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)

In a first part of this report, purification and characterization of several nucleased from lysates of Haemophilus influenzae are described. The enzymes bind to DNA with agarose columns and are removed by elution with phosphate buffer. Among the considered enzymes, the exonucleases 1 and 3, and endonuclease, a DNA polymerase and a restriction enzyme were recovered mixed by raising the phosphate concentration from 0.1 to 0.3 M, while the ATP-dependent DNAase recovered well purified, by raising the phosphate concentration to 0.45 M. After a rechromatography, on a second DNA with agarose column, of the peak of the ATP-dependent DNAase, the specific activity tested with 3H-labeled DNA was 125 units/mg of protein, representing a 300-fold purification of the original crude extract. In a second part, we have investigated the inactivation, at various pH, of transforming DNA of Haemophilus influenzae wild strain Rd with the different eluted fractions of the column, in order to determine the importance of contamination with other enzymatic activities, and also in order to confirm the nature of theisolated enzymes with a biological method. Finally, with enzymatic extracts of mutant strain Rd com minus 56, a strain which integrates shorter than normal pieces of DNA and which is suspected to possess and "activated specific endonuclease" able to recognize even small conformational modifications in paired structures, we tried to detect this activity on artificially constructed heteroduplex regions in DNA.
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PMID:Studies on deoxyribonucleases from Haemophilus influenzae on DNA agarose affinity chromatography. Two-step purification of ATP-dependent deoxyribonuclease. 23 41

DNA extracted from Dane particles has been characterized by gel electrophoresis and restriction enzyme cleavage with endonuclease R-HaeIII (from Hemophilus aegyptius). Dane particle DNA is proposed to be a double-stranded circular DNA approximately 3600 nucleotides in length containing a single-stranded gap of 600-2100 nucleotides. The endogenous DNA polymerase (DNA nucleotidyl-transferase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase; EC 2.7.7.7) reaction appears to repair this single-stranded gap.
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PMID:Genome of hepatitis B virus: restriction enzyme cleavage and structure of DNA extracted from Dane particles. 106 Jan 40

The double-stranded form of adeno-associated virus (AAV) DNA has about 20 sites sensitive to endonuclease R.Hae III from Haemophilus aegypitus; the fragments produced fall into about 13 size classes, 8 of which contain single fragments. The location of the Hae III-produced AAV fragments relative to the three EcoR1 fragments was determined. Using revised figures for the molecular weights of the Hae III cleavage products of phiX174 replicative form DNA, we calculated that AAV DNA contains about 4,000 nucleotides. After Hae III digestiion of duplex DNA terminally labeled with 32P using polynucleotide kinase, the majority of fragments containing a 5' 32P label were about 40 nucleotides in length, and fragments of similar size were generated from each end, suggesting that the Hae site closest to the end is within the terminal repetition. Two more-slowly-migrating cleavage products also bore 5' 32P end label. These three terminally labeled species were also generated from single-stranded AAV DNA by digestion with Hae III, and evidence that one may have a nonlinear ("rabbit-ear") structure is presented. The predominant 5' terminal base was identified as thymine for both the plus and minus strands of AAV. Single-stranded AAV molecules could not be efficiently covalently circularized by incubation with polynucleotide ligase or ligase plus T4 DNA polymerase.
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PMID:Multiple structures of adeno-associated virus DNA: analysis of terminally labeled molecules with endonuclease R-Hae III. 127 22

Haemophilus influenzae was found to produce a DNA polymerase that was similar to polymerase I of Escherichia coli. E. coli polA mutants were used as backgrounds for the selection of H. influenzae polA suppressor genes. Six different H. influenzae fragments were isolated that could suppress E. coli polA mutations. None of the suppressors appeared to encode the H. influenzae equivalent of the E. coli polA gene. One type of clone, represented by pGW41, caused a polymerase I activity to appear in a suppressed polA1 mutant. Plasmids from the pGW41 class contained two genes (pol-2 and pol-3) that were both required for polA suppression. Mutated nonsuppressing derivatives of the pGW41 class were used to create H. influenzae mutants that were deficient in polymerase I.
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PMID:Isolation of Haemophilus influenzae genes that suppress Escherichia coli polA mutations. 329 1

