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)

Japanese quail oviduct total cDNA was synthesized from total mRNA by the classical method, using AMV reverse transcriptase, the Klenow fragment of DNA polymerase I and S1 nuclease. The results of the synthesis of total ss-cDNA partially differed from those published for the synthesis of chicken total ss-cDNA. The presumed causes of the differences in the complete reverse transcription of mRNAcon and incomplete reverse transcription of mRNAlys and a large part of mRNAov in our case are discussed. An atypical strategy was used for cloning full-length cDNAov. The total cDNA was dC-tailed, then fractionated by analytical agarose electrophoresis and 4 cDNA fractions of different lengths were isolated from the gel using DEAE cellulose membranes. A cDNA fraction about 1500-2500 bp long containing full-length cDNAov was annealed with dG-tailed PstI-linearized plasmid pBR322 and cloned into competent E. coli DHl cells. Seventy-two clones were screened for the presence of full-length cDNAov, initially by insert size and then by means of hybrid-arrested translation. Four clones containing 1900-1980 bp cDNAov were obtained. The cDNA ends in one of these clones were sequenced. Comparison of these sequences with those of chicken mRNAov indicated that almost full-length cDNAov's had been cloned. They lacked a small number of nucleotides at their 5' ends, which had probably been split off during the degradation of the hairpin loop by S1 nuclease. A sequence of 134 bases from the 5' end of mRNAov is presented and compared with the known sequence of chicken mRNAov. The advantages of the cloning strategy employed, in particular, its cloning efficiency and the possibility of simultaneously identifying clones of also other oviduct cDNA species (in this work: cDNAY and, tentatively, cDNAcon), are discussed.
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PMID:Cloning of Japanese quail ovalbumin cDNA in E. coli. 265 90

We have mapped the termini and determined the relative abundance and ribosome density of the major cytoplasmic transcript of the DNA polymerase (pol) gene of herpes simplex virus type 1. Nuclease protection and primer extension analyses located the 5' end of the major pol transcript at two closely spaced sites 51 and 57 nucleotides to the left of a BamHI site at map position 0.413. S1-sensitive sites corresponding to additional minor transcripts were found to map further upstream within a palindromic sequence that contains a viral replication origin. The major 3' end was found to map 90 nucleotides upstream of a KpnI site at map position 0.439. Quantitative S1 nuclease assays revealed that pol transcripts were nearly as abundant as transcripts encoded by the viral thymidine kinase gene. However, relatively few pol transcripts were found on large polysomes at 5.5 h after infection, when pol transcripts were most abundant. This was in marked contrast to the polyribosome distribution of transcripts from the thymidine kinase gene and the major DNA-binding protein gene. These results and sequence features of the pol transcript suggest that pol expression is regulated, in part, at the level of translation.
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PMID:Analysis of the transcript of the herpes simplex virus DNA polymerase gene provides evidence that polymerase expression is inefficient at the level of translation. 283 6

The influence of a C----G transversion at position 1 of the 30-base pair replication origin of bacteriophage phi X174 replicative form I DNA (phi X RFI) was examined in the RF----single-stranded circular DNA replication pathway catalyzed by the combined action of the purified phi X A protein, the Escherichia coli DNA polymerase III holoenzyme, rep helicase, and single-stranded DNA binding protein (Eisenberg, S., Scott, J.F., and Kornberg, A. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 1594-1597; Reinberg, D., Zipursky, S.L., and Hurwitz, J. (1981) J. Biol. Chem. 256, 13143-13151). RFI DNA containing this transversion was cleaved to RFII by the phi X A protein as effectively as DNA containing the wild-type origin. The altered duplex DNA, however, supported replication at a slower rate (3- to 4-fold) than the wild-type DNA due to a defect in the termination and reinitiation reactions catalyzed by the phi X A protein. This defect resulted in the accumulation of DNA products containing long single strands covalently joined to the mutant DNA. These single strands were susceptible to nuclease S1 and exonuclease VII attack. The defect in the template DNA containing C----G transversion was not corrected when this mutant origin was placed on the same strand with a wild-type origin. This double-origin DNA was also replicated poorly and led to the accumulation of large products, in contrast to the products formed with RFI DNA containing two wild-type 30-base pair replication origins on the same strand.
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PMID:Studies on the role of the phi X174 gene A protein in phi X viral strand synthesis. I. Replication of DNA containing an alteration in position 1 of the 30-nucleotide icosahedral bacteriophage origin. 297 11

