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Biomedical subjects

A Matsukage

Publications and source records attributed to A Matsukage.

At least 55 records · Page 3Linked to original sources
PubMed1997

Use of a fusion protein to obtain crystals suitable for X-ray analysis: crystallization of a GST-fused protein containing the DNA-binding domain of DNA replication-related element-binding factor, DREF.

Crystals of glutathione-S-transferase (GST)-fused protein containing the DNA-binding domain of DNA replication-related element-binding factor, DREF, were obtained under crystallization conditions similar to those for GST. Preliminary X-ray crystallographic analysis revealed that crystals of the GST-fused protein belong to space group P6(1)22 or P6(5)22 with unit cell dimensions a = b = 140.4 A, c = 93.5 A and gamma = 120 degrees, having one molecule in the crystallographic asymmetric unit. The crystals diffract to 2.5 A resolution. The cell dimensions are related to those of GST crystals thus far reported. Crystallization of the DNA-binding domain that was cleaved from the fused protein by thrombin was also carried out using several methods under numerous conditions, but efforts to produce well-ordered large crystals were unsuccessful. A possible application of GST-fusion proteins for small target proteins or domains to obtain crystals suitable for X-ray structure determination is proposed.

Crystallography, X-Ray↗
PubMed1997

Involvement of the DNA replication-related element (DRE) and DRE-binding factor (DREF) in transcriptional regulation of the Bombyx mori PCNA gene.

The promoter region of the Bombyx mori gene encoding the proliferating cell nuclear antigen (PCNA), and its activating factor(s) were analyzed to ascertain similarities with Drosophila regulatory elements. Full promoter activity was established to reside within the region from -466 to +347 base pairs with respect to the transcription initiation site. Within this region, we found a sequence similar to the DNA replication-related element (DRE), 5'-TATCGATA, which is a promoter-activating sequence common to promoters of the Drosophila genes for DNA replication-related factors, including PCNA. A mutation in the DRE-like sequence of the B. mori PCNA gene promoter caused reduction of the promoter activity and also binding to the recombinant Drosophila DRE-binding factor (DREF). Furthermore, a factor(s) binding to the DRE sequence was detected in extracts of B. mori BmN4 cells. Monoclonal antibodies against Drosophila DREF inhibited the binding activity of the factor, as shown by gel mobility shift assays, and allowed specific detection of a 100 kDa protein on immuno Western blot analysis. These results suggest that the B. mori DREF homolog binds to DRE to regulate transcription of the PCNA gene.

Amino Acid Sequence↗
PubMed1996

Transcriptional regulation of the Drosophila CycA gene by the DNA replication-related element (DRE) and DRE binding factor (DREF).

The Drosophila gene for cyclin A is expressed in dividing cells throughout development. This expression pattern is similar to those of genes related to DNA replication, suggesting involvement of some common control mechanism(s). In the upstream region (-71 to -64 with respect to the transcription initiation site) of the CycA gene, we found a sequence identical to the DNA replication-related element (DRE; 5'-TATCGATA), which is important for high level expression of replication-related genes such as those encoding DNA polymerase alpha and proliferating cell nuclear antigen. Transient expression assays with chloramphenicol acetyltransferase (CAT) were carried out to examine the function of the DRE sequence of the CycA gene. Deletion or base substitution mutations resulted in an extensive reduction in CAT expression. Furthermore, monoclonal antibodies against DRE binding factor (DREF) diminished or supershifted the complex of the DREF and DRE-containing fragment. The results indicate that the Drosophila CycA gene is under the control of a DRE/DREF system, as are DNA replication-related genes.

Animals↗
PubMed1996

Fatty acids selectively inhibit eukaryotic DNA polymerase activities in vitro.

The in vitro relationship between eukaryotic DNA polymerases and fatty acids was investigated. Some fatty acids strongly inhibited the activities of DNA polymerase alpha and/or beta in vitro. The kinetics of inhibition by linoleic acid showed that DNA polymerase alpha was non-competitively inhibited with respect to the DNA template and substrate (dTTP), while DNA polymerase beta was inhibited competitively with both DNA and substrate.

Animals↗
PubMed1996

DNA replication-related elements cooperate to enhance promoter activity of the drosophila DNA polymerase alpha 73-kDa subunit gene.

