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Biomedical subjects

A Matsukage

Publications and source records attributed to A Matsukage.

At least 109 records · Page 6Linked to original sources
PubMed1989

Difference in the expression level of DNA polymerase beta among mouse tissues: high expression in the pachytene spermatocyte.

The expression level of DNA polymerase beta was determined in various mouse tissues. Northern blot hybridization analysis using rat cDNA as a probe revealed that the mRNA of about 1.5 kb for this enzyme is present in all kinds of tissues examined, but its content widely varies among tissues; the most abundant DNA polymerase beta mRNA was present in the testis, which was followed by brain, thymus, and spleen. The mRNA content was low in heart, kidney, and liver. In testis and brain, two minor species of transcripts of 3.3 and 6.2 kb were detected in addition to that of 1.5 kb. DNA polymerase beta activities in these tissues were closely correlated with the mRNA content, indicating that the expression of this enzyme is mainly regulated by the level of the mRNA. A survey of DNA polymerase beta mRNA levels in the testes at successive postnatal developmental stages and in isolated spermatogenic cells indicated that DNA polymerase beta mRNA was most abundant in spermatocytes at early pachytene. Since meiotic recombination occurs in this period, DNA polymerase beta may be involved in the repair-type DNA synthesis associated with the recombination process.

Animals↗
PubMed1989

Dual cis-acting negative regulatory elements located upstream of the mouse DNA polymerase beta gene.

A 2,200 base pair (bp) fragment containing the 5'-flanking region of the mouse DNA polymerase beta gene was placed adjacent to and upstream of the chloramphenicol acetyl transferase (CAT)-coding region of the CAT vector. A transient expression assay of CAT activity in mouse NIH/3T3 cells transfected with this recombinant plasmid or a set of its 5'-deletion derivatives was carried out to identify a cis-acting regulatory element(s) for DNA polymerase beta gene expression. Depending on the extent of the deletion, CAT activity was dramatically increased, indicating the existence of a negative regulatory region which could be divided into two distinct domains: removal of the first domain (NRE-I), nucleotides -1860 to -1580 (+1 denotes the position of 5'-most proximal transcription initiation site), caused two to three-fold stimulation of CAT activity, and removal of the second domain (NRE-II), nucleotides -828 to -456, stimulated CAT expression another two to three-fold. When an 1,864-bp segment containing these negative regulatory elements (-2190 to -327) was inserted in the plasmid carrying the simian virus 40 early promoter and enhancer-directed CAT gene, it inhibited the CAT expression relatively independently of the orientation of insertion and the distance from the promoter-enhancer. We also mapped the promoter element of the DNA polymerase beta gene to within a 133-bp DNA fragment from nucleotide position -100 to +33. Either the NRE-I region or the NRE-II region alone can inhibit DNA polymerase beta gene promoter function.

Animals↗
PubMed1988

Mouse DNA polymerase beta gene promoter: fine mapping and involvement of Sp1-like mouse transcription factor in its function.

The promoter of mouse DNA polymerase beta gene was analyzed by combining 5'-upstream region of this gene with chloramphenicol acetyltransferase (CAT) gene and by introduction of the recombinant plasmid DNA into mouse NIH/3T3 cells. Serial deletion of the mouse DNA sequence revealed that the promoter function resides within a 33 base pair region from the nucleotide position -48 to -15 with respect to the transcription initiation site, and is highly active without enhancer sequence. The promoter region was separated into two subregions: one (-48 to -35) contains a GC-box and the other contains a 10 base pair palindrome, whose sequence is similar to one of promoter consensus sequences found in a number of promoters including adenovirus promoters. The DNA polymerase beta promoter-directed CAT expression was competitively inhibited by the simultaneous transfection of plasmid DNA containing SV40 early promoter sequence. The viral sequences which are competitive to the GC-box of DNA polymerase beta gene promoter were the GC-boxes of SV40 promoter. Therefore, it is concluded that transcription of mouse DNA polymerase beta gene is regulated by mouse trans-acting factors equivalent to human Sp1 which is known to be trans-acting protein factor acting on SV40 GC-box sequences.

Animals↗
PubMed1988

Expression of active rat DNA polymerase beta in Escherichia coli.

A recombinant plasmid for expression of rat DNA polymerase beta was constructed in a plasmid/phage chimeric vector, pUC118, by an oligonucleotide-directed mutagenesis technique. The insert contained a 1005 bp coding sequence for the whole rat DNA polymerase beta. The recombinant plasmid was designed to use the regulatory sequence of Escherichia coli lac operon and the initiation ATG codon for beta-galactosidase as those for DNA polymerase beta. The recombinant clone, JMp beta 5, obtained by transfection of E. coli JM109 with the plasmid, produced high levels of DNA polymerase activity and a 40-kDa polypeptide that were not detected in JM109 cell extract. Inducing this recombinant E. coli with isopropyl beta-thiogalactopyranoside (IPTG) yielded amounts of 40-kDa polypeptide as high as 19.3% of total protein. Another recombinant clone, JMp beta 2-1, which was constructed by an oligonucleotide-directed mutagenesis to use the second ATG codon for the initiation codon, thus deleting the first 17 amino acid residues from the amino terminus, produced neither high DNA polymerase activity nor the 40-kDa polypeptide. The evidence suggests that this amino-terminal structure is important for stability of this enzyme in E. coli. The DNA polymerase was purified to homogeneity from the IPTG-induced JMp beta 5 cells by fewer steps than the procedure for purification of DNA polymerase beta from animal cells. The properties of this enzyme in activity, chromatographic behavior, size, antigenicity, and also lack of associated nuclease activity were indistinguishable from those of DNA polymerase beta purified from rat cells, indicating the identity of the overproduced DNA polymerase in the JMp beta 5 and the rat DNA polymerase beta.

Amino Acid Sequence↗
PubMed1988

Effects of 1-beta-D-arabinofuranosylcytosine incorporation on elongation of specific DNA sequences by DNA polymerase beta.

1-beta-D-Arabinofuranosylcytosine (ara-C) is an effective antileukemic agent which acts as an inhibitor of DNA synthesis. The precise mechanism responsible for this inhibitory effect, however, remains unclear. The present work has examined the effects of the triphosphate derivative, ara-CTP, on purified DNA polymerase beta. These studies were performed on M13 phage DNA templates of defined sequence. The results demonstrate that ara-C is incorporated into DNA by DNA polymerase beta. The results also demonstrate that the incorporated ara-C residue acts as a relative chain terminator. Moreover, the relative chain terminating effects of ara-C are sequence specific. In this regard, DNA strand elongation was progressively slowed at sequences of two, three, and four contiguous sites for cytosine incorporation. We also demonstrate that the inhibitory effects of ara-C are reversed by competition with deoxycytidine-triphosphate for incorporation into the DNA strand. Taken together, these findings are consistent with structural differences of the incorporated arabinosyl moiety which alter reactivity of the 3'-terminus and thereby inhibit chain elongation. These findings also provide new insights regarding the inhibitory effects of ara-C on elongation of specific DNA sequences.

Arabinofuranosylcytosine Triphosphate↗
PubMed1988

Identification and subcellular localization of the polypeptide for chick DNA primase with a specific monoclonal antibody.

