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F Hirose

Publications and source records attributed to F Hirose.

At least 37 records · Page 2Linked to original sources
PubMed1997

DNA polymerase epsilon from Drosophila melanogaster.

We identified a DNA polymerase species in Drosophila melanogaster embryos, and purified it. This polymerase shared some common properties with DNA polymerase epsilon from mammals and yeast as follows; it has a preference for poly(dA)/oligo(dT) as a template/primer, it is highly processive in DNA synthesis, it co-fractionates with 3'-5' exonuclease activity, it is sensitive to aphidicolin and is resistance to ddTTP. The polymerase activity was inhibited in the immuno-precipitation assay with anti-pol-epsilon antibodies, which were produced against a polypeptide coded on the cDNA of a putative Drosophila pol-epsilon we isolated previously. Using these antibodies, Western blot analysis revealed that this polymerase is a 250kDa polypeptide, which is the same size as observed in mammals and yeast. These results indicate that Drosophila produces the epsilon-class of DNA polymerase, and like mammals or yeast, possesses the 5 typical classes of DNA polymerases (alpha to epsilon) in its embryos.

Animals↗
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

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

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

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↗
PubMed1994

Repression of regulatory factor for Drosophila DNA replication-related gene promoters by zerknüllt homeodomain protein.

The expression of the chloramphenicol acetyltransferase gene under control of the 1152-base pair 5'-flanking region (-1107 to +45 nucleotide positions with respect to the major transcription initiation site) of the Drosophila DNA polymerase alpha gene was repressed by cotransfection into Drosophila Kc cells with a zerknüllt (zen)-expressing plasmid as previously observed with the proliferating cell nuclear antigen (PCNA) gene promoter. The expression of the zen resulted in reduction of the abundance of mRNA for the transfected chloramphenicol acetyltransferase gene and also mRNAs for both DNA polymerase alpha and PCNA. Results obtained using various deletion derivatives of the promoter region and chemically synthesized oligonucleotides of the DNA replication-related element (DRE), a positive cis-acting element found in both DNA polymerase alpha and PCNA genes, revealed that the DRE sequences are responsible to repression by Zen protein. The nuclear extract of Kc cells transfected by the zen-expressing plasmid contained lesser amounts of the DRE-binding factor (DREF) than that of untransfected or mutant zen-transfected cells. These results suggest that the Zen protein represses expression of DNA replication-related genes by reducing DREF, although the detailed mechanism of the repression remains to be elucidated.

Animals↗
PubMed1993

Novel 8-base pair sequence (Drosophila DNA replication-related element) and specific binding factor involved in the expression of Drosophila genes for DNA polymerase alpha and proliferating cell nuclear antigen.

Upstream regions containing a novel common 8-base pair (bp) palindromic sequence, 5'-TATCGATA (Drosophila DNA replication-related element (DRE)), are required for the high expression of Drosophila genes for DNA polymerase alpha and the proliferating cell nuclear antigen (PCNA) (an auxiliary protein for DNA polymerase delta). Three DREs and one DRE are present in the DNA polymerase alpha gene (nucleotides-217, -83, and -30 with respect to the transcription initiation site) and in the PCNA gene (nucleotide-100), respectively. Deletions or 2-bp insertional mutations of DRE sequences led to an extensive reduction of promoter activities of both genes. Chemically synthesized oligonucleotides containing DRE sequences greatly stimulated the activity of the heterologous promoter of the Drosophila metallothionein gene, in addition to the promoter of the PCNA gene, when they were placed upstream from these promoters in a normal or a reverse orientation. The stimulatory effect increased synergistically and depended on the number of DREs. DRE activated the promoter when placed within 1.4 kilobases upstream from the promoter, but was much less active when placed 2.5 kilobases or more apart from the promoter. Using a gel mobility shift assay method, we obtained evidence for a protein factor (DREF) in the nuclear extract of cultured Drosophila cells (Kc cells), and this factor specifically binds to DREs of both genes. DNase I footprinting analysis indicated that DREF binds to the 24-bp DRE region of the DNA polymerase alpha gene in which 8-bp palindromic sequences are centered. A UV cross-linking experiment revealed that a polypeptide of approximately 90 kDa in the nuclear extract interacts directly with the DRE sequence. Using DRE-conjugated latex particles, DREF was affinity-purified from the Kc cell nuclear extract. By comparing results obtained by SDS-polyacrylamide gel electrophoresis and gel mobility shift experiments, we concluded that DREF is associated with the 86-kDa polypeptide. On gel filtration chromatography, a single peak of DREF activity was recovered in fractions corresponding to a molecular mass of 170 kDa, and the 86-kDa polypeptide was detected only in the corresponding fractions; thus, active DREF is probably a homodimeric form of the 86-kDa polypeptide. DREF may play important roles in coordinating expressions of Drosophila DNA replication-related genes.

Animals↗
PubMed1992

Activation of the mouse DNA polymerase beta gene promoter by adenovirus type 12 E1A proteins.

A plasmid carrying the 5'-flanking region (-1852 to +33 with respect to the transcription initiation site) of the mouse DNA polymerase beta gene fused with the chloramphenicol acetyltransferase (CAT) gene was cotransfected into mouse N18TG2 cells with adenovirus type 12 E1 genes-expressing plasmids. Expression of E1A gene products resulted in the elevation of the CAT expression by 3 to 7 folds, but that of E1B gene product was much less effective. RNase protection analysis revealed that the activation by E1A was at the transcription process. Both the 13S E1A and the 12S E1A activated the DNA polymerase beta gene promoter, indicating that the activation domain of E1A is in a common region(s) of 13S and 12S E1A products. The major target sequence of E1A was mapped within the 10 base pair-region (-30 to -20) of the DNA polymerase beta gene promoter, which overlapped with the palindromic sequence known as the ATF(CREB)/E4F-binding consensus. The results suggest that the palindromic sequence is essential for E1A-induced transcriptional activation of the mouse DNA polymerase beta gene.

Adenoviridae↗
PubMed1992

Activation of the mouse proliferating cell nuclear antigen gene promoter by adenovirus type 12 E1A proteins.

A plasmid carrying the 5'-flanking region (-1584 to +47 with respect to the transcription initiation site) of the mouse proliferating cell nuclear antigen (PCNA) gene was fused with the chloramphenicol acetyltransferase (CAT) gene, and then cotransfected into mouse N18TG2 cells with expression plasmids for the adenovirus type 12 E1 genes. Expression of E1A gene products elevated the CAT expression by 5- to 9-fold, but expression of the E1B gene product did not. RNase protection analysis revealed that the activation of the PCNA gene promoter by E1A was at the transcription step. Both the 13S E1A and the 12S E1A activated the PCNA gene promoter, indicating that the activation domain of E1A resides in a common region(s) of 13S and 12S E1A products. The major target region of E1A was mapped within the 68 base-pair region (-21 to +47) of the PCNA gene, which includes consensus sequences for transcription factors PEA3 and E2F, although the upstream region (-83 to -21) including ATF(CREB)-binding consensus had an additional effect in the transactivation.

Adenovirus Early Proteins↗