An analysis was carried out on the promoter region of the Drosophila DNA polymerase alpha 73-kDa subunit gene and the factor(s) activating the promoter. Transcription initiation sites were newly identified in the region downstream of the previously determined sites. Full promoter activity resided within the region from -285 to +129 base pairs with respect to the newly determined major site. Within this region, we found three sequences identical or similar to the DNA replication-related element (DRE), 5'-TATCGATA, which is known as a common promoter-activating element for the Drosophila DNA polymerase alpha 180-kDa subunit gene and the proliferating cell nuclear antigen gene. These sites were located at positions -77 to -70 (DREalpha-I), -44 to -37 (DREalpha-II), and +3 to +10 (DREalpha-III). Footprinting analysis using the recombinant DRE-binding factor (DREF) or Kc cell nuclear extract demonstrated that DREF can bind to all three DRE-related sites. Introduction of mutation in even one of the three DRE-related sequences caused extensive reductions of the promoter activity and also the DREF-binding activity of the promoter-containing fragment. The results indicate that the three DREF-binding sites cooperate to enhance promoter activity of the DNA polymerase alpha 73-kDa subunit gene.

Animals↗
PubMed1996

Isolation and characterization of cDNA for DREF, a promoter-activating factor for Drosophila DNA replication-related genes.

DREF, a transcription regulatory factor which specifically binds to the promoter-activating element DRE (DNA replication-related element) of DNA replication-related genes, was purified to homogeneity from nuclear extracts of Drosophila Kc cells. cDNA for DREF was isolated with the reverse-transcriptase polymerase chain reaction method using primers synthesized on the basis of partial amino acid sequences and following screening of cDNA libraries. Deduced from the nucleotide sequences of cDNA, DREF is a polypeptide of 701 amino acid residues with a molecular weight of 80,096, which contains three characteristic regions, rich in basic amino acids, proline, and acidic amino acids, respectively. Deletion analysis of bacterially expressed DREF fused with glutathione S-transferase (GST-DREF) indicated that a part of the N-terminal basic amino acid region (16-115 amino acids) is responsible for the specific binding to DRE. A polyclonal and four monoclonal antibodies were raised against the GST-DREF fusion protein. The antibodies inhibited specifically the transcription of DNA polymerase alpha promoter in vitro. Cotransfection experiments using Kc cells demonstrated that overproduction of DREF protein overcomes the repression of the proliferating cell nuclear antigen gene promoter by the zerknüllt gene product. These results confirmed that DREF is a trans-activating factor for DNA replication-related genes. Immunocytochemical analysis demonstrated the presence of DREF polypeptide in nuclei after the eighth nuclear division cycle, suggesting that nuclear accumulation of DREF is important for the coordinate zygotic expression of DNA replication-related genes carrying DRE sequences.

Amino Acid Sequence↗
PubMed1996

Roles of multiple promoter elements of the proliferating cell nuclear antigen gene during Drosophila development.

The expression of genes involved in DNA replication is closely correlated with the proliferating state of cells and is repressed with the progression of differentiation during development. Promoter regions of the Drosophila proliferating cell nuclear antigen (PCNA) gene and the DNA polymerase alpha gene contain a common 8-base pair promoter element (DRE: DNA replication-related element). The examination of a common expression mechanism for DNA replication-related genes, which is regulated positively by growth signals and negatively by differentiation signals would be of interest. We generated PCNA-LacZ fusion genes in which the 5'-flanking sequence of the PCNA gene has been mutated. An examination of the expression of these fusion genes, introduced into flies by germ-line transformation, led to the identification of another distinct regulatory element, URE (upstream regulatory element), within the region from -168 to -119 with respect to the transcription initiation site. During embryogenesis, the region containing the DRE sequence (-108 to -91) greatly stimulated the PCNA gene minimal promoter (-86 to +130), when it was placed upstream of the promoter in both normal and reverse orientations. Addition of the URE sequence further stimulated the promoter activity twofold. During larval stages, both DRE and URE were indispensable to the promoter activity, since neither of the sequences alone activated the minimal promoter. Demonstration of beta-galactosidase activity indicated URE plays an essential role in various larval tissues such as salivary gland and imaginal disc. While the minimal promoter region alone directed maternal expression of lacZ in ovaries of adult females, both DRE and URE further stimulated promoter activity. These results show several elements of the PCNA gene promoter play roles during Drosophila development.

