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E Akaboshi

Publications and source records attributed to E Akaboshi.

18 recordsLinked to original sources
PubMed1999

Recognition of sequence-directed structure of the ssDNA backbone by nucleases.

Escherichia coli endonuclease I and exonuclease VII appear to recognize sequence-dependent conformations in the ssDNA backbone. ssDNAs, containing either A- and/or T-tract or a CAP binding region, were digested with these nucleases under conditions which minimize the formation of secondary structures. The digestion patterns were examined in relation to previous results of biochemical and crystallographic studies on dsDNA, and showed broad agreement. Endonuclease I cleaved ssDNA at sites corresponding to bent sites in dsDNA.

Bacterial Proteins↗
PubMed1994

Cloning of the cDNA and genomic DNA that correspond to the recA-like gene of Drosophila melanogaster.

We have isolated a cDNA homologous to the yeast DMC1 and RAD51 genes from Drosophila melanogaster. The DMC1 and RAD51 genes of Saccharomyces cerevisiae are known to play crucial roles during meiosis and during both meiosis and mitosis, respectively, and their gene products are homologous to each other and to the RecA protein of Escherichia coli. The cDNA cloned here contains an open reading frame that encodes 336 amino acids. Sequence analysis of the corresponding genomic DNA fragment showed one short intron existing in the coding region as in the DMC1 gene, but not in the RAD51 gene. By in situ hybridization to the salivary gland chromosomes, the recA-like gene was cytologically mapped to 99D of the third chromosome.

Amino Acid Sequence↗
PubMed1990

RecA-ssDNA interaction: induced strand cleavage by hydroxyl radical at a defined distance from the 5' end.

Interaction of the RecA protein with single-stranded DNA (ssDNA) was analyzed by challenge with the hydroxyl radical, which can cleave the DNA backbone. We found that RecA protein induces cleavage by the radical at a defined distance from the 5' end. The cleavage was at the 11th nucleotide in many oligodeoxynucleotides. Cleavage may be intermittent since a second cleavage was induced at the 22nd or 21st site. This specific cleavage was observed under optimal conditions for filament formation, homologous pairing and strand exchange. Specificity in cleavage was, however, decreased by replacement of ATP by adenosine 5'-(gamma-thio)triphosphate (ATP gamma S), replacement of RecA protein by a mutant (RecA1) protein, or an increase in Mg2+ concentration. We propose that RecA protein induces a special structural alteration, such as bending, perhaps sequentially, on ssDNA and that this altered site plays an important role in homologous pairing and strand exchange.

Adenosine Triphosphate↗
PubMed1990

DNase I footprinting analysis of RecA protein polymerized on DNA during strand exchange reaction between a gapped circle and a linear duplex.

RecA protein mediates homologous pairing and strand exchange reactions between a circular duplex with a single strand gap and a linear duplex. We have used the DNase I footprinting method to analyze processes involving four strands during these reactions. We asked how the length of DNA protected by RecA protein changes as these reactions proceed. We compared two kinds of gapped DNAs. We found that RecA protein polymerizes rapidly in the forward direction (5' to 3' with respect to the single strand). We found, however, that polymerization in the reverse direction was more prominent with a duplex carrying a longer gap than one carrying a shorter gap. DNase I footprints showing protection by RecA protein were obtained only at limited nuclease concentrations, which in turn depended on the position of the end label and the stage of the strand exchange reaction. As judged by the concentrations of DNase I good for footprinting, the extent of protection by RecA protein was greatest for (+) single-stranded DNA at its first binding site, next highest for heteroduplex containing this (+) strand, and least for the gapped homoduplex DNA. These differences in DNase I sensitivity can be explained in terms of differences in the accessibility of various strands on the basis of a three-dimensional model for the strand exchange reaction.

Binding Sites↗
PubMed1989

Proteins induced by DNA-damaging agents in cultured Drosophila cells.

In Drosophila cultured cells, the effects of several DNA-damaging agents on the expression of proteins were investigated. Poly(A+) RNA prepared from both untreated cells and cells treated with DNA-damaging agents was translated in vitro. The translation products were analyzed by two-dimensional electrophoresis. Methyl methanesulfonate, the most potent agent used, induced about 25 proteins, some new and some enhanced pre-existing proteins. Angelicin plus near UV irradiation, 4-nitroquinoline N-oxide and ethyl methanesulfonate were efficient inducers. Mitomycin C, UV irradiation and hydrogen peroxide were poor inducers, inducing only a few proteins at low levels. A tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, and a DNA gyrase inhibitor, nalidixic acid, also were used. In this system they were weak inducers of new proteins. Several of the new or enhanced proteins were common to several agents, but others were agent specific. The distribution of mutagen-induced proteins was compared with that of proteins induced in cells heated at 37 degrees C. Some of the proteins induced by DNA-damaging agents were found to overlap heat-shock proteins. These results suggest that there are sets of induced genes that are regulated differently.

