芝田硫化叶菌新型α-淀粉酶基因在大肠杆菌的克隆和表达

A novel α-amylase gene was amplified from Sulfolobus shibatae by using PCR technique.The amplified 1.7kb DNA fragment was inserted into an expression vector pBV220 to yield the recombinant plasmid pSBAM. The novel α-amylase gene in pSBAM was expressed in E. coli. The production of the novel α-amylase activity reached over 8 units/100mL of the culture. The molecular weight of this enzyme was about 61kD by SDS-PAGE. The expressed novel α-amylase protein in E.coli DHSα accounted for about 20 % of the total protein in the recombinant cell. The cooperative action of the novel α-amylase and the maltooligosyltrehalose synthase from Sulfolobus shibatae was investigated and trehalose was detected by using HPLC analysis when using amylose and partial starch hydrolysates as substrates.     

芝田硫化叶菌新型α-淀粉酶基因在大肠杆菌的克隆和表达

A novel α-amylase gene was amplified from Sulfolobus shibatae by using PCR technique.The amplified 1.7kb DNA fragment was inserted into an expression vector pBV220 to yield the recombinant plasmid pSBAM. The novel α-amylase gene in pSBAM was expressed in E. coli. The production of the novel α-amylase activity reached over 8 units/100mL of the culture. The molecular weight of this enzyme was about 61kD by SDS-PAGE. The expressed novel α-amylase protein in E.coli DHSα accounted for about 20 % of the total protein in the recombinant cell. The cooperative action of the novel α-amylase and the maltooligosyltrehalose synthase from Sulfolobus shibatae was investigated and trehalose was detected by using HPLC analysis when using amylose and partial starch hydrolysates as substrates.     

酿酒酵母嗜杀质粒(dsRNA)的提取与分离纯化

对酿酒酵母嗜杀质粒的提取、电泳检测及纯化方法进行了研究。以改进的Fried和Fink方法在不预破细胞壁的情况下,巯基乙醇在碱性条件(pH9. 3)预处理后,直接以SDS-苯酚抽提完整细胞分离到了嗜杀质粒(dsRNA) 。在此基础上, 以电泳透析方法获得了纯化的M2和L双链RNA,琼脂糖凝胶电泳测定分子大小分别为1.5kb和4.0kb。 Abstract:The method of extraction and purification of the double-stranded RNA killer plasmids from Saccharomyces cerevisiae was studied.With the improved method of Fried and Fink we directly extracted the intact cells with SDS-phenol after pre-treating the cells with ß-mercaptoethanol under analkli condition.The result was detected and confirmed by the gel electrophoresis.On this basis we acquired the purified M2 and L-dsRNA by means of electrophoretic dialysis,their molecular lengths being 1.5kb and 4.0kb respectively.     

酿酒酵母嗜杀质粒(dsRNA)的提取与分离纯化

对酿酒酵母嗜杀质粒的提取、电泳检测及纯化方法进行了研究。以改进的Fried和Fink方法在不预破细胞壁的情况下,巯基乙醇在碱性条件(pH9. 3)预处理后,直接以SDS-苯酚抽提完整细胞分离到了嗜杀质粒(dsRNA) 。在此基础上, 以电泳透析方法获得了纯化的M2和L双链RNA,琼脂糖凝胶电泳测定分子大小分别为1.5kb和4.0kb。 Abstract:The method of extraction and purification of the double-stranded RNA killer plasmids from Saccharomyces cerevisiae was studied.With the improved method of Fried and Fink we directly extracted the intact cells with SDS-phenol after pre-treating the cells with ß-mercaptoethanol under analkli condition.The result was detected and confirmed by the gel electrophoresis.On this basis we acquired the purified M2 and L-dsRNA by means of electrophoretic dialysis,their molecular lengths being 1.5kb and 4.0kb respectively.     