Animals↗
PubMed1995

The DRE sequence TATCGATA, a putative promoter-activating element for Drosophila melanogaster cell-proliferation-related genes.

We have confirmed that the DNA replication-related element (DRE) consisting of an 8-bp palindrome, TATCGATA, and not neighboring sequences, are responsible for activating promoters of the Drosophila melanogaster (Dm) PCNA (proliferating cell nuclear antigen)- and DNA polymerase alpha-encoding genes in both cultured cell and transgenic fly systems. We have so far found 153 copies of DRE in the Dm gene database. 73 of them are concentrated within the 600-bp upstream regions from the transcription start points of 61 genes. Interestingly, many of these genes are involved in either DNA replication, transcription, translation, signal transduction, cell cycle or other putative regulatory functions, and are possibly related to cell proliferation. It seems likely that DRE is an element common to the regulation of cell-proliferation-related genes, although their expression patterns may be different depending on which of regulatory elements other than the DRE are combined.

Animals↗
PubMed1995

Essential role of E2F recognition sites in regulation of the proliferating cell nuclear antigen gene promoter during Drosophila development.

We have found sequences similar to the transcription factor E2F recognition site within the Drosophila proliferating cell nuclear antigen (PCNA) gene promoter. These sequences are located at positions -43 to -36 (site I). and -56 to -49 (site II) with respect to the cap site Glutathione S-transferase (GST)-E2F and GST-DP fusion proteins cooperate and bind to the potential E2F sites in the PCNA promoter in vitro. A binding factor(s) to these sequences that has similar binding specificity to that of E2F was detected in nuclear extracts of Drosophila Kc cells. Furthermore, transient expression of target site for the activation coincided with the E2F sites. These results indicate that the PCNA gene is a likely target gene of E2F. Examination of lacZ expression from PCNA-lacZ fusion genes carrying mutations in either or both of two E2F sites introduced into flies by germ line transformation revealed that site II plays a major role in the PCNA promoter activity during embryogenesis and larval development, although both sites are required for optimal promoter activity. However, for maternal expression in ovaries, either one of the two sites is essentially sufficient to direct optimal promoter activity. These results demonstrate, for the first time, an essential role for E2F sites in regulation of PCNA promoter activity during development of a multicellular organism.

Animals↗
PubMed1995

A nucleotide sequence essential for the function of DRE, a common promoter element for Drosophila DNa replication-related genes.

Promoter regions of the Drosophila proliferating cell nuclear antigen (PCNA) gene and the DNA polymerase alpha 180-kDa catalytic subunit gene contain a common 8 base pair (bp) promoter element, 5'-TATCGATA (DRE, Drosophila DNA replication-related element). We have generated various base substitutions and internal deletions in and around DRE (nucleotide positions -93 to -100 with respect to the transcription initiation site) of the PCNA gene in vitro and subsequently examined their effects on the binding to DREF (DRE-binding factor) and PCNA gene promote activity in cultured Drosophila Kc cells as well as in living flies. Gel mobility shift assays using nuclear extracts of Kc cells with and without competitor DNA fragments carrying the mutations indicated that the 10-bp sequence from positions -91 to -100 is essential for complex formation with DREF. Transient expression assays of chloramphenicol acetyl-transferase (CAT) in Kc cells transfected with PCNA promoter-CAT fusion genes carrying the mutations revealed that the 8-bp sequence from -93 to -100 is essential for activation of the promoter in Kc cells. Examination of lacZ expression from PCNA promoter-lacZ fusion genes carrying the mutations, introduced into flies by germ-line transformation, revealed that the 8-bp sequence is also important for DRE function during development. However, we obtained two exceptional mutations in the 8-bp sequence that did not or only marginally affected the PCNA gene promoter activity in transgenic flies. Both of these mutations effectively reduced the promoter activity in CAT transient expression assay in Kc cells and the binding to DREF in vitro. Therefore, the 8-bp sequence requirement for DRE function appears to be less stringent in living flies than in the cultured cell or in vitro cases.

