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Biomedical subjects

A Matsukage

Publications and source records attributed to A Matsukage.

At least 91 records · Page 5Linked to original sources
PubMed1991

Inactivation of DNA polymerase beta by in vitro phosphorylation with protein kinase C.

The Mr = 38,300 polypeptide of the purified recombinant rat DNA polymerase beta served as an excellent substrate for protein kinase C (PKC) in vitro but not for the catalytic subunit of cAMP-dependent protein kinase. The phosphorylation by PKC resulted in inactivation of DNA polymerase beta activity, and recovery was achieved by dephosphorylation with alkaline phosphatase. Since the phosphorylated DNA polymerase beta was retained with use of a single-stranded DNA-cellulose column, inactivation might occur at a site different from that for the DNA binding. Amino acid sequence analysis of the phosphopeptides revealed that the phosphorylated sites were 2 serine residues at positions 44 and 55 from the NH2 terminus, either or both of which might be involved in the catalytic activity of DNA polymerase beta. Thus, the inactivation of the DNA repair enzyme, DNA polymerase beta, by PKC may be an important process in the modification of DNA metabolism in the nucleus through signal transduction processes.

Alkaline Phosphatase↗
PubMed1991

Aspartic acid residues at positions 190 and 192 of rat DNA polymerase beta are involved in primer binding.

The sequence Gly-Asp-Met-Asp, spanning positions 189-192 of rat DNA polymerase beta, is similar to the sequence motif Gly-Asp-Thr-Asp that is highly conserved in a number of replicative DNA polymerases from eukaryotic cells, viruses, and phages. The role of this sequence in the catalytic function of rat DNA polymerase beta was investigated by individually changing each amino acid in this region by site-directed mutagenesis. The mutant enzymes DE190 and DE192, in which aspartic acid residues at positions 190 and 192, respectively, were replaced by glutamic acid, showed about 0.1% activity of the wild-type enzyme. On the other hand, the replacement of Gly-189 by alanine or Met-191 by isoleucine or threonine only slightly affected the enzyme activity. A gel mobility shift assay showed that DNA complexes with enzyme DE190 and especially with DE192 were less stable than the corresponding complex with the wild-type enzyme. Kinetic analysis with these mutant enzymes indicate that their Km's for primer DNA were about 10-fold higher than that of the wild type, while Km's for deoxyribonucleoside triphosphate were not changed. Since neither DE190 nor DE192 had any significant alteration in secondary structure, our results suggest that both Asp-190 and Asp-192 are located in the active site and are involved in the interaction of DNA polymerase beta with primer.

Amino Acid Sequence↗
PubMed1991

Molecular cloning and structural analysis of mouse gene and pseudogenes for proliferating cell nuclear antigen.

We have isolated clones containing the entire mouse proliferating cell nuclear antigen (PCNA) gene of 3890 bp and flanking sequences using a rat PCNA cDNA as a probe. The mouse gene has 6 exons whose sequences and junction points of exons with introns are extensively homologous to the human gene while sizes and nucleotide sequences of introns are much less conserved than exons. By a transient expression assay of chloramphenicol acetyltransferase, the promoter of this gene is localized within 200 bp upstream of the transcription initiation site. We have also isolated two processed pseudogenes. Homology between the first one (psi PCNA-I) and the exons of the PCNA gene was 76.8% in the region so far sequenced. The second one (psi PCNA-II) consists of a region highly homologous to the entire exons of the PCNA gene, and only 9 out of total 1256 bp are different from the corresponding exon sequence of the gene. The 5'-flanking region of the psi PCNA-II did not function as an active promoter. Surveys in various wild and laboratory mice genomes suggest that the psi PCNA-II was generated through the reverse transcription process of the PCNA mRNA about 5 x 10(5) years ago in the domesticus subspecies of Mus musculus, the house mouse. The psi PCNA-II is tentatively mapped in the chromosome 17 of the C57BL mouse.

Amino Acid Sequence↗
PubMed1991

Organization of mouse DNA polymerase beta gene silencer elements and identification of the silencer-binding factor(s).