Animals↗
PubMed1985

Cloning and sequence analysis of porcine myoglobin cDNA.

Porcine myoglobin cDNA clones have been isolated from a cDNA library prepared from enriched heart-myoglobin mRNA. Sequence analysis revealed 59 nucleotides (nt) in the 5'-untranslated, 462 nt in the amino acid (aa)-coding, and 590 nt in the 3'-untranslated regions. The myoglobin cDNA showed a high G + C content (60%). When the nt sequence of the porcine myoglobin cDNA is compared with those of seal and human myoglobin cDNAs deduced from the corresponding genomic myoglobin genes [Blanchetot et al., Nature 301 (1983) 732-734; Weller et al., EMBO J. 3 (1984) 439-446; Akaboshi, Gene 33 (1985) 241-249], a high degree of homology is observed in the 5'-untranslated region and in parts of the 3'-untranslated region, as well as in the coding region.

Amino Acid Sequence↗
PubMed1985

Cloning of the human myoglobin gene.

Genomic clones that contain the human myoglobin gene were isolated by cross-hybridization to the porcine myoglobin cDNA from human genomic libraries. The myoglobin gene is about 10.5 kb long and contains two introns as in the case with hemoglobin genes. Diverse tandem repetitive sequences with 45 FokI sites are located 1100-1750 bp upstream from the putative cap site of myoglobin mRNA. Two kinds of direct repeats (116 and 58 bp) are contained in this repetitive region. In addition, a purine-rich sequence starting from the FokI site is found around 68-114 bp upstream of a putative initiation site of transcription. Another 33-bp sequence is repeated four times in the first intron and bordered by a 9-bp direct repeat, a structure that is common with the eukaryotic transposable elements.

Animals↗
PubMed1985

Structural analysis of the umu operon required for inducible mutagenesis in Escherichia coli.

The nucleotide sequence of a 2.8-kilobase fragment of the Escherichia coli chromosome containing the umuDC genes has been determined. The DNA sequence specifies two open reading frames of 417 and 1266 nucleotides encoding proteins with calculated molecular weights of 15,063 and 47,677, respectively. From these and the previous results of genetic and biochemical studies on the cloned genes, we conclude that the former is the umuD and the latter is the umuC gene. In vitro transcription of the regulatory region of the umu operon revealed that (i) upstream of the coding region there is a promoter-operator complex having the consensus sequence, CTGTATATAAAAACAG, of an SOS box, (ii) transcription of the umu operon begins at an adenine residue in the SOS box, and (iii) the LexA protein binds to the operator region, with an equilibrium dissociation constant (Kd) of 0.2, thereby repressing the transcription of the umuDC genes. These results indicate that the expression of the umu operon is under the coordinated control of the recA lexA gene products.

Amino Acid Sequence↗
PubMed1983

Instability of bacteriophage lambda initiator O and P proteins in DNA replication.

Lambda dv plasmids having an amber mutation in an initiator gene, O or P, were constructed from mutant lambda phages by recombinant DNA techniques and several properties of such derivatives were investigated. These plasmids are perpetuated in suppressor-plus (amber-permissive) cells, but not in non-suppressor cells. The plasmid copy number in the suppressor-plus cells was low as compared to that of the plasmid without the amber mutation. In cells carrying a thermosensitive suppressor 2, raising the temperature is expected to block new production of amber proteins, but should not affect conservation of the protein made prior to heating. It was observed, however, that replication of the plasmids carrying an amber mutation in the O or P gene was abolished soon after raising the temperature, suggesting that neither of the initiator proteins can continue functioning unless replenished. Pulse-chase experiments demonstrated that O protein decays with a half-life of 8 min. Several lines of evidence suggest that this degradation occurs independently of the protein function. On the other hand, P protein was not degraded under the same experimental conditions. These observations are discussed in connection with functional instability of the initiator molecules. It appears that they do not work catalytically.

Bacteriophage lambda↗
PubMed1979

Kynurenine hydroxylase in Musca domestica L.

1. Kynurenine hydroxylase activity was assayed in extracts of Musca domestica L. 2. During the life cycle of the fly, there is a peak of enzyme activity shortly after pupation. 3. Eye-color mutants, rb2, rb1; rb2 and cm, show reduced activity during pupal stages. 4. An ocra mutant lacks this activity. 5. The rb1 and rb2 mutations were separated from the rb1; rb2 strain. From measurements of the xanthommatin content, the rb1 mutation was suggested to enhance the expression of the rb2 gene.

Amino Acids↗