Polypeptides responsible for activities of chick embryo DNA primase and DNA polymerase alpha were identified using monoclonal antibodies specific to these two enzymes. The 4-8H antibody neutralized DNA polymerase alpha activity measured on activated DNA template and also ribonucleoside triphosphate-dependent DNA synthesis on single-stranded DNA template (DNA primase-DNA polymerase alpha combined activity) to a partial extent (about 30%), but did not affect DNA primase activity. The 4-2D antibody, although it did not affect DNA polymerase alpha activity, did neutralize both DNA primase activity and DNA primase-DNA polymerase alpha combined activity extensively (up to 70%). Immunoblotting analysis of the DNA primase-DNA polymerase alpha complex showed that 4-2D and 4-8H antibodies recognize 60-kDa and 160-180-kDa polypeptides, respectively. An immunoaffinity column made of either of these antibodies retained DNA primase-DNA polymerase alpha complex. When the enzyme was eluted from the 4-8H column with alkaline solution, DNA primase was eluted prior to DNA polymerase alpha. In the case of 4-2D antibody column chromatography, the elution order of two enzymes was reversed. Results indicate that two enzymes in the complex which was retained in the antibody column were dissociated by lower alkaline pH than that dissociated the antigenic enzymes from the corresponding antibodies. In both cases, the fractions with DNA primase activity contained exclusively 60-kDa polypeptide, while those with DNA polymerase alpha contained 160-180-kDa polypeptides. Thus, DNA primase resided in 60-kDa polypeptide and was recognized by 4-2D antibody while DNA polymerase alpha resided in 160-180-kDa polypeptides and was recognized by 4-8H antibody. Immunofluorescence made with the DNA primase-specific 4-2D antibody as well as with 4-8H antibody appeared in granular structures which were tightly bound to the nuclear matrix. These nuclear fluorescences were much reduced in quiescent cells. Furthermore, since the fluorescence made by these antibodies was induced by adding serum to the quiescent cells in serum-deprived cultures, the expression of DNA primase and its organization in the structures on the nuclear matrix are regulated in correlation to the proliferating stage of cells, as observed with DNA polymerase alpha.

Animals↗
PubMed1988

Assignment of the human cytochrome c1 gene to chromosome 8.

We previously isolated a cDNA clone for human cytochrome c1. The insert DNA of approximately 950 bp from this clone was used as a probe to identify the cytochrome c1 gene. High molecular weight DNAs extracted from a panel of 14 independent human-mouse somatic cell hybrids were digested with BamHI and analyzed by Southern blot hybridization. The results indicated that the gene for human cytochrome c1 is located on chromosome 8.

Animals↗
PubMed1987

Homology between mammalian DNA polymerase beta and terminal deoxynucleotidyltransferase.

Nucleotide sequence analysis of the cDNA and the genomic clones for rat DNA polymerase beta revealed the existence of a 1,005-base pair open reading frame capable of encoding a Mr = 38,269 polypeptide of 335 amino acid residues. The region of 174 amino acid residues between the 42nd and 215th residues of the DNA polymerase beta polypeptide has extensive amino acid sequence homology with the region between the 195th and 366th residues of human terminal deoxynucleotidyltransferase. The two enzymes share extensive homology not only in primary structures but also in the computer-derived higher structures in these particular regions. The genes for DNA polymerase beta and terminal deoxynucleotidyltransferase are proposed to be derived from a common ancestral DNA polymerase gene.

Amino Acid Sequence↗
PubMed1987

Murine DNA polymerase beta gene: mapping of transcription initiation sites and the nucleotide sequence of the putative promoter region.

The nucleotide sequence of the region (total, 2,512 base pairs [bp]) from intron 2 to the 5'-flanking region was determined for the mouse DNA polymerase beta genomic clone, and the 300-bp region from intron 1 to the 5'-flanking region was also sequenced for the rat clone. At 51 bp upstream from the ATG codon which was previously suggested to be the translation initiation codon for the rat cDNA sequence, we found another ATG in the same reading frame in both mouse and rat genes. Three major transcription initiation sites (cap sites) each for rat and mouse DNA polymerase beta mRNAs were localized precisely by primer extension analysis at 51, 41, and 0 bp upstream from the first ATG codon, suggesting that this codon is used for translation initiation. The 400-bp region around exon 1 was extremely G + C rich (about 70%). Although neither a TATA box nor a CAAT box was found within the 500-bp region upstream of the 5'-most cap site, triple repeats of 5'-CCGCCC were found within the 100-bp region flanking the cap site.

Amino Acid Sequence↗
PubMed1987

Purification and partial amino acid sequence of human platelet membrane glycoproteins IIb and IIIa.