We have examined a cDNA displacement synthesis procedure in which the extent of precursor incorporation and the unusual kinetics of displacement synthesis suggest a unique replicative form of DNA and the occurrence of multiple rounds of displacement synthesis, leading to amplification of mRNA sequences. Globin double-stranded DNA containing a hairpin loop was extended by the addition of a homopolymer to the 3' end. This was followed by displacement synthesis with the Klenow fragment of DNA polymerase I that was primed by an oligonucleotide hybridized to the homopolymer. Thus, the hairpin cDNA was copied to form an open duplex with an inverted repetition of globin sequences. These molecules can then serve as templates for additional synthesis which would be primed from oligomers bound the homopolymer. Globin cDNA sequences appear to be amplified 10-fold or more by this procedure. Globin cDNA obtained by displacement synthesis was similar in size to the original template. However, displaced molecules associate to the extent that they are not readily resolved by electrophoresis or sedimentation under nondenaturing conditions. Restriction endonuclease digests of 32P-labeled displaced strands gave fragment patterns similar to rabbit globin cDNA hairpin molecules. S1 nuclease studies demonstrated that displaced complexes and replication intermediates are partially single stranded, which might account for their aggregation properties.
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PMID:Displacement synthesis of globin complementary DNA: evidence for sequence amplification. 298 27

An easy and quick method to synthesize large cDNA molecules and to clone them with very high efficiency in the expression vector lambda gt11 is described. The technique employs RNase H and Escherichia coli DNA ligase treatment during second-strand synthesis, followed by repair of the ds cDNA extremities by S1 nuclease and PolIk (Klenow fragment) treatment. This treatment allows efficient addition of suitable linkers and results in a 100-fold increase in the yield of cloned cDNA, when compared with other published techniques. Using 75 ng of poly(A)+ RNA from CHO cells, we have prepared a library of 1.1 X 10(7) clones. This library was screened with polyclonal antibodies raised against a 100-kDal nucleolar protein of CHO cells. Five recombinants were isolated with inserts of 500-2500 bp. The average size of cDNA obtained by this method is considerable: the 2500-bp cDNA represents 90% of the mRNA coding for the 100-kDal protein.
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PMID:A powerful method for the preparation of cDNA libraries: isolation of cDNA encoding a 100-kDal nucleolar protein. 299 85

The properties and sources of all known restriction endonucleases and methylases are listed. The enzymes are cross-indexed (Table I), classified according to their recognition sequence homologies (Table II), and characterized within Table II by the cleavage and methylation positions, the number of recognition sites on the double-stranded DNA of the bacteriophages lambda, phi X174 and M13mp7, the viruses Ad2 and SV40, the plasmids pBR322 and pBR328, and the microorganisms from which they originate. Other tabulated properties of the restriction endonucleases include relaxed specificities (integrated into Table II), the structure of the generated fragment ends (Table III), and the sensitivity to different kinds of DNA methylation (Table V). In Table IV the conversion of two- and four-base 5'-protruding ends into new recognition sequences is compiled which is obtained by the fill-in reaction with Klenow fragment of the Escherichia coli DNA polymerase I or additional nuclease S1 treatment followed by ligation of the modified fragment termini [P3]. Interconversion of restriction sites generates novel cloning sites without the need of linkers. This should improve the flexibility of genetic engineering experiments. Table VI classifies the restriction methylases according to the nature of the methylated base(s) within their recognition sequences. This table also comprises restriction endonucleases which are known to be inhibited or activated by the modified nucleotides. The detailed sequences of those overlapping restriction sites are also included which become resistant to cleavage after the sequential action of corresponding restriction methylases and endonucleases [N11, M21]. By this approach large DNA fragments can be generated which is helpful in the construction of genomic libraries. The data given in both Tables IV and VI allow the design of novel sequence specificities. These procedures complement the creation of universal cleavage specificities applying class IIS enzymes and bivalent DNA adapter molecules [P17, S82].
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PMID:Specificity of restriction endonucleases and methylases--a review. 303 Aug 90