Different portions of the 5'-upstream region of the mouse DNA polymerase beta gene were combined with bacterial chloramphenicol acetyltransferase (CAT) gene of the CAT vector. Transfection of these recombinant plasmids into mouse NIH/3T3 cells has revealed that each of the previously identified two negatively acting regions (silencers I and II) of this gene consists of multiple sub-domains. The distal silencer (silencer I) at around -1.5 kb consists of four sub-domains (-1852 to -1667, -1663 to -1616, -1564 to -1525 and -1355 to -1257). The promoter-proximal silencer (silencer II) at around -0.5 kb consists of two functional domains (-681 to -523 and -490 to -447) separated by a neutral region of 33 base pairs. Silencer II functioned efficiently when silencer I was deleted. Conversely, the distal silencer I functioned efficiently when silencer II was deleted. Thus, these silencers functioned redundantly to each other in NIH/3T3 cells. Nucleotide sequence analysis revealed no extensive sequence similarity between these two silencers. Significant sequence similarity is present between a distal portion of silencer II and the c-myc gene silencer, and also between a proximal portion of silencer II and the mouse F9 cell-specific silencer. A protein factor(s) that specifically bound to the silencer elements was detected in nuclear extracts of NIH/3T3 cells and mouse liver in which DNA polymerase beta was expressed at a rather low level. The same binding factor(s) can bind to both silencer I and II regions, although its affinity for silencer II is much higher than that for silencer I.

Animals↗
PubMed1991

Repression of the Drosophila proliferating-cell nuclear antigen gene promoter by zerknüllt protein.

A 631-bp fragment containing the 5'-flanking region of the Drosophila melanogaster proliferating-cell nuclear antigen (PCNA) gene was placed upstream of the chloramphenicol acetyltransferase (CAT) gene of a CAT vector. A transient expression assay of CAT activity in Drosophila Kc cells transfected with this plasmid and a set of 5'-deletion derivatives revealed that the promoter function resided within a 192-bp region (-168 to +24 with respect to the transcription initiation site). Cotransfection with a zerknüllt (zen)-expressing plasmid specifically repressed CAT expression. However, cotransfection with expression plasmids for a nonfunctional zen mutation, even-skipped, or bicoid showed no significant effect on CAT expression. RNase protection analysis revealed that the repression by zen was at the transcription step. The target sequence of zen was mapped within the 34-bp region (-119 to -86) of the PCNA gene promoter, even though it lacked zen protein-binding sites. Transgenic flies carrying the PCNA gene regulatory region (-607 to +137 or -168 to +137) fused with lacZ were established. When these flies were crossed with the zen mutant, ectopic expression of lacZ was observed in the dorsal region of gastrulating embryos carrying the transgene with either construct. These results indicate that zen indirectly represses PCNA gene expression, probably by regulating the expression of some transcription factor(s) that binds to the PCNA gene promoter.

Amino Acid Sequence↗
PubMed1991

Distribution of PCNA in Drosophila embryo during nuclear division cycles.

An immunocytochemical method using a specific antibody was employed to detect the proliferating cell nuclear antigen (PCNA) in Drosophila embryos during the first 13 nuclear division cycles. Strong nuclear staining with the anti-PCNA antibody was observed at interphase throughout 13 cycles. Metaphase chromosomes were not stained throughout these cycles. The chromosomal (nuclear) staining reappeared at anaphase until cycle 10 and at telophase in cycle 11. During cycles 12 and 13, nuclear staining was detected exclusively at interphase. Relatively uniform staining of syncytial cytoplasm was observed throughout mitotic phases until cycle 9. In the following cycles, strong staining in both the central yolk mass and the cortical layer of cytoplasm was detected at metaphase and telophase. During interphase of cycles later than the 9th, staining in the central yolk mass got much fainter and that in the cortical cytoplasm completely disappeared. These results suggest that the PCNA dissociates from chromosomes at metaphase; then in later mitotic phases, it is transported from the syncytial cytoplasm into nuclei to participate in formation of the active DNA-replication enzyme complexes.

