Mitsuoki Morimyo

The construction of an EST database for Bombyx mori and its application

To build a foundation for the complete genome analysis of Bombyx mori, we have constructed an EST database. Because gene expression patterns deeply depend on tissues as well as developmental stages, we analyzed many cDNA libraries prepared from various tissues and different developmental stages to cover the entire set of Bombyx genes. So far, the Bombyx EST database contains 35,000 ESTs from 36 cDNA libraries, which are grouped into ≈11,000 nonredundant ESTs with the average length of 1.25 kb. The comparison with FlyBase suggests that the present EST database, SilkBase, covers >55% of all genes of Bombyx. The fraction of library-specific ESTs in each cDNA library indicates that we have not yet reached saturation, showing the validity of our strategy for constructing an EST database to cover all genes. To tackle the coming saturation problem, we have checked two methods, subtraction and normalization, to increase coverage and decrease the number of housekeeping genes, resulting in a 5–11% increase of library-specific ESTs. The identification of a number of genes and comprehensive cloning of gene families have already emerged from the SilkBase search. Direct links of SilkBase with FlyBase and WormBase provide ready identification of candidate Lepidoptera-specific genes.

Introduction of a foreign gene into medakafish using the particle gun method

We developed a procedure to introduce a foreign gene into fertilized eggs of medakafish (Oryzias latipes) using the particle gun method, which is one of the easiest and most reliable techniques for gene transfer. A plasmid construct with the green fluorescence protein (GFP) gene driven by the madakafish beta-actin gene promoter was successfully introduced into eggs, and the expression of GFP was observed in 20% of the primary transfectant (chimera) fish. In addition, germ line transmission of GFP was observed in 13% of the GFP-positive primary transfectant fish. The new application described here should enable us to investigate gene expression using the fish model on a routine basis without high technical sophistication. J. Exp. Zool. 287:285-293, 2000.

Transcription Dependence and the Roles of Two Excision Repair Pathways for UV Damage in Fission Yeast Schizosaccharomyces pombe

Fission yeasts Schizosaccharomyces pombe possess two types of excision repair systems for UV-induced DNA damage, nucleotide excision repair (NER) and UV-damaged DNA endonuclease (UVDE)-dependent excision repair (UVER). Despite its high efficiency in damage removal, UVER defects have less effect on UV survival than NER defects. To understand the differential roles of two pathways, we examined strand-specific damage removal at the myo2 and rpb2 loci. Although NER removes cyclobutane pyrimidine dimers from the transcribed strand more rapidly than from the nontranscribed strand, UVER repairs cyclobutane pyrimidine dimers equally on both strands and at a much higher rate than NER. The low rate of damage removal from the nontranscribed strand in the absence of UVER indicates inefficient global genome repair (GGR) in this organism and a possible function of UVER as an alternative to GGR. Disruption of rhp26, the S. pombe homolog of CSB/RAD26, eliminated the strand bias of NER almost completely and resulted in a significant increase of UV sensitivity of cells in a uvdeΔ background. We suggest that the combination of transcription-coupled repair of NER and rapid UVER contributes to UV survival in growing S. pombe cells, which is accomplished by transcription-coupled repair and GGR in other organisms.

Evidence that the geneuvrB is indispensable for a polymerase I deficient strain ofEscherichia coli K-12

SummaryConclusive evidence in presented to show that the geneuvrA is dispensable, but theuvrB is indispensable for anEscherichia coli strain carrying genepolA1. We constructed strains E139 (sup-126 polA1 uvrB59) and E159 (sup-126 polA1 uvrA43) where mutationspolA1, uvrB59 anduvrA43 are amber mutations and mutationsup-126 is an amber suppressor mutation effective at 30°C but not at 42°C. At 42°C, strain E139 is inviable but strain E159 viable whereas both are viable at 30°C. Revertants of E139 viable at 42°C occurred spontaneously at a frequency of about 3×10-4. One of the revertants was shown to be caused by suppressor mutation, designatedspu, rather than back mutation of the geneuvrB59 orpolA1 or amber suppressor mutation. Viabilities of the revertants varied from 10-3 to 1.0 at 42°C compared with those at 30°C. At 42°C, all the revertants with normal viabilities at 42°C were non-filamentous in contrast to the filamentous character of E139. However, strain E159 was viable at 42°C despite its filamentous character. We conclude that the geneuvrB is involved not only in excision repair but also in normal growth in apolA background.

CLONING AND SEQUENCING FOR THE LARGEST SUBUNIT OF CHINESE HAMSTER RNA POLYMERASE II GENE : IDENTIFICATION OF A MUTATION RELATED TO ABNORMAL INDUCTION OF SISTER CHROMATID EXCHANGES

In order to analyze the mutation sites related to abnormal induction of sister chromatid exchanges (SCEs) in the RNA polymerase II largest subunit (RpII LS) gene of the Chinese hamster CHO-KI cell mutant, we have completely sequenced the whole region of the RpII LS cDNAs obtained from normal and mutant cells. By comparing both sequences, a mutation that results in an amino acid (aa) change in the RpII LS gene was found. This aa change was Pro (CCC) to Ser (TCC) at position 1006. Multiple alignment for aa sequences of RpII LS from various species revealed that this Pro residue was highly conserved throughout the eukaryotes. Considering the differences in physico-chemical properties between Pro and Ser residues, the Pro-->Ser substitution may alter the RpII LS structure.

Mouse cdc21 only 0.5 kb upstream from dna-pkcs in a head-to-head organization: an implication of co-evolution of ATM family members and cell cycle regulating genes.

The catalytic subunit of the DNA-dependent protein kinase (DNAPKcs) is a member of the ATM family, which in turn is a branch of the phosphatidylinositol 3-kinase (PI3K) superfamily (Hartley et al. 1995). The ATM family consists of relatively large proteins, all of which have motifs found in PI3Ks’ catalytic domains at their carboxy-terminal region. Many of the family members have been found to be involved in DNA repair, cell-cycle checkpoint control, and cell-cycle transition control (Zakian 1995). Furthermore, the ATM gene itself was demonstrated to be the responsible gene for the genetic disorder ataxia telangiectasia (AT) with a wide spectrum of clinical manifestations including hypersensitivity to ionizing radiation and radiomimetic drugs (Savitsky et al. 1995). The hypersensitivity is due to the dysfunction of radiation-induced checkpoint control in AT cells (Shiloh 1995). DNA-PKcs with Ku components catalyzes double-stranded broken DNA-dependent phosphorylation of proteins (Gottlieb and Jackson 1993), suggesting that DNA-PK is a surveyor of damaged DNA. Although several biochemical and mapping studies suggested that the mouse geneDna-pkcsmight be thescid-responsible gene, the exact nature of the mutation remained unknown. Very recent reports described a candidate mutation of the gene (Blunt et al. 1996; Danska et al. 1996), and we made the definitive identification of the scid mutation inDna-pkcs gene (Araki et al. 1997). The last study proved that the truncation of the DNA-PK csin the carboxy-terminal kinase domain is indeed responsible for the scid mutation. In the following course of studying the mouse promoter region of Dna-pkcs, we obtained several genomic clones that corresponded to the 5 8 end portion of the gene. Nucleotide sequencing of these clones confirmed a homology stretch to the previously determined 5 8 end sequence of the cDNA of Dna-pkcs. The first exon with the putative initial methionine of Dna-pk cs (Araki et al. 1997) was identified, and the successive upstream sequence was also determined (Fig. 1; DDBJ/EMBL/GenBank accession number AB000629). The TFD transcription factor DNA binding site database search with this sequence suggested a variety of ciselemental motifs for transcription factors. In Fig. 1, only basic motifs such as two Sp1 boxes and three CCAAT boxes are indicated. The nucleotide sequence databases search unexpectedly revealed a complete homology stretch to Cdc21(Kimura et al. 1995) in the promoter region. This homologous sequence is also indi-

Expression of the bombyx mori β-tubulin-encoding gene in testis

Abstract We have isolated a β-tubulin-encoding cDNA clone of Bombyx mori from testes and determined the nucleotide sequence. Northern analyses showed that its expression is testis-specific and most active in the pupal stage.