DNA polymerase beta (beta-pol) is a housekeeping enzyme considered to be involved in DNA repair in vertebrate cells. We cloned a fragment of genomic DNA spanning the first two exons of the human beta-pol gene and approximately 11 kilobases of the flanking region. The segment just 5' of the transcription start site can direct expression of the bacterial chloramphenicol acetyltransferase (CAT) gene in HeLa cells. A sequence containing only 113 base pairs of flanking DNA has promoter activity, and various constructs containing up to 4.8 kilobases of flanking sequence are expressed at a similar level, indicating that with this assay the important regulatory elements are located within or proximal to the approximately 100-bp core promoter. S1 nuclease mapping was used to show that transcription of the transfected genes is initiated at the same position as the endogenous beta-pol gene. The region upstream of the transcription start site is G + C rich and contains neither CAAT nor TATA boxes, but does have three decanucleotide elements matching high affinity binding sites for the RNA polymerase II transcription factor Sp1. Extending 5' from position -39 and surrounded by Sp1 consensus binding elements, there is a 10-nucleotide sequence with perfect dyad symmetry, GTGACGTCAC. Similar sequences are found in a number of cellular and viral promoters, including several adenovirus promoters. Experiments to test whether the core beta-pol promoter is activated by the adenovirus early region products showed that cotransfection with an adenovirus expression plasmid strongly activates expression of the beta-pol promoter.
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PMID:Human beta-polymerase gene. Structure of the 5'-flanking region and active promoter. 318 28

Replication of human adenovirus (Ad) DNA requires three virus-encoded proteins that are coordinately transcribed from a single promoter at early times after infection. The mRNAs for two of these proteins, the precursor to the terminal protein (pTP) and the Ad DNA polymerase (Ad Pol), share several exons, including one encoded near Ad genome coordinate 39. The positions of the splice points of these mRNAs have been mapped by S1 nuclease mapping, by RNA sequencing, and by cDNA cloning. As a result of RNA splicing events, a short open reading frame (ORF) encoded at genome coordinate 39 is connected to the beginning of both the pTP and Ad Pol coding sequences; inclusion of this upstream ORF is essential for expression of functional pTP and Ad Pol proteins.
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PMID:The precise structure and coding capacity of mRNAs from early region 2B of human adenovirus serotype 2. 340 47

A method is described for the preparation of deletions that extend in one direction from a fixed point. The method is based on the ability of deoxynucleoside [1-thio]triphosphates to be incorporated into DNA by DNA polymerase I, Klenow fragment, and the fact that alpha-thiophosphate-containing phosphodiester bonds are resistant to hydrolysis by the 3'-to-5' exonucleolytic activity of phage T4 DNA polymerase. Therefore, linear duplex DNA molecules blocked at one 3'-terminus with a thiophosphate were prepared and then degraded from the other end with the exonuclease. Digestion for different lengths of time followed by treatment with nuclease S1 and ligation allowed the preparation and recovery of a nested set of deletion mutants. Importantly, it was observed that a significant fraction of deletion mutants of recombinant M13 phages carrying the target gene in the same orientation as 'lacZ alpha' yielded phage that produced lacZ alpha-complementing activity. Nucleotide (nt) sequencing showed that these phages carried in-frame fusions between the target gene and 'lacZ alpha'. The deletion mutagenesis procedure is applied to the nt sequencing of a gnd gene from a natural isolate of Escherichia coli.
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PMID:A method for unidirectional deletion mutagenesis with application to nucleotide sequencing and preparation of gene fusions. 355 83

The genomic double-stranded DNA of mycobacteriophage I3, when denatured with alkali, heat, formamide or dimethylsulfoxide, breaks down to heterogeneous-sized single-strand (ss) fragments smaller than the expected intact unit genome length suggesting the presence of random ss interruptions on both the strands. The occurrence of the interruptions at random is also demonstrated by two-dimensional gel electrophoresis of the restriction fragments of I3 DNA. These interruptions have no adverse effect on the phage infectivity or DNA transfectivity. Studies with nuclease BAL 31 and end-labeling analysis confirm the presence of random interruptions. Detailed analysis using T4 DNA ligase, nuclease S1 and DNA polymerase I Klenow fragment revealed that the interruptions are in the form of small gaps rather than single phosphodiester bond breaks. The average length of the gap is about 10 nucleotides long and there are 13 to 14 such gaps per DNA molecule.
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PMID:Presence of random single-strand gaps in mycobacteriophage I3 DNA. 378 Dec 46


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