To determine the orientation of transcription of the E and L strands of DNA from simian virus 40 (SV40), we used linear DNA prepared by cleavage of superhelical viral DNA by endonuclease R.R(1) from Escherichia coli as a primer.template for DNA polymerase. The resulting molecules, which were labeled only at the 3' end of each DNA strand, were then cleaved with Hemophilus parainfluenzae endonuclease Hpa I. The ensuing four DNA fragments, whose locations on the viral genome are known, were separated by electrophoresis, denatured, and hybridized to asymmetric SV40 complementary RNA. From the pattern of hybridization of the fragments containing the labeled 3' ends, we conclude that transcription of SV40 proceeds in a clockwise direction on the L strand and in a counterclockwise direction on the E strand as drawn on the conventional SV40 map. To map the "early" and "late" regions of the viral genome, we extracted RNA from lytically infected cells and hybridized it to the separated strands of the four fragments of (32)P-labeled SV40 DNA. Early after infection, RNA complementary to part of the E strand of the contiguous fragments A and C was detected. Late polysomal RNA was complementary to part of the L strand sequences of fragments A and C and to the total L strand sequence of fragments B and D.
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PMID:Transcription of simian virus 40. 3. Mapping of "early" and "late" species of RNA. 412 25

Linear phiX174 single-stranded DNA can be isolated from phiX phage particles produced under various conditions. About half of the linear strands have a dGMP residue at the 5' end, the remaining have roughly comparable amounts of dCMP, dTMP, and dAMP. The linear strands can be converted to covalently closed circular molecules by polynucleotide ligase, but only after they have been incubated with T4 DNA polymerase and deoxynucleoside triphosphates. Experiments with endonuclease R, the restriction enzyme from Haemophilus influenzae, indicated that the nucleotides incorporated into the DNA during this reaction were found predominantly in a limited region of the genome. The results suggest that the normal intermediate in single-stranded phiX174 DNA synthesis may be a single-stranded linear molecule which is shorter than unit length and is intrinsically capable of circularization.
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PMID:Mechanism of replication of single-stranded PhiX174 DNA. VII. Circularization of the progeny viral strand. 459 Oct 49

In this study, we have developed a chemically sensitive and specific polymerase chain reaction (PCR) assay to detect the presence of Streptococcus pneumoniae genomic DNA. The target DNA sequence was a 322-base pair segment of the S. pneumoniae DNA polymerase I gene (pol I). PCR products of pure cultures of a set of pneumococcal serotypes commonly associated with human infection could be amplified in water and in blood cultures of clinical isolates containing S. pneumoniae. We were able to detect 2 fg of purified S. pneumoniae DNA. There were no false-positive reactions when the assay was performed on samples containing the following clinically encountered bacteria: Haemophilus influenzae type B, Neisseria meningitidis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas spp. nontypeable H. influenzae, Staphylococcus aureus, coagulase-negative staphylococci, and Streptococcus pyogenes. The addition of EDTA and citrate-anticoagulated whole blood to the PCR reaction mixture inhibited the PCR assay, whereas the addition of lithium heparin, sodium heparin, and sodium polyanetholesulfonate-anticoagulated whole blood to PCR reaction mixture did not interfere with the ability to detect the presence of S. pneumoniae DNA.
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PMID:Development of a polymerase chain reaction assay to detect the presence of Streptococcus pneumoniae DNA. 770 31