Anaphase↗
PubMed1990

Striking similarity of the distribution patterns of the poly(ADP-ribose) polymerase and DNA polymerase beta among various mouse organs.

The expression level of poly(ADP-ribose) polymerase mRNA as well as the level of enzymatic activity were examined in various mouse organs by northern blot and activity gel analyses. High levels of the mRNA expression and enzymatic activity were observed in testis, thymus, spleen, and brain. On the other hand, low levels of the mRNA expression and enzymatic activity were observed in liver and kidney. These findings suggest that the expression of the poly(ADP-ribose) polymerase is mainly regulated by transcription. Striking similarity was observed between the patterns of organ distribution of enzymatic activities of poly(ADP-ribose) polymerase and DNA polymerase beta in various mouse organs.

Animals↗
PubMed1990

Rat DNA polymerase beta gene can join in excision repair of Escherichia coli.

Though DNA polymerase I (poll) of Escherichia (E.) coli is understood to play a role in repair synthesis of excision repair, it is still obscure whether DNA polymerase beta (pol beta) plays a similar role in eukaryotic cells. To estimate the role of pol beta in excision repair processes, we inserted the rat pol beta gene into several mutant E. coli defective in a diverse set of enzymatic activities of poll. UV resistance was seen only when the 5'----3' exonuclease (exo) activity of poll molecules remained. Therefore it is suggested that 5'----3' exo activity as well as pol beta activity are essential for repair synthesis of excision repair in eukaryotic cells.

Animals↗
PubMed1990

Site-directed mutagenesis of recombinant rat DNA polymerase beta: involvement of arginine-183 in primer recognition.

By site-directed mutagenesis using synthetic oligonucleotides, amino acid residues 181Phe-Arg-Arg183 of recombinant rat DNA polymerase beta were replaced by other amino acids to clarify the roles of these residues in the DNA synthesizing reaction. Replacement of Phe-181 by alanine reduced the enzyme activity only 30%. Replacement of Arg-182 by alanine and glutamine resulted in reduction of the activity by about 67% and 95%, respectively. The Arg-182----Gln replacement increased the binding strength to single-stranded DNA but did not significantly change the Km's for the primer and dTTP, suggesting that Arg-182 is involved in modulation of binding to the template rather than to the primer or deoxyribonucleoside triphosphate. Replacement of Arg-183 by Gln resulted in reduction of the activity by about 95%, and this change, although causing little change in binding strength to single-stranded DNA, resulted in a 3-4-fold increase in the Km's for the primer and deoxyribonucleoside triphosphate. A more dramatic change was observed when Arg-183 was replaced by Ala, which resulted in a 99.98% reduction of enzyme activity. Although the Km for deoxyribonucleoside triphosphate of this mutant enzyme was hardly changed, that for the primer increased 159-fold. Therefore, it is concluded that Arg-183 occupies an important part of the primer recognition site of DNA polymerase beta.

Amino Acid Sequence↗
PubMed1990

Coprecipitation of 3'----5' exonuclease and DNA polymerase gamma with anti-DNA polymerase gamma antibody.

Highly purified preparations of chick embryo DNA polymerase gamma contained 3'----5' exonuclease activity which might be responsible for the exonucleolytic proofreading during DNA synthesis [Kunkel, T.A. & Soni, A. (1988) J. Biol. Chem. 262, 4450-4459]. A rabbit antibody produced against highly purified chick DNA polymerase gamma precipitated 3'----5' exonuclease activity to the same extent as DNA polymerase gamma activity. Furthermore, the antibody neutralized the two enzyme activities to an equal extent. However, the exonuclease activity was more resistant than DNA polymerase gamma activity to thermal treatment at 50 degrees C, although both activities were partially protected with polynucleotides. The results obtained suggest that these two enzymes are associated as a single enzyme complex or that the two activities reside in a single molecule, and the active site of DNA polymerase gamma and 3'----5' exonuclease are, although not identical, closely correlated.

Animals↗
PubMed1990

Drosophila proliferating cell nuclear antigen (cyclin) gene: structure, expression during development, and specific binding of homeodomain proteins to its 5'-flanking region.

The genomic and cDNA clones for a Drosophila melanogaster proliferating cell nuclear antigen (PCNA) (cyclin) were isolated and sequenced. The coding sequence for a 260-amino-acid residue polypeptide was interrupted by a single short intron of 60 base pairs (bp), and about 70% of the deduced amino acid sequence of the Drosophila PCNA was identical to the rat and human PCNA polypeptides, with conserved unique repeats of leucine in the C-terminal region. Genomic Southern blot hybridization analysis indicates the presence of a single gene for PCNA per genome. The PCNA mRNA was detected at a high level in adult ovaries, unfertilized eggs, and early embryos and at low levels in the other developmental stages. The major transcription initiation site (cap site) was localized at 89 bp upstream from the ATG codon. Neither a TATA box nor a CAAT box was found within the 600-bp region upstream of the cap site. Clusters of 10 bp of sequence similar to the binding sites for Drosophila proteins containing homeodomains were found in the region from -127 to -413. DNase I footprint analysis revealed that the Drosophila homeodomain proteins coded by even-skipped and zerknüllt genes can specifically bind to these sites. These results suggest that the expression of the PCNA gene is under the control of genes coding for homeodomain proteins.

Amino Acid Sequence↗
PubMed1990

Chromosomal assignment of the gene for the ubiquinone-binding protein of human mitochondrial cytochrome bc1 complex.

The ubiquinone-binding protein (QP-C) is a nuclear-encoded subunit of the cytochrome bc1 complex in the mitochondrial respiratory chain and is thought to be involved in the electron transfer reaction coupled to energy transduction. We recently isolated a nuclear gene for human QP-C and used, in the present study, its fragment as a probe for Southern blot analysis of EcoRI-digested DNAs prepared from 14 human-mouse somatic cell hybrids. The results indicated that the human QP-C gene is located on chromosome 8.

Animals↗
PubMed1990

DNA replication can overcome the silencer function on transcription.

Several cis-acting transcriptional silencer elements that are dispersed over a long (more than 1 kb) region upstream of the mouse DNA polymerase-beta gene repress transcription not only of this gene but also of heterologous promoter-enhancers of other genes. The effect of DNA replication on the functions of these silencer elements was examined by combining them with transiently replicating plasmids carrying the chloramphenicol acetyltransferase (CAT) gene and the replication origins (ori) of polyoma virus and SV40 DNA. The silencers functioned efficiently when these plasmids were transfected into mouse NIH 3T3 and monkey CV1 cells. These cells lack T antigen and therefore do not allow replication of the plasmids. The silencers also functioned when the plasmids contained nonfunctional SV40 ori. In contrast, the negative effect of the silencer was not observed when the plasmids were transfected into cells producing large T antigens, such as MOP8 cells and COS1 cells. The observed differences in the silencer function between the permissive and nonpermissive cells was not due to differences in plasmid copy numbers in the cells. The results indicate that ongoing DNA replication can completely overcome the negative effects of the silencer elements on transcription.

Animals↗
PubMed1989

Repression of polyoma virus DNA replication by 5'-flanking region of mouse DNA polymerase beta gene containing transcriptional silencer elements.

Dual cis-acting silencer elements are located upstream of the mouse DNA polymerase beta gene (Yamaguchi, M., Hayashi, Y., and Matsukage, A. (1989) J. Biochem. (Tokyo) 105, 79-83). In order to examine possible involvement of transcriptional silencer elements in the regulation of DNA replication, we have utilized a transient replication system of the plasmid DNA carrying replication origin of polyoma virus DNA in mouse MOP8 cells, which is constitutively producing polyoma virus large T-antigen. The polyoma virus origin of DNA replication is composed of three cis-acting genetic elements called alpha, beta, and core, in which alpha and beta elements correspond to enhancer domains. When the 5'-flanking regions of the DNA polymerase beta gene containing silencer elements were placed at the late gene border of alpha element, they effectively repressed the DNA replication. However, when placed at the early gene border of core element, it only marginally repressed the DNA replication. These results suggest that the silencer elements at cis position repress polyoma virus DNA replication by impeding the enhancer function that activates the DNA replication.

Animals↗
PubMed1989

Paradoxical effect of Simian virus 40 enhancer on the function of mouse DNA polymerase beta gene promoter.

Simian virus (SV) 40 enhancer (nucleotide position 108 to 294) was combined with chloramphenicol acetyltransferase (CAT) plasmid whose expression is under control of mouse DNA polymerase beta gene promoter. Although the SV40 enhancer stimulated the transient CAT-expression directed by the DNA polymerase beta gene promoter two to three fold in human HeLa cells, it repressed the CAT-expression by 50 to 60% in mouse NIH/3T3 cells. The repression was observed relatively independently on the orientation of the insertion and the distance from the promoter. These properties of the enhancer are very similar to those of so-called transcriptional silencer element. In both HeLa and NIH/3T3 cells, the SV40 enhancer stimulated effectively its own early gene promoter-directed CAT-expression. In mouse immature T-cell line RV-1 in which the SV40 promoter-enhancer did not function, no effect of the SV40 enhancer sequence on the DNA polymerase beta promoter-directed CAT-expression was observed. Thus, it is suggested that both cell type-specific trans-acting factor(s) and the specific combination with the promoter sequence turn the properties of the SV40 enhancer into those of a silencer.

Animals↗
PubMed1989

Sequence-specific inhibition of DNA strand elongation by incorporation of 9-beta-D-arabinofuranosyladenine.

9-beta-D-Arabinofuranosyladenine (ara-A) is an inhibitor of DNA replication with antitumor and antiviral activity. The molecular basis for this inhibitory effect has remained unclear. The present work has examined the effects of 9-beta-D-arabinofuranosyladenine-triphosphate on DNA polymerase-beta. These studies were performed on different M13 phage DNA templates. The findings demonstrate that 9-beta-D-arabinofuranosyladenine is incorporated into the elongating DNA strand by DNA polymerase-beta. The findings also demonstrate that the incorporated 9-beta-D-arabinofuranosyladenine residue acts as a relative chain terminator. Furthermore, the relative chain-terminating effects of this agent are sequence specific and reversed by competition with deoxyadenosine-triphosphate for incorporation into the DNA strand. These findings are in concert with hydrogen bonding differences of the incorporated arabinosyl moiety which alters reactivity of the chain terminus and thereby inhibits elongation. These findings are also in agreement with recent studies of 1-beta-D-arabinofuranosylcytosine and provide insights into the sequence specific effects of these agents.

Base Sequence↗
PubMed1989

Cell-type-specific expression of mouse DNA polymerase beta-gene is regulated by silencer elements.

RNA blot hybridization analysis revealed that the steady-state level of DNA polymerase beta-mRNA in mouse neuroblastoma N18TG2 cells was approximately five-fold higher than that in NIH/3T3 cells. In order to examine the function of DNA polymerase beta-gene silencers in these two cell lines, we employed a chloramphenicol acetyltransferase (CAT)-transient expression assay using the CAT plasmids containing the silencers linked to various promoter-enhancers. In NIH/3T3 cells, DNA polymerase beta-gene silencers effectively repressed the function of its own promoter and those of several other heterologous promoter-enhancers. In contrast, the silencers only marginally affected the CAT expression directed by DNA polymerase beta-gene promoter and heterologous promoter-enhancers in N18TG2 cells. The extent of the increase of CAT expression by removing silencer elements in NIH/3T3 cells was very similar to the ratio of DNA polymerase beta-mRNA content in N18TG2 cells to that in NIH/3T3 cells. These results indicate that cell-type-specific expression of DNA polymerase beta-gene is primarily controlled by the function of its silencer elements.

Animals↗