Screening and identification of receptor antagonist for shiga toxin from random peptides displayed on filamentous bacteriophages

The bacteriophage clones which can bind with shiga toxin B subunit (StxB) and inhibit cytotoxicity of shiga toxin were obtained by using antibody capturing method from a 15-mer random peptide library displayed on the surface of bacteriophage fd. Among them, one peptide encoded by the random DNA region of a selected bacteriophage (A12) was synthesized and tested in vitro and in vivo, where the peptide competed with the receptor of shiga toxin to bind StxB, and inhibited the cytotoxicity and enterotoxicity of shiga toxin. The peptide can also block other apparently unrelated StxB binding bacteriophage (A3), which suggests that there are overlapping StxB interaction sites for those ligands with different sequences. The results provide a demonstration of bacteriophage display to screen peptide ligands for a small and/or unable biotinylated molecule by antibodies-capturing strategy, and take the lead for the development of receptor antagonists for shiga toxin.     

Investigation of a toxic water-bloom of Microcystis aeruginosa (Cyanophyceae) in Lake Akersvatn,Norway

During the summer and fall of 1984 and 1985, the eutrophic Lake Akersvatn in south-eastern Norway, used as reserve drinking water reservoir, was found to produce heavy water-blooms of the colonial blue-green alga Microcystis aeruginosa. Samples of the water-bloom were found to be toxic using the mouse bioassay. No toxin was found free in the water as detected by HPLC and mouse bioassay. The toxic cells (minimum lethal dose 8–15 mg/kg body weight in mice) and purified toxin (minimum lethal dose 50 μg/kg body weight in mice) showed signs of poisoning in laboratory rats and mice identical to that of other hepatotoxin-producing M. aeruginosa blooms and strains reported from other parts of the world. The toxin has chemical properties similar to the cyclic heptapeptide reported for a South African M. aeruginosa toxin. The toxin from Lake Akersvatn bloom material has a molecular weight of 994. The toxic bloom of M. aeruginosa persisted from August to November in 1984 and reappeared in July of 1985. While water from Lake Akersvatn was not used for municipal water supply during this period, the presence of toxic blue-green algae in a drinking water reservoir indicates the need to develop monitoring and detection methods for toxic cells and toxin(s).     

Structure and function ofsawB, a gene involved in differentiation ofStreptomyces ansochromogenes

A partial DNA library of Streptomyces ansochromogenes 7100 was constructed by using plasmid plJ702 as vector and white mutant W19 as recipient. About 3 000 clones were obtained, two of which gave rise to the grey phenotype as wild type 7100. The plasmids were isolated from two transformants. The result indicated that the 5.2 kb and 5.8 kb DNA fragments were inserted into plJ702. The resulting recombinant plasmids were designated as pNL-1 and pNL-2 respectively. The 1.25 kb Pstl l-Apa l DNA fragment from pNL-1 was recognized as its complementarity to W19 strain. The nucleotide sequence of the 3.0 kb Pst I DNA fragment including 1.25 kb was determined and analyzed. The result indicated that this DNA fragment contains one complete open reading frame (ORF1) which encodes a protein with 295 amino acid residues, and this gene was designated as sawB. The deduced protein has 81% amino acid identities in comparison with that encoded by whiH in Streptomyces coelicolor. The function of sawB gene was studied by usi     

Intercontinental and intracontinental biogeography patterns and methods

The study of biogeography has benefited from the exponential increase of DNA sequence data from recent molecular systematic studies, the development of analytical methods in the last decade concerning divergence time estimation and geographic area analyses, and the availability of large-scale distributiofi data of species in many groups of organisms. The underlying principle of divergence time estimation from DNA and protein data is that sequence divergence depends on the product of evolutionary rate and time. With their molecular clock hypothesis, Zuckerkandl and Pauling （1965） separated rates of molecular evolution from time by incorporating fossil evidence. Originally,     

Epidermolytic toxin serotype B of Staphylococcus aureus is plasmid-encoded

Abstract The gene coding for epidermolytic toxin serotype B ( etb ) was cloned in a plasmid expression vector in Escherichia coli . Its expression was dependent on the vector plasmid's trc promoter. A polypeptide immunochemically indistinguishable from the purified staphylococcal toxin and with the same molecular weight was predominantly localized in the periplasm of E. coli . The etb gene resides on a 1.7 kb Hin dIII fragment of the 42 kb plasmid pTC142 present in the parental Staphylococcus aureus strain.     

