Isolation and characterization of a type II restriction endonuclease from Streptococcus thermophilus

The alpha-toxin (phospholipase C) of Clostridium perfringens has been reported to contain catalytically essential zinc ions. We report here that histidine residues are essential for the co-ordination of these ion(s). Incubation of alpha toxin with diethylpyrocarbonate, a histidine modifying reagent, did not result in the loss of phospholipase C activity unless the protein was first incubated with EDTA, suggesting that zinc ions normally protect the susceptible histidine residues. When the amino acid sequences of three phospholipase C's were aligned, essential zinc binding histidine residues in the non-toxic B. cereus phospholipase C were found in similar positions in the toxic C. perfringens enzyme and the weakly toxic C. bifermentans phospholipase C.     

Who is the mother of the potato? — restriction endonuclease analysis of chloroplast DNA of cultivated potatoes

Summary Chloroplast DNA from 44 lines of 16 wild and 7 cultivatedSolanum species were compared by restriction endonuclease analysis. Seven chloroplast genome types were identified among them by 5 restriction enzymes: Type A (S. tuberosum ssp.andigena andS. maglia); Type S (S. goniocalyx, S. phureja, S. stenotomum, S. ×chaucha and a line of ssp.andigena); Type C (S. acaule, S. bukasovii, S. canasense, S. multidissectum andS. ×juzepczukii); Type T (S. tuberosum ssp.tuberosum); Type W (other wild species); Type W (S. chacoense f.gibberulosum) and Type W (S. tarijense). From this cytoplasmic identification, it was concluded thatS. goniocalyx, S. phureja, S. ×chaucha and ssp.andigena were derived fromS. stenotomum or its primitive type, which may have originally evolved itself fromS. canasense. The chloroplast genome of the European potato, however, was introduced from the Chilean potato, which might have been primarily constructed with the nuclear genome from ssp.andigena and with cytoplasm from other species. The cytoplasmic donor of the Chilean potato could not be determined.Contribution from the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, Japan, No. 479. This work was done at Kyoto University when the author was a graduate student at Kobe University     

Site-directed mutagenesis of the T4 endonuclease V gene: mutations which enhance enzyme specific activity at low salt concentrations

Previous structure/function analyses of the DNA repair enzyme, T4 endonuclease V, have suggested that the extreme carboxyl portion of the enzyme is associated with pyrimidine dimer-specific binding (Recinos and Lloyd, and Stump and Lloyd, Biochemistry 27:1832-1838 and 1839-1843, 1988, respectively). Within the final 11 amino acids there are 5 aromatic, 2 basic, and no acidic residues and it has been proposed that these residues stack with and electrostatically interact with the kinked DNA at the site of a pyrimidine dimer. The role of the tyrosine residue at position 129 has been investigated by oligonucleotide site-directed mutagenesis in which the codon for Tyr-129 has been altered to reflect conservative changes of Trp and Phe and more dramatic changes of Ser, a stop codon, deletion of the codon or introduction of a frameshift. Both changes to the aromatic amino acids resulted in proteins which accumulated well in E. coli and not only significantly enhanced the UV survival of repair-deficient cells but also complemented a defective denV gene within UV-irradiated T4 phage. Partially purified preparations of the Tyr-129----Trp and Tyr-129----Phe mutants were assayed for their ability to processively incise UV-irradiated plasmid DNA (a nicking reaction carried out at low 25 mM salt concentrations). The mutant enzymes Tyr-129----Phe and Tyr-129----Trp displayed a 1000% and 500% enhanced specific nicking activity, respectively. These reactions were also shown to be completely processive. Assays performed at higher (100 mM) salt concentrations reduced the specific activities of the mutant enzymes approximately to that of wild type for the Tyr-129----Phe mutant and to 20% that of wild type for the Tyr-129----Trp mutant.     

Ⅱ型限制性核酸内切酶NcrⅠ的研究

 在肉色诺卡氏菌C-212株Nocardia carnea C-212中筛选到一种Ⅱ型限制性核酸内切酶NcrⅠ,经与BglⅡ的λDNA降解物的酶谱比较,以及酶识别特异性和切割位点的检测,证明了NcrⅠ是已知的限制酶BglⅡ的同切限制酶,而且其切割位点也与BglⅡ相同,其为:     

Isolation and characterization of restriction endonuclease EclHKI from an Enterobacter cloacae strain

An Enterobacter cloacae strain, producing restriction enzyme EclHKI, was isolated from a decaying potato. The enzyme is an isoschizomer of Eam1105I, which recognizes and cleaves GACNNN!NNGTC. EclHKI was produced at high activity (4×10⁴ U/g wet cells) and was purified from contaminants which interfere with restriction digestion by passing the cell lysate through DEAE-Sephacel and heparin columns. Activity was optimal at 37°C in a medium salt buffer.H.-Y. Chan, Y.-C. Chan and P.-C. Shaw are with the Department of Biochemistry, The Chinese University of Hong Kong, Hong Kong: K.-M. Kam is with the Institute of Pathology, Sai Ying Pun Jockey Club Clinic, Hong Kong.     

SruI restriction endonuclease from Selenomonas ruminantium

Abstract Sru I, specific restriction endonuclease, has been characterized from Selenomonas ruminantium isolated from the rumen of fallow deer. Results from the study demonstrate that S. ruminantium 18D possesses a type II restriction endonuclease, which recognizes the sequence 5'-TTT↓AAA-3'. The recognition sequence of Sru I was identified using digestions on pBR322, pBR328, pUC18, M13mp18RF, pACYC184 and λDNA. The cleavage patterns obtained were compared with computer-derived data. Sru I recognises the palindromic hexanucleotide sequence and cleaves DNA after the third T in the sequence, producing blunt ends. The purification and characterization of restriction endonuclease Sru I presented here is the first described for Selenomonas ruminantium spp. and demonstrates that this microorganism pocesses a DNA-cleaving enzyme with the same specificity as Dra I or Aha III.     

GLOBAL POPULATION STRUCTURE AND NATURAL HISTORY OF THE GREEN TURTLE (CHELONIA MYDAS) IN TERMS OF MATRIARCHAL PHYLOGENY

To address aspects of the evolution and natural history of green turtles, we assayed mitochondrial (mt) DNA genotypes from 226 specimens representing 15 major rookeries around the world. Phylogenetic analyses of these data revealed (1) a comparatively low level of mtDNA variability and a slow mtDNA evolutionary rate (relative to estimates for many other vertebrates); (2) a fundamental phylogenetic split distinguishing all green turtles in the Atlantic-Mediterranean from those in the Indian-Pacific Oceans; (3) no evidence for matrilineal distinctiveness of a commonly recognized taxonomic form in the East Pacific (the black turtle C.m. agassizi or C. agassizi); (4) in opposition to published hypotheses, a recent origin for the Ascension Island rookery, and its close genetic relationship to a geographically proximate rookery in Brazil; and (5) a geographic population substructure within each ocean basin (typically involving fixed or nearly fixed genotypic differences between nesting populations) that suggests a strong propensity for natal homing by females. Overall, the global matriarchal phylogeny of Chelonia mydas appears to have been shaped by both geography (ocean basin separations) and behavior (natal homing on regional or rookery-specific scales). The shallow evolutionary population structure within ocean basins likely results from demographic turnover (extinction and colonization) of rookeries over time frames that are short by evolutionary standards but long by ecological standards.     

Activity of single-stranded DNA endonucleases in mung bean is associated with cell division

A single-strand-specific endonuclease from mung bean sprouts is widely usedin molecular biology. However, the biological role of this enzyme is unknown. We studied the spatial and temporal activity of single-stranded DNA endonucleases in mung bean seedling by following enzyme activity that linearizes supercoiled plasmid DNA, a characteristic of this type of enzyme. The formation of a linear molecule from supercoiled DNA was found to occur in two distinguishable steps. The first, which involves introducing a nick into the supercoiled DNA and relaxing it, is very rapid and complete within a few seconds. The second step of cleaving the opposite strand to generate a unit-length linear duplex DNA is a relatively slow process. Analysis of the DNA cleavage sites showed the nuclease preferentially cuts supercoiled DNA at an AT-rich region. Varying levels of nuclease activity could be detected in different tissues of the mung bean seedling. The highest activity was in the root tip and was correlated with histone H1 kinase activity. This implies a link between nuclease activity and cell division. Induction of cell division in mung bean hypocotyls with auxin promoted formation of root primordia and considerably increased the activity of single-stranded DNA endonucleases. The nuclease activity and histone H1 kinase activity were reduced in mung bean cuttings treated with hydroxyurea, but not in cuttings treated with oryzalin. The potential function of single-stranded DNA endonucleases is discussed.     

Isolation of restriction enzyme EcoVIII, an isoschizomer of HindIII, produced by Escherichia coli E1585-68

A restriction endonuclease designated EcoVIII, an isoschizomer of HindIII, was isolated from Escherichia coli E1585-68 and purified by dextran-polyethylene glycol (DPG) phase partition, ammonium sulfate precipitation, phospho- and DEAE-cellulose chromatography, and hydroxylapatite chromatography. The purified EcoVIII was stable during the purification procedure and its high specific activity required 10 mM Mg²⁺. Unlike HindIII, Eco VIII exhibited a high specific activity even at low pH (pH 6.3) and showed the highest activity at 48° C. Transformation of purified plasmid DNA from E. coli E1585-68 into K-12 indicated that the EcoVIII gene was carried on a multicopy 4.4-kb miniplasmid. EcoVIII seems to be preferable to HindIII for its production and use because of easier handling of producer cells and a wider range of activity.