Animals↗
PubMed1995

Misincorporation of dAMP opposite 2-hydroxyadenine, an oxidative form of adenine.

Nucleotide incorporation opposite an oxidative form of adenine, 2-hydroxyadenine (2-OH-Ade) was investigated. When a primed template with 2-OH-Ade was treated with an exonuclease-deficient Klenow fragment of Escherichia coli DNA polymerase I (KFexo-), recombinant rat DNA polymerase beta (pol beta) or calf thymus DNA polymerase alpha (pol alpha), incorporation of dTMP and dAMP was observed. In addition, KFexo- inserted dGMP as well. A steady-state kinetic study indicated that the insertion of dAMP and dTMP opposite the DNA lesion occurred with similar frequency with KFexo- and pol beta. Insertion of dTMP opposite 2-OH-Ade was favored to that of dAMP by pol alpha. Chain extension from the A.2-OH-Ade pair is less favored than that from the T.2-OH-Ade pair by all three DNA polymerase. Analysis of full-length products of in vitro DNA synthesis showed that dTMP and dAMP were incorporated by DNA polymerases and that exonuclease-proficient and -deficient Klenow fragments also inserted dGMP opposite 2-OH-Ade. These results suggest that formation of 2-OH-Ade from A in DNA will induce A-->T and A-->C transversions in cells.

Animals↗
PubMed1995

Expression patterns of DNA replication enzymes and the regulatory factor DREF during Drosophila development analyzed with specific antibodies.

Specific antibodies were prepared against Drosophila DNA polymerase epsilon and DREF, a regulatory factor for DNA replication-related genes. Using these antibodies together with those for DNA polymerase alpha and proliferating cell nuclear antigen (PCNA), we examined expression patterns and sub-cellular distributions of these proteins during Drosophila development. DNA polymerase alpha, epsilon and PCNA proteins were maternally stored in unfertilized eggs and maintained at high levels during embryogenesis. With distinct nuclear localization, proteins were observed in embryos at interphase stages throughout the 13 nuclear division cycles, suggesting that they all participate in rapid nuclear DNA replication during these cycles. In contrast, maternal storage of a DREF protein was relatively low and its level increased throughout embryogenesis. Strong nuclear staining with the anti-DREF antibody was not observed until the nuclear division cycle 8. Immunostaining of various larval tissues from transgenic flies carrying the PCNA gene promoter-lacZ fusion gene revealed co-expression of DREF, PCNA and lacZ, suggesting that DREF regulates the expression of PCNA gene in these tissues. In addition, we detected a relatively high level of DREF in adult males as well as females. Since DNA polymerase alpha, epsilon and PCNA are hardly detectable in adult males, DREF very likely regulates genes other than those closely linked to DNA replication in adult males.

Animals↗
PubMed1995

Distribution of PCNA during postblastoderm cell division cycles in the Drosophila melanogaster embryo: effect of a string- mutation.

We used immunocytochemical methods and a specific antibody to identify proliferating cell nuclear antigen (PCNA) in Drosophila embryos during the postblastoderm cell division cycles. The strong nuclear staining observed during interphase disappeared at prophase, staining remained nil throughout the remaining mitotic (M) phases and reappeared in nuclei at the next interphase. As the embryos with the homozygous string- mutation sustained the strong staining signal in nuclei throughout embryonic development, disappearance of the staining signal probably depends on string function and is coupled with the onset of mitosis. When cells in embryos were arrested at M phase following treatment with TN-16 or Vinblastine, the staining signal with the anti-PCNA antibody was lost. However, Western blots showed that the level of PCNA protein in M phase-arrested embryos did not decrease. Therefore, the disappearance of staining signals is apparently due to reorganization of PCNA protein in the multiprotein complex in nuclei, rendering it inaccessible to the antibody, rather than to the degradation of PCNA protein. In contrast to findings in developing embryos, cultured Drosophila Kc cells stained strongly with the anti-PCNA antibody, during both interphase and the M phase. In genetic crossing experiments of transgenic flies carrying the lacZ gene under the control of the PCNA gene regulatory region (-607 to +137 with respect to the transcription initiation site) with string- mutant files, the PCNA gene promoter seems to function in a manner independent of cell cycle progression or of functions of the string gene.