Cloning and expression of the toxin B gene ofClostridium difficile

Results from our cloning studies on toxin A indicated that the gene for toxin B resided approximately 1 kb upstream of the toxin A gene. Clone pCD19, which contains the 5-end of the toxin A gene and a small open reading frame, was found to contain 1.2 kb of DNA which, when subcloned, expressed a nontoxic peptide that reacted with toxin B antibodies. The rest of the toxin B gene was located on the 6.8 kb cloned fragment of plasmid pCD19L. The two fragments overlapped 0.8 kb. Lysates containing protein expressed by the 6.8 fragment were cytotoxic and lethal, and were neutralized by toxin B antibody. The two fragments were ligated to give the complete toxin B gene. The protein expressed by the complete gene was cytotoxic and lethal, and showed complete immunological identity with toxin B. Further analysis of the expressed protein and the toxin B gene confirmed our earlier findings showing that toxin B has a molecular weight of 240,000 or greater.     

Cloning and partial sequencing of the proteinase gene complex from Lactococcus lactis subsp. lactis UC317.

The proteinase genes from Lactococcus lactis subsp. lactis UC317 were identified on a plasmid, pCI310, which is a deletion derivative of a cointegrate between pCI301, the 75 kb Lac Prt plasmid from UC317 and the 38.5 kb cryptic plasmid from that strain. The prt genes were cloned using a replacement cloning strategy whereby fragments from pCI310 were exchanged with the equivalent fragments in pNZ521, which contains the cloned proteinase genes from L. lactis subsp. lactis SK112. This generated two plasmids which encoded a cell-envelope-associated and a secreted proteinase, respectively. Specific regions of the UC317 structural prtP gene known to encode seven of the amino acids essential for substrate cleavage specificity were sequenced and compared with the known sequences of prt genes from L. lactis strains SK112, Wg2 and NCDO763. In spite of various differences that were detected in the nucleotide sequence of this region, it appears that these seven amino acids in strains UC317 and NCDO763 are identical, and represent a combination of three of the amino acids from SK112 and four from Wg2. These results indicate that the UC317 proteinase is a natural hybrid of the SK112 and Wg2 proteinases.     

DNA cleavage by homo- and heterotetranuclear Cu(II) and Mn(II) complexes with tetrathioether-tetrathiol moiety

Novel homotetranuclear Cu(II) and heteronuclear Cu(II)-Mn(II) complexes with tetrathioether-tetrathiol moiety have been prepared and their DNA relaxation activities with plasmid pCYTEXP (5kb) were electrophoretically established. The cleavage products analyzed by neutral agarose gel electrophoresis indicated that the interaction of the metal complexes with supercoiled plasmid DNA yielded linear, nicked or degraded DNA. The relaxation activities of both homo- and heterotetranuclear (SK4) complexes are time- and concentration-dependent. The findings suggest that SK4 with potent nucleolytic activity is a good nuclease substitute in the presence of cooxidant. Furthermore, the observation of induction of DNA into smaller fragments by SK4 is also significant.     

Identification of novel Clostridium perfringens type E strains that carry an iota toxin plasmid with a functional enterotoxin gene

Clostridium perfringens enterotoxin (CPE) is a major virulence factor for human gastrointestinal diseases, such as food poisoning and antibiotic associated diarrhea. The CPE-encoding gene (cpe) can be chromosomal or plasmid-borne. Recent development of conventional PCR cpe-genotyping assays makes it possible to identify cpe location (chromosomal or plasmid) in type A isolates. Initial studies for developing cpe genotyping assays indicated that all cpe-positive strