Katsuichiro Okazaki

TIP49b, a New RuvB-like DNA Helicase, Is Included in a Complex Together with Another RuvB-like DNA Helicase, TIP49a

We previously reported that TIP49a is a novel mammalian DNA helicase showing structural similarity with the bacterial recombination factor RuvB. In this study, we isolated a newTIP49a-related gene, termed TIP49b, from human and yeast cells. TIP49b also resembled RuvB, thus suggesting that TIP49a and TIP49b are included in a gene family. Like TIP49a, TIP49b was abundantly expressed in the testis and thymus. Enzyme assays revealed that TIP49b was an single-stranded DNA-stimulated ATPase and ATP-dependent DNA helicase. Most of the enzymatic properties of TIP49b were the same as those of TIP49a, whereas the polarity of TIP49b DNA helicase activity (5′ to 3′) was the opposite to that of TIP49a. TIP49b and TIP49a bound to each other and were included in the same complex of ∼700 kDa in a cell. We found thatTIP49b was an essential gene for the growth ofSaccharomyces cerevisiae, as is the TIP49agene, suggesting that TIP49b does not complement the TIP49a function and vice versa. From these observations, we suggest that TIP49b plays an essential role in the cellular processes involved in DNA metabolism.

Possible Antitumor Promoters in Spinacia oleracea (Spinach) and Comparison of their Contents among Cultivars

Spinach leaves were found to contain two potent antitumor promoters as detected by the activity against tumor promoter-induced Epstein-Barr virus activation. The active components were identified as 1-O-α-linolenoyl-2-O-(7Z,10Z,13Z)-hexadecatrienoyl-3- O-β-D-galactopyranosyl-sn-glycerol and 1,2-di-O-α-linolenoyl-3-O-β-D-galactopyranosyl-sn-glycerol by spectroscopic data and some chemical and enzymatic reactions. Their contents significantly varied with the cultivar and with the culture conditions.

Molecular Cloning and Expression of the Gene Encoding Family 19 Chitinase from Streptomyces sp. J-13-3

The gene encoding chitinase from Streptomyces sp. (strain J-13-3) was cloned and its nucleotide structure was analyzed. The chitinase consisted of 298 amino acids containing a signal peptides (29 amino acids) and a mature protein (269 amino acids), and had calculated molecular mass of 31,081 Da. The calculated molecular mass (28,229 Da) of the mature protein was almost same as that of the native chitinase determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometer. Comparison of the encoded amino acid sequences with those of other chitinases showed that J-13-3 chitinase was a member of the glycosyl-hydrolase family 19 chitinases and the mature protein had a chitin binding domain (65 amino acids) containing AKWWTQ motif and a catalytic domain (204 amino acids). The J-13-3 strain had a single chitinase gene. The chitinase (298 amino acids) with C-terminal His tag was overexpressed in Escherichia coli BL21(DE3) cells. The recombinant chitinase purified from the cell extract had identical N-terminal amino acid sequence of the mature protein in spite of confirmation of the nucleotide sequence, suggesting that the signal peptide sequence is successfully cut off at the predicted site by signal peptidase from E. coli and will be a useful genetic tool in protein engineering for production of soluble recombinant protein. The optimum temperature and pH ranges of the purified chitinase were at 35–40°C and 5.5–6.0, respectively. The purified chitinase hydrolyzed colloidal chitin and trimer to hexamer of N-acetylglucosamine and also inhibited the hyphal extension of Tricoderma reesei.

Isolation and some cultural conditions of Streptomyces species which produce enzymes lysing Aspergillus niger cell wall

Abstract As the result of screening microorganisms having lytic activity towards Aspergillus niger cell wall, two Streptomyces strains were isolated. One, termed J-28, was identified as Streptomyces cinereoruber and the other, termed J-13-3, was an unknown species belonging to Streptomyces . These strains inducibly produced chitinase and α-glucanase in a medium containing A. niger cell wall; specifically, strain J-28 chiefly produced chitinase and strain J-13-3 mainly α-glucanase. The best medium for chitinase production by the former was 0.5% A. niger cell wall, 0.5% yeast extract, 0.2% K 2 HPO 4 , 0.1% MgSO 4 ·7H 2 O and 0.01% FeSO 4 ·7H 2 O, while for α-glucanase production by the latter it was the same medium except for 1% instead of 0.5% A. niger cell wall, both in shaking cultures. Reaction products in chitin hydrolysis with chitinase were N , N ′-diacetyl chitobiose and chitin oligomers and those of nigeran with α-glucanase were maltose and nigeran oligomers.

Purification and properties of chitinase from Streptomyces cinereoruber

Abstract A chitinase (EC 3.2.1.14) was purified from the culture filtrate of Streptomyces cinereoruber, selected as a microorganism which produces enzymes lysing Aspergillus niger cell wall, by fractional precipitation with ammonium sulfate and column chromatographies on DEAE-cellulose, Sephadex G-100 and CM-Sephadex C-50. The final preparation was homogenous in polyacrylamide gel disc electrophoresis. The molecular weight of the enzyme was about 19,000 daltons and its isoelectric point was pH 8.6. The optimum pH and temperature for chitinase activity were 4.5 and at 50°C, respectively. The enzyme was stable in the pH range from 4.0 to 10.0. The activity was inhibited by Ag+, Hg+, Hg2+ and p-chloromercuribenzoate. Paper chromatographic analysis demonstrated that the hydrolytic products of colloidal chitin and chitotriose with the enzyme were N-acetylglucosamine and chitobiose. The lysis of A. niger cell wall with the enzyme is discussed.

Purification and Properties of Chitinase from Arthrobacter sp. NHB-10.

A chitinase was purified from the culture filtrate of nigeran-degrading Arthrobacter sp. NHB-10 by precipitation with ammonium sulfate and column chromatographies on DEAE-Sephadex A-50 and Superose 12. The final preparation was homogenous in polyacrylamide gel electrophoresis. The molecular weight of the purified enzyme was 30,000 and its isoelectric point was 6.8. The optimum pH and temperature for the enzyme activity were 5.0 and 45°C, respectively. The enzyme was stable from pH 3 to 7 and up to 55°C. The enzyme activity was inhibited by Hg(2+) and p-chloromercuribenzoic acid. Two internal amino acid sequences of the enzyme were AGPQLLTGYY and IGGVMT.

Cloning and characterization of the gene encoding endo-β-1,3-glucanase from Arthrobacter sp. NHB-10

The gluA gene, encoding an endo-β-1,3-glucanase from Arthrobacter sp. (strain NHB-10), was cloned and analyzed. The deduced endo-β-1,3-glucanase amino acid sequence was 750 amino acids long and contained a 42 amino acid signal peptide with a mature protein of 708 amino acids. There was no similarity to known endo-β-1,3-glucanases, but GluA was partially similar to two fungal exo-β-1,3-glucanases in glycoside hydrolase (GH) family 55. Of five possible residues for catalysis and two motifs in two β-helix heads of GH family 55, three residues and one motif were conserved in GluA, suggesting that GluA is the first bacterial endo-β-1,3-glucanase in GH family 55. Significant similarity was also found to two proteins of unknown function from Streptomyces coelicolor A3(2) and S. avermitilis.

Enhanced expression of PP1γ1, a catalytic subunit isoform of protein phosphatase type 1, in invasive ductal carcinoma of the breast

Breast cancer is one of the most common malignancies of women. Assessing the biological parameters of malignant tumors may facilitate predictions of clinical outcome. The expression of the three catalytic subunits of protein phosphatase (PP) type 1, PP1 alpha, PP1 gamma 1 and PP1 delta, as well as the one catalytic subunit of PP type 2, PP2AC, were examined in ten cases of mammary dysplasia, ten cases of fibroadenoma and 12 cases of invasive ductal carcinoma, using immunohistochemical analysis. Moreover, we measured the S-phase fraction of the cell cycle for use as a marker value of cell growth, using flow cytometric analysis. The percentage of proliferating cells that stained positive with antisera against PP1 gamma 1 was significantly higher in invasive ductal carcinoma than in mammary dysplasia and fibroadenoma. Furthermore, invasive ductal carcinoma showed a markedly high number of tumor cells in the S-phase of the cell cycle, as compared to mammary dysplasia and fibroadenoma. Our results indicate that PP1 gamma 1 may be involved in the accelerated growth of malignant cells in breast tumors.

Mariner-based transposon mutagenesis for Bacteroides species

Bacteroides is one of the most predominant groups of human gut microbiota. Recent metagenomic analyses and studies on gnotobiotic mice demonstrated the tight association of Bacteroides with epithelial function, the gut immune system and systemic metabolism in the host. The mariner family transposon shows relatively low target site specificity and has hosts ranging from prokaryotes to eukaryotes. Thereby, random mutagenesis using the mariner family transposon is expected to identify key molecules for human‐Bacteroides symbiosis. In this study, we constructed the plasmid pMI07 to deliver the gene cassette (ermF/ITR), which harbors the erythromycin resistant marker (ermF) and the inverted repeat sequences (ITRs) recognized by Himar1 transposase, to Bacteroides via electrotransformation. pMI07 successfully delivered ermF/ITR to the Bacteroides genomes and generated thousands of insertion mutants/μg of pMI07 in B. thetaiotaomicron, B. fragilis, B. ovatus, and also, although to a lesser extent, B. vulgatus. Analyses of the ermF/ITR insertion sites in B. thetaiotaomicron and B. vulgatus revealed that the cassette targeted the dinucleotide TA and integrated into the genomes in an unbiased manner. The data reported here will provide useful information for transposon mutagenesis in Bacteroides species, which will enable identification of the genes responsible for their unique phenotypes.

Purification and Antifungal Activity of Recombinant Chitinase from Escherichia coli Carrying the Family 19 Chitinase Gene of Streptomyces sp. J-13-3

A recombinant chitinase was purified from the cell extract of Escherichia coli JM109 transformed by plasmid pUC19 carrying the gene encoding family 19 chitinase of Streptomyces sp. J-13-3 by column chromatography on DEAE-Sepharose, CM-Sepharose, and Bio-Gel P-100. The final preparation was homogenous in polyacrylamide gel electrophoresis. The molecular weight of the purified enzyme was estimated to be 32,000. The recombinant chitinase hydrolyzed the trimer to hexamer of N-acetylglucosamine and had the identical N-terminal amino acid sequence of the mature protein, indicating removal of the signal sequence by E. coli signal peptidase. The fungal growth in well (200 μl of medium) of microplate by measurement of absorbance at 595 nm indicated that the chitinase (10 μg) completely and half inhibited growth of Trichoderma reesei and Aspergillus niger respectively.