Animals↗
PubMed1995

Role of homeodomain protein binding region in the expression of Drosophila proliferating cell nuclear antigen gene: analysis with transgenic flies.

The regulatory region of Drosophila proliferating cell nuclear antigen (PCNA) gene consists of a promoter region (-168 to +24 with respect to the transcription initiation site) and an upstream region containing three homeodomain protein binding sites (HDB) (-357 to -165). The PCNA gene regulatory regions with HDB (-607 to +137) or without HDB (-168 to +137) were fused with the lacZ and transgenic flies were established by P-element-mediated transformation. Male transgenic flies were crossed with wild-type females, and zygotic expression of the lacZ was monitored by quantitative beta-galactosidase assay, at various stages of development. Expression of the lacZ was high in embryos, first and second instar larvae, and adult females, and low at other stages of development. Only a marginal difference in expression was observed between flies carrying the homeodomain protein binding region and those not carrying it. Spatial pattern of the lacZ expression in the embryo visualized by immunostaining with the anti-lacZ antibody was similar to the distribution of the endogenous PCNA protein. Here, too, only a marginal difference was observed between transgenic flies carrying two different constructs of the PCNA lacZ. In genetic crossing experiments of transgenic flies with those carrying mutation in homeobox genes, no significant change in the lacZ expression pattern was observed. However, when male transgenic flies were crossed with female flies homozygous for a torso gain-of-function allele, repression of the lacZ expression was observed in the central region of the embryo. Because these local changes in the lacZ expression depend on the homeodomain protein binding region, unidentified homeodomain proteins are probably involved. Our results suggest that the promoter region is practically sufficient for expression of the PCNA gene and that the homeodomain protein binding region functions as a silencer when torso is activated ectopically.

Animals↗
PubMed1994

D-type cyclin-binding regions of proliferating cell nuclear antigen.

Proliferating cell nuclear antigen (PCNA) is an auxiliary protein for DNA polymerase delta and is required for both DNA replication and DNA repair. PCNA forms complexes with D-type cyclins, candidate G1 cyclins in mammalian cells. To better understand the functions of the complexes, we examined interactions between PCNA and D-type cyclins, using in vitro-translated mouse PCNA and mouse cyclin D1 or D3 fused to glutathione S-transferase (GST). Analysis of a set of deletion mutants of PCNA revealed that either the N-terminal (residues 2-64) or the C-terminal (residues 197-228) region is necessary for association with D-type cyclins. The cyclin binding of the chimeric protein of the N-terminal (residues 1-68) or the C-terminal (residues 195-261) region of PCNA and rat DNA polymerase beta which does not bind to the cyclins by itself supports this notion. The purified recombinant mouse PCNA expressed in Escherichia coli bound to the D-type cyclin-GST fusion proteins, thereby suggesting that PCNA binds directly to D-type cyclins, without the requirement of other cellular factors. This is apparently the first report on the structure-function relationship of PCNA which may link DNA replication and DNA repair with cell cycle control.

Animals↗
PubMed1994

Differential effect of p53 on the promoters of mouse DNA polymerase beta gene and proliferating-cell-nuclear-antigen gene.

A plasmid carrying the 5' flanking region of the mouse proliferating-cell-nuclear-antigen (PCNA) gene or DNA polymerase beta gene was fused with the chloramphenicol acetyltransferase (CAT) gene, then cotransfected into mouse N18TG2 cells with the expression plasmid for the p53 gene. Expression of the wild-type p53 repressed the CAT expression directed by the PCNA gene promoter, while it had little effect on the DNA polymerase beta gene promoter. RNase protection analysis revealed that the repression of the PCNA gene promoter by p53 was at the transcription step. Analysis with various deletion mutants in the PCNA gene promoter revealed that a specific sequence is not required for the repression, suggesting that p53 represses the PCNA gene promoter by interacting with some components of the basic transcription machinery. By analysis with various deletion mutants in the DNA polymerase beta gene promoter, we identified the unique 10-bp palindromic sequence (-24 to -15), in the presence of which p53 was not able to repress the promoter activity. This sequence conferred resistance to p53 repression onto the PCNA gene promoter, when it was placed 21-bp upstream from the transcription-initiation site.

Animals↗