[Location of RNA-dependent DNA polymerase within the endogenous template complex and discrimination of RNA- and DNA-dependent synthesis of DNA in Escherichia coli cells by alkylating dTTP gamma-amide]. 619 21


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