The glycoprotein (GP) IIb-IIIa complex was isolated from human platelet membranes by immunoaffinity chromatography using a monoclonal antibody specific for GP IIb-IIIa. GP IIb and IIIa were further separated in the presence of sodium dodecyl sulfate (SDS) by gel filtration high-performance liquid chromatography (HPLC). Two cycles of this procedure yielded almost complete separation of homogeneous preparations of GP IIb and IIIa. Each protein was then digested with lysyl endopeptidase (Achromobacter protease I), which cleaves at the carboxyl side of lysine residues, and the resulting oligopeptides from GP IIb and IIIa were fractionated with HPLC using a C18 reverse-phase column. Comparison of the elution profiles showed no obvious homology between the two proteins. Amino acid sequences of selected oligopeptides from each glycoprotein were determined using a gas-phase protein sequencer. Sixty amino acid residues (26 residues for IIb and 34 residues for IIIa) were identified.

Amino Acid Sequence↗
PubMed1986

Poly(ADP-ribosyl)ation of DNA polymerase beta in vitro.

DNA polymerase beta purified from bovine thymus is markedly inhibited when incubated in a reconstituted poly(ADP-ribosyl)ating reaction system. Analyses of the reaction product synthesized in this system by SDS-polyacrylamide gel electrophoresis and subsequent fluorography of the gel indicated that ADP-ribose is covalently attached to DNA polymerase beta molecule (Mr = 44,000).

DNA Polymerase I↗
PubMed1986

Differentiation of lens and neural cells in chicken embryos is accompanied by simultaneous decay of DNA replication machinery.

DNA polymerase alpha was detected in cells of developing chicken embryos by an immunofluorescent method using a monoclonal antibody specific for the high molecular weight polypeptide of chicken DNA polymerase alpha, and DNA polymerase beta was detected using a rabbit anti-chicken DNA polymerase beta antibody. In lens tissue of the 3- to 4-day chicken embryo, fluorescence with anti-DNA polymerase alpha antibody was detected in nuclei of lens epithelial cells but not in nuclei of lens fiber cells which had differentiated from epithelial cells. The localization of cells containing DNA polymerase alpha coincided with the distribution of cells capable of DNA replication as detected by [3H]thymidine autoradiography. Similar results were obtained during the differentiation of neural matrix cells to neuroblasts in the developing neural tube. In contrast to DNA polymerase alpha, DNA polymerase beta was detected in nuclei of both undifferentiated and differentiated cells of these tissues. Since the disappearance of DNA polymerase alpha was very rapid after the onset of differentiation, the DNA replication machinery in which DNA polymerase alpha plays a central role is thought to decay almost simultaneously with the onset of cellular differentiation in these tissues.

Animals↗
PubMed1986

Structure of rat DNA polymerase beta revealed by partial amino acid sequencing and cDNA cloning.

A cDNA library of newborn rat brain poly(A)+ RNA in phage lambda gt11 was screened with a polyclonal antibody against chicken DNA polymerase beta. One positive phage was isolated and purified after testing 2 X 10(7) recombinants. This phage, designated lambda pol beta-10, contained an 1197-base-pair cDNA insert that corresponded to a mRNA with a poly(A) sequence at the 3' terminus and a single, long open-reading frame of 957 bases. The open-reading frame, starting 44 residues from the 5' end of the cDNA, predicted a 36,375-Da protein of 318 amino acids. Comparison of this deduced amino acid sequence with the partial sequence obtained with purified polymerase beta revealed a match of six tryptic peptides, involving a total of 47 amino acid residues. This confirmed the identity of the cDNA. Blot-hybridization analysis of newborn rat brain poly(A)+ RNA revealed a mRNA species of approximately the same size as the cDNA insert; in addition, a second mRNA species approximately equal to 4000 bases long was detected. Computer-derived secondary structure analysis of the enzyme predicted seven regions of alpha-helix distributed throughout and three regions of beta-sheet.