ALTERATION OF THE LARGEST SUBUNIT OF RNA POLYMERASE II AND ITS EFFECT ON CHROMOSOME STABILITY IN SCHIZOSACCHAROMYCES POMBE

Abstract A mutation in the RNA polymerase II largest subunit (RpII LS) that is related to abnormal induction of sister chromatid exchange has previously been described the CHO-K1 cell mutant tsTM4. To elucidate the molecular basis of this effect we introduced the mutation into the homologous site in the Schizosaccharomyces pombe rpb1 gene, which encodes RpII LS. Since the tsTM4 mutant exhibited a decrease in the rate of DNA synthesis in cells arrested in S phase at the nonpermissive temperature, we focussed on the study of growth, the cell cycle, and chromosome stability at various temperatures. First, we examined the effects of the mutation on haploid yeast cells. The mutant showed slower growth than the wild type, but cell growth was not arrested at the nonpermissive temperature. When growing cells were shifted to the nonpermissive temperature, an accumulation of cells in G1 and/or G0 was observed. Tetrad analysis suggested that these phenotypes were associated with the mutation. In diploid cells, chromosome instability was detected by loss of intragenic complementation between two alleles of the ade6 gene. An abnormal fraction of cells containing an intermediate DNA content was also observed by FACS analysis. The accumulation of this fraction may reflect the fact that a large number of cells are in S phase or have an abnormal DNA content as a result of chromosome instability. These observations demonstrate that the S. pomberpb1 mutant exhibits a phenotype very similar to that of the CHO-K1 cell mutant tsTM4.

CHO · K1 cell mutants sensitive to active oxygen-generating agents. I. Isolation and genetic studies

Nine mutants isolated from CHO.K1 cells with increased sensitivity to the lethal effect of plumbagin (PG), a powerful superoxide generator, were classified into five groups, A-E, according to their sensitivity to PG and methyl viologen (MV). Two mutants of group B (Pa13 and Pb4) were sensitive to both drugs, and two mutants of group C (Pa14 and Pa15) were moderately sensitive to PG and extremely sensitive to MV. To mitomycin C (MMC) these mutants showed cross-sensitivity; especially Pa13 and Pb4 (group B) were highly sensitive to MMC. Genetic complementation analyses of these four mutants were carried out using MV sensitivity. Sensitivity group B was divided into two complementation group, I and II. Pa14 and Pa15 belonged to the same complementation group III. These four mutants were also classified into three complementation groups for MMC sensitivity. Because Pa13 and Pb4 were also sensitive to cis-diamminedichloroplatinum(II), they may have a defect in the repair of DNA crosslinks induced by these agents. A complementation group IV (Pa2 and Pa8) was also suggested based on the studies of MMC sensitivity.

Genetic Analysis of ultraviolet-sensitive mutants of Escherichia coli K12; URT−43 (urt-1) and KMBL99dar3, carrying temperature-sensitive mutations at the locus uvrA

Abstract URT- 43 ( urt-1 ) is a thermosensitive ultraviolet-sensitive mutant of Escherichia coli K12. A second mutant KMBL 99 dar3 also has properties almost identical with those of URT-43. To perform complementation analysis with respect to ultraviolet sensitivity, merodiploid strains carrying urt-1 or dar3 on the host chromosome and uvrA6 on an F′ episome were constructed. Neither the mutation urt-1 nor dar3 complemented the uvrA6 mutation. Transductional crosses with P1 phage were carried out among mutants uvr6, urt-1 and dar3 : the pairwise combination all gave rise to only 0.1 ∼ 0.6% ultraviolet-resistant transductants among those selected for the nearby malB marker. It is thus concluded that both urt-1 and dar3 mutations are located within the uvrA cistron.