Orbit, a novel microtubule-associated protein essential for mitosis in Drosophila melanogaster.

We describe a Drosophila gene, orbit, that encodes a conserved 165-kD microtubule-associated protein (MAP) with GTP binding motifs. Hypomorphic mutations in orbit lead to a maternal effect resulting in branched and bent mitotic spindles in the syncytial embryo. In the larval central nervous system, such mutants have an elevated mitotic index with some mitotic cells showing an increase in ploidy. Amorphic alleles show late lethality and greater frequencies of hyperploid mitotic cells. The presence of cells in the hypomorphic mutant in which the chromosomes can be arranged, either in a circular metaphase or an anaphase-like configuration on monopolar spindles, suggests that polyploidy arises through spindle and chromosome segregation defects rather than defects in cytokinesis. A role for the Orbit protein in regulating microtubule behavior in mitosis is suggested by its association with microtubules throughout the spindle at all mitotic stages, by its copurification with microtubules from embryonic extracts, and by the finding that the Orbit protein directly binds to MAP-free microtubules in a GTP-dependent manner.

Targeted expression of the DNA binding domain of DRE-binding factor, a Drosophila transcription factor, attenuates DNA replication of the salivary gland and eye imaginal disc.

The promoters of Drosophila genes encoding DNA replication-related proteins contain transcription regulatory elements consisting of an 8-bp palindromic DNA replication-related element (DRE) sequence (5'-TATCGATA). The specific DRE-binding factor (DREF), a homodimer of the polypeptide with 709 amino acid residues, is a positive trans-acting factor for transcription of DRE-containing genes. Both DRE binding and dimer formation are associated with residues 16 to 115 of the N-terminal region. We have established transgenic flies expressing the full-length DREF polypeptide or its N-terminal fragment (amino acid residues 1 to 125) under the control of the heat shock promoter, the salivary gland-specific promoter, or the eye imaginal disc-specific promoter. Heat shock induction of the N-terminal fragment during embryonic, larval, or pupal stages caused greater than 50% lethality. This lethality was overcome by coexpression of the full-length DREF. In salivary glands of the transgenic larvae expressing the N-terminal fragment, this fragment formed a homodimer and a heterodimer with the endogenous DREF. Ectopic expression of the N-terminal fragment in salivary gland cells reduced the contents of mRNAs for the 180-kDa subunit of DNA polymerase alpha and for dE2F and the extent of DNA endoreplication. Ectopic expression of the N-terminal fragment in the eye imaginal discs significantly reduced DNA replication in cells at the second mitotic wave. The lines of evidence suggest that the N-terminal fragment can impede the endogenous DREF function in a dominant negative manner and that DREF is required for normal DNA replication in both mitotic cell cycle and endo cycle.

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.

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.

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.

Ectopic expression of DREF induces DNA synthesis, apoptosis, and unusual morphogenesis in the Drosophila eye imaginal disc: possible interaction with Polycomb and trithorax group proteins.

The promoters of Drosophila genes encoding DNA replication-related proteins contain transcription regulatory element DRE (5'-TATCGATA) in addition to E2F recognition sites. A specific DRE-binding factor, DREF, positively regulates DRE-containing genes. In addition, it has been reported that DREF can bind to a sequence in the hsp70 scs' chromatin boundary element that is also recognized by boundary element-associated factor, and thus DREF may participate in regulating insulator activity. To examine DREF function in vivo, we established transgenic flies in which ectopic expression of DREF was targeted to the eye imaginal discs. Adult flies expressing DREF exhibited a severe rough eye phenotype. Expression of DREF induced ectopic DNA synthesis in the cells behind the morphogenetic furrow, which are normally postmitotic, and abolished photoreceptor specifications of R1, R6, and R7. Furthermore, DREF expression caused apoptosis in the imaginal disc cells in the region where commitment to R1/R6 cells takes place, suggesting that failure of differentiation of R1/R6 photoreceptor cells might cause apoptosis. The DREF-induced rough eye phenotype was suppressed by a half-dose reduction of the E2F gene, one of the genes regulated by DREF, indicating that the DREF overexpression phenotype is useful to screen for modifiers of DREF activity. Among Polycomb/trithorax group genes, we found that a half-dose reduction of some of the trithorax group genes involved in determining chromatin structure or chromatin remodeling (brahma, moira, and osa) significantly suppressed and that reduction of Distal-less enhanced the DREF-induced rough eye phenotype. The results suggest a possibility that DREF activity might be regulated by protein complexes that play a role in modulating chromatin structure. Genetic crosses of transgenic flies expressing DREF to a collection of Drosophila deficiency stocks allowed us to identify several genomic regions, deletions of which caused enhancement or suppression of the DREF-induced rough eye phenotype. These deletions should be useful to identify novel targets of DREF and its positive or negative regulators.