Amino Acid Sequence↗
PubMed1986

Assignment of the gene for human DNA polymerase beta (POLB) to chromosome 8.

A cloned cDNA segment of 442 bp which contains the region coding the N-terminal sequence of rat DNA polymerase beta polypeptide was used as a probe in Southern hybridization analysis to identify the gene for this enzyme. This probe hybridized with DNA from mouse and human in addition to rat DNA, and the BamHI fragment containing DNA polymerase beta gene sequence was unique in size in each species: 15.8 kb for rat DNA, 8.7 kb for mouse DNA and 10.1 kb for human DNA. DNA extracted from a panel of 12 independent human-mouse somatic cell hybrids was analyzed by Southern blot hybridization with the cloned cDNA probe. The results indicate that the gene for the human DNA polymerase beta sequence (POLB) is located on chromosome 8.

Animals↗
PubMed1985

Monoclonal antibody specific for chicken DNA polymerase alpha associated with DNA primase.

Four monoclonal antibodies against chicken DNA polymerase alpha were obtained from mouse hybridomas (see ref. 1). Two of them, 4-2D and 4-8H, recognized different epitopes of the DNA polymerase alpha-DNA primase complex as determined by a competitive enzyme-linked immunosorbent assay. Antibody 4-8H partially (about 30%) neutralized the combined activity of primase-DNA polymerase alpha as well as the DNA polymerase alpha activity. In contrast, antibody 4-2D did not neutralize DNA polymerase alpha activity, but neutralized the primase-DNA polymerase alpha activity extensively (up to 80%). Furthermore, although an immunoaffinity column made with 4-8H antibody retained virtually all of the DNA polymerase alpha with and without associated primase, a column made with 4-2D antibody did not bind DNA polymerase alpha without the primase, but retained the enzyme associated with the primase. These results indicate that 4-8H monoclonal antibody is specific for DNA polymerase alpha and 4-2D monoclonal antibody is specific for the primase or a special structure present in the primase-DNA polymerase alpha complex.

Animals↗
PubMed1985

Inhibition of DNA polymerase alpha, DNA polymerase beta, terminal deoxynucleotidyl transferase, and DNA ligase II by poly(ADP-ribosyl)ation reaction in vitro.

Incubation of DNA polymerase alpha, DNA polymerase beta, terminal deoxynucleotidyl transferase, or DNA ligase II in a reconstituted poly(ADP-ribosyl)ating enzyme system markedly suppressed the activity of these enzymes. Components required for poly(ADP-ribose) synthesis including poly(ADP-ribose) polymerase, NAD+, DNA, and Mg2+ were all essential for the observed suppression. Purified poly(ADP-ribose) itself, however, was slightly inhibitory to all of these enzymes. Furthermore, the suppressed activities of DNA polymerase alpha, DNA polymerase beta, and terminal deoxynucleotidyl transferase were largely restored (3 to 4-fold stimulation was observed) by a mild alkaline treatment, a procedure known to hydrolyze alkaline-labile ester linkage between poly(ADP-ribose) and an acceptor protein. All of these results strongly suggest that the four nuclear enzymes were inhibited as a result of poly(ADP-ribosyl)ation of either the enzyme molecule itself or some regulatory proteins of these enzymes.

Animals↗
PubMed1985

Increase in DNA polymerase alpha in nuclei of chicken kidney cells after avian adenovirus infection.

DNA polymerase activity which sedimented at about 7s in the presence of 0.5M KC1 increased in cultured kidney cells after infection with a chicken embryo lethal orphan virus, an avian adenovirus. Elevated levels of antigenic material to a monoclonal antibody specific for the large subunit of chicken DNA polymerase alpha were also detected in nuclei of virus-infected cells by an immunofluorescence method. Since the elevations of DNA polymerase activity and of nuclear immunofluorescence was not inhibited with 1-beta-D-arabinofuranosylcytosine, the early viral gene(s) seems to be responsible for the induction of the synthesis and nuclear accumulation of DNA polymerase alpha.

Adenoviridae Infections↗