Synonymous codon usage patterns of bacteriophage and host genomes were compared. Two indexes, G + C base composition of a gene (fgc) and fraction of translationally optimal codons of the gene (fop), were used in the comparison. Synonymous codon usage data of all the coding sequences on a genome are represented as a cloud of points in the plane of fop vs. fgc. The Escherichia coli coding sequences appear to exhibit two phases, "rising" and "flat" phases. Genes that are essential for survival and are thought to be native are located in the flat phase, while foreign-type genes from prophages and transposons are found in the rising phase with a slope of nearly unity in the fgc vs. fop plot. Synonymous codon distribution patterns of genes from temperate phages P4, P2, N15 and lambda are similar to the pattern of E. coli rising phase genes. In contrast, genes from the virulent phage T7 or T4, for which a phage-encoded DNA polymerase is identified, fall in a linear curve with a slope of nearly zero in the fop vs. fgc plane. These results may suggest that the G + C contents for T7, T4 and E. coli flat phase genes are subject to the directional mutation pressure and are determined by the DNA polymerase used in the replication. There is significant variation in the fop values of the phage genes, suggesting an adjustment to gene expression level. Similar analyses of codon distribution patterns were carried out for Haemophilus influenzae, Bacillus subtilis, Mycobacterium tuberculosis and their phages with complete genomic sequences available.
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PMID:Comparison of synonymous codon distribution patterns of bacteriophage and host genomes. 1004 80

The complete 4312-bp sequence of the pEC156 plasmid from Escherichia coli E1585-68, which carries genes encoding the EcoVIII restriction-modification (R-M) system, an isoschizomer of HindIII from Haemophilus influenzae, has been determined. Two clustered and convergently oriented open reading frames, large enough to encode genes of the EcoVIII R-M system, were found. The transcriptional start points were mapped by the primer extension method. The relative molecular masses of the EcoVIII endonuclease and EcoVIII methyltransferase deduced from the nucleotide sequence are 35,554 and 33,910, respectively. Nucleotide sequence analysis of pEC156 suggests that this plasmid is a ColE1-type replicon. It consists of an origin of replication and two untranslated genes encoding RNA I and RNA II, both involved in the regulation of plasmid DNA replication. The replication region also contains the gene encoding a 64-aa Rom-like protein. Inactivation of the putative rom gene by insertion of a kanamycin-resistance cassette resulted in 4.5-fold increase in pEC156-derived plasmid copy number in E. coli cells. All of these elements (RNA I, RNA II, and rom) reveal a high level of similarity to ColE1 homologs. The replication of all ColE1-type plasmids is dependent on the activity of E. coli DNA polymerase I. It was shown that a pEC156 derivative (pIB8) carrying an antibiotic resistance gene indeed failed to replicate in an E. coli polA12(ts) mutant at 43 degrees C, and its copy number was reduced in the E. coli pcnB80 mutant. These results prove that pEC156 is a ColE1-type replicon.
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PMID:Characterization of pEC156, a ColE1-type plasmid from Escherichia coli E1585-68 that carries genes of the EcoVIII restriction-modification system. 1159 Nov 38

We present here computer generated model of N-terminal fragment, amino acids (aa) 36-245, of a Plasmodium vivax heat shock metalloprotease called PVHSP28, whose gene was cloned and characterised earlier. The fragment showed homology with HSPs from many organisms, including Escherichia coli and Haemophilus influenzae. PVHSP28 had the signature sequence 'HEXXH' and 'EXXXD' of Zinc metalloproteases. Being the first malarial HSP possessing metalloprotease activity, PVHSP28 is an ideal target for the design of new anti-malarial drugs. However, except for a small region (aa 62-132) which had 24.6% sequence similarity with 1TAQ (a DNA polymerase), it did not show sequence similarity with any published structures in protein data bank. Hence it could not be modelled using any automated modeling programs. We modelled 36-245 aa of PVHSP28 using predicted secondary structure as well as experimentally determined and predicted properties of the protein on the basis of its amino acid sequence, using various Internet tools and in-house package MODEL. The model was energy minimised using Sander's module of AMBER 5.0, working on a Silicon Graphics machine, with all atom force field.
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PMID:Computer modeling of small heat-shock metalloprotease of the human malaria parasite Plasmodium vivax. 1169 26


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