E2F-dependent transcription of the raf proto-oncogene during Drosophila development.

D-raf, a Drosophila homolog of the raf proto-oncogene, has diverse functions throughout development and is transcribed in a wide range of tissues, with high levels of expression in the ovary and in association with rapid proliferation. The expression pattern resembles those of S phase genes, which are regulated by E2F transcription factors. In the 5'-flanking region of D-raf, four sequences (E2F sites 1-4) similar to the E2F recognition sequence were found, one of them (E2F site 3) being recognized efficiently by Drosophila E2F (dE2F) in vitro. Transient luciferase expression assays confirmed activation of the D-raf gene promoter by dE2F/dDP. Expression of Draf-lacZ was greatly reduced in embryos homozygous for the dE2F mutation. These results suggest that dE2F is likely to be an important regulator of D-raf transcription.

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.

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.

Inhibitory effect of 1-beta-D-arabinofuranosylthymine 5'-triphosphate and 1-beta-D-arabinofuranosylcytosine 5'-triphosphate on DNA polymerases from murine cells and oncornavirus.

This report compares the effect of the newly synthesized 1-beta-D-arabinofuranosylthymine 5'-triphosphate with 1-beta-D-arabinofuranosylcytosine 5'-triphosphate on the activity of DNA polymerases from mouse cells and oncornavirus. 1-beta-D-Arabinofuranosylthymine 5'-triphosphate inhibited all the activities of DNA polymerase alpha, beta, and gamma and viral DNA polymerase. The mode of inhibition of 1-beta-D-arabinofuranosylthymine 5'-triphosphate as well as 1-beta-D-arabinofuranosylcytosine 5'-triphosphate was competitive to the deoxynucleoside triphosphate with the same base. The inhibition constant (Ki) and the mode of inhibition of nucleotide incorporation varied with changes in the combination of the inhibitor, substrate(s), and enzyme species.

Inhibitory effects of 9-beta-D-arabinofuranosylguanine 5'-triphosphate and 9-beta-D-arabinofuranosyladenine 5'-triphosphate on DNA polymerases from murine cells and oncornavirus.

The effects of the newly synthesized compound 9-beta-D-arabinofuranosylguanine 5'-triphosphate (ara-GTP) on the activity of DNA polymerases from mouse cells and oncornavirus were compared with those of 9-beta-D-arabinofuranosyladenine 5'-triphosphate. Ara-GTP did not replace deoxyguanosine 5'- triphosphate as substrate for these DNA polymerases but inhibited the activities of DNA polymerase alpha, beta, and gamma and viral DNA polymerase. DNA polymerase alpha was more sensitive than DNA polymerases beta and gamma and viral DNA polymerase to inhibition by ara-GTP. The inhibitions by ara-GTP and 9-beta-D-arabinofuranosyladenine 5'-triphosphate were due to competition or partial competition 5'-triphosphate were due to competition or partial competition with deoxynucleoside triphosphate with the same base. The inhibition constant (Ki) and the mode of inhibition of nucleotide incorporation varied depending on the combination of inhibitor, substrate(s), and enzyme species.

Ectopic expression of human p53 inhibits entry into S phase and induces apoptosis in the Drosophila eye imaginal disc.

Transgenic flies in which ectopic expression of human p53 was targeted to the Drosophila eye imaginal disc were established. On sectioning of adult fly eyes which displayed a severe rough eye phenotype, most ommatidia were found to be fused and irregular shapes of rabdomeres were observed. In addition, many pigment cells were lost. In the developing eye imaginal disc, photoreceptor cell differentiation was initiated normally despite the ectopic expression of p53. However, expression of p53 inhibited cell cycle progression in eye imaginal disc cells and the S phase zone (the second mitotic wave) behind the morphogenetic furrow was almost completely abolished. Furthermore, expression of p53 induced extensive apoptosis of eye imaginal disc cells, and co-expression of baculovirus P35 in the eye imaginal disc suppressed the p53-induced rough eye phenotype. These results are consistent with the known functions of human p53 and indicate the existence of signaling systems with elements corresponding to human p53 in Drosophila eye imaginal disc cells. Genetic crosses of transgenic flies expressing p53 to a collection of Drosophila deficiency stocks allowed us to identify several genomic regions, deletions of which caused enhancement or suppression of the p53-induced rough eye phenotype. The transgenic flies established in this study should be useful to identify novel targets of p53 and its positive or negative regulators in Drosophila.

The biochemical inhibition mode of bredinin-5'-monophosphate on DNA polymerase beta.

We reported previously [T. Horie, Y. Mizushina, M. Takemura, F. Sugawara, A. Matsukage, S. Yoshida, K. Sakaguchi, Int. J. Mol. Med., 1 (1998) 83-90.] that a 5'-monophosphate form (breMP) of bredinin, which has been used clinically as an immunosuppressive drug, selectively suppressed the activities of mammalian DNA polymerase alpha (pol. alpha) and beta (pol. beta). In a preliminary study of the action mode, for pol. beta, breMP acted by competing with, unexpectedly, not only the substrate but also with the template-primer. The mode might be attributable to the structure and function of pol. beta itself. We therefore investigated the biochemical inhibition mode of pol. beta in more detail by using two pol. beta fragments which were proteolytically separated into the template-primer-binding domain and the catalytic domain. BreMP inhibited only the catalytic activity of the catalytic domain fragment, and could not bind to the template-primer-binding domain fragment, suggesting that it directly competes with the substrate at its binding site of the catalytic domain, and indirectly, but simultaneously and competitively disturbs the template-primer incorporation into the template-primer-binding domain.

The inhibitory action of fatty acids on DNA polymerase beta.

We found previously that long-chain fatty acids could inhibit eukaryotic DNA polymerase activities in vitro [1,2]. The purpose of the present study was to investigate the mode of this inhibition in greater detail. Among the C18 to C24 fatty acids examined, the strongest inhibitor was a C24 fatty acid, nervonic acid (NA), and the weakest was a C18 fatty acid, linoleic acid (LA). We analyzed the inhibitory effect of these two fatty acids and their modes of action. For DNA polymerase beta (pol. beta), NA acted by competing with both the substrate- and template-primer, but for DNA polymerase alpha (pol. alpha) or human immunodeficiency virus type 1 reverse transcriptase (HIV-1 reverse transcriptase or HIV-RT), NA acted non-competitively. NA-binding to pol. beta could be stopped with a non-ionic detergent, but the binding to pol. alpha or HIV-RT could not. The inhibition mode of LA showed the same characteristics, except that the minimum inhibitory dose of the longer chain was much lower. We also tested the effects of NA and LA using pol. beta and its proteolytic fragments, as described by Kumar et al. [3,4]. Both of the fatty acids were found to bind to the 8 kDa DNA-binding domain fragment, and to suppress binding to the template-primer DNA. We found that 10,000 times more of either fatty acid was required for it to bind to the 31 kDa catalytic domain or inhibit the DNA polymerase activity. The possible modes of inhibition by these long-chain fatty acids are discussed, based on the present findings.

Differential sensitivity of low molecular weight DNA polymerase to sulfhydryl-blocking reagents.

Activity of a 2.5 S mouse myeloma DNA polymerase (termed DNA polymerase II) measured with either poly(rA) or poly(dA) as template did not require sulfhydryl-reducing reagents, but was sensitive to inhibition by p-hydroxymercuribenzoate and the sulfhydryl-alkylating reagent, N-ethylmaleimide; however, the activity was much more sensitive to inhibition by p-hydroxymercuribenzoate than by the sulfhydryl-alkylating reagent. The p-hydroxymercuribenzoate inhibition appeared to involve the mercurial portion of the p-hydroxymercuribenzoate molecule because HgCl2 was an equally effective inhibitor, while p-hydroxybenzoate had little effect upon enzyme activity. The p-hydroxymercuribenzoate inhibition was reversed by an equal concentration of the sulfhydryl-reducing reagent, dithiothreitol.

Identification of a polypeptide component of mouse myeloma DNA polymerase gamma.

Mouse myeloma DNA polymerase gamma was extensively purified to a final specific activity of 156 000 units (nmol dTMP incorporation per h) per mg protein on (rA)n.(dT)12-18 as a template primer. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of protein fractions obtained by DNA-cellulose column chromatography revealed that the amount of a polypeptide of Mr = 47 000 changed proportionally with DNA polymerase gamma activity. A minor polypeptide of Mr = 140 000 also seemed to change with the enzyme activity, but other polypeptides did not. Analysis by 125I-labeled peptide mapping indicates that the Mr 47 000 polypeptide in the mouse myeloma DNA polymerase gamma preparation is structurally related to the Mr 47 000 polypeptide of chick embryo DNA polymerase gamma (Yamaguchi, M., Matsukage, A. and Takahashi, T. (1980) J. Biol. Chem. 255, 7002-7009). An antibody against chick embryo DNA polymerase gamma cross-reacted with the mouse enzyme, indicating a structural relationship between avian and murine enzymes. Since the Mr 47 000 polypeptide accounts for 31.4% of total protein in the purified preparation, the specific activity is estimated to be about 490 000 units per mg of the Mr 47 000 polypeptide. The rate of poly(dT) elongation by the purified enzyme was 1 260 per nucleotides per min. This value is in the same range as the turnover number (1 530 nucleotides per min per enzyme molecule) which is calculated from the 'expected' specific activity with respect to the Mr 47 000 polypeptide and the molecular weight (Mr = 188 000 on the assumption of a tetrameric structure of the Mr 47 000 polypeptide). Results indicate that the Mr 47 000 polypeptide is a component of the mouse myeloma DNA polymerase gamma.

Transcription of the catalytic 180-kDa subunit gene of mouse DNA polymerase alpha is controlled by E2F, an Ets-related transcription factor, and Sp1.

We have isolated a genomic DNA fragment spanning the 5'-end of the gene encoding the catalytic subunit of mouse DNA polymerase alpha. The nucleotide sequence of the upstream region was G/C-rich and lacked a TATA box. Transient expression assays in cycling NIH 3T3 cells demonstrated that the GC box of 20 bp (at nucleotides -112/-93 with respect to the transcription initiation site) and the palindromic sequence of 14 bp (at nucleotides -71/-58) were essential for basal promoter activity. Electrophoretic mobility shift assays showed that Sp1 binds to the GC box. We also purified a protein capable of binding to the palindrome and identified it as GA-binding protein (GABP), an Ets- and Notch-related transcription factor. Transient expression assays in synchronized NIH 3T3 cells revealed that three variant E2F sites near the transcription initiation site (at nucleotides -23/-16, -1/+7 and +17/+29) had no basal promoter activity by themselves, but were essential for growth-dependent stimulation of the gene expression. These data indicate that E2F, GABP and Sp1 regulate the gene expression of this principal replication enzyme.

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.

Identification of ter94, Drosophila VCP, as a modulator of polyglutamine-induced neurodegeneration.

We have successfully generated a Drosophila model of human polyglutamine (polyQ) diseases by the targeted expression of expanded-polyQ (ex-polyQ) in the Drosophila compound eye. The resulting eye degeneration is progressive and ex-polyQ dosage- and ex-polyQ length-dependent. Furthermore, intergenerational changes in repeat length were observed in homozygotes, with concomitant changes in the levels of degeneration. Through genetic screening, using this fly model, we identified loss-of-function mutants of the ter94 gene that encodes the Drosophila homolog of VCP/CDC48, a member of the AAA+ class of the ATPase protein family, as dominant suppressors. The suppressive effects of the ter94 mutants on ex-polyQ-induced neurodegeneration correlated well with the degrees of loss-of-function, but appeared not to result from the inhibition of ex-polyQ aggregate formation. In the ex-polyQ-expressing cells of the late pupa, an upregulation of ter94 expression was observed prior to cell death. Co-expression of ter94 with ex-polyQ severely enhanced eye degeneration. Interestingly, when ter94 was overexpressed in the eye by increasing the transgene copies, severe eye degeneration was induced. Furthermore, genetical studies revealed that ter94 was not involved in grim-, reaper-, hid-, ced4-, or p53-induced cell death pathways. From these observations, we propose that VCP is a novel cell death effector molecule in ex-polyQ-induced neurodegeneration, where the amount of VCP is critical. Control of VCP expression may thus be a potential therapeutic target in ex-polyQ-induced neurodegeneration.

Crystallization of 31 kDa C-terminal fragment of rat DNA polymerase beta.

A 248 residue C-terminal fragment of rat DNA polymerase beta (335 amino acid residues), a eukaryotic DNA repair enzyme, has been crystallized from polyethylene glycol 6000 solution. The crystals are orthorhombic, space group P2(1)2(1)2 with cell dimensions a = 120.3 A, b = 64.2 A, c = 39.4 A, and contain a single 31 kDa fragment in an asymmetric unit. The crystals diffract to 2.8 A resolution with laboratory X-ray source, and to 2.3 A resolution with synchrotron X-ray source, and are suitable for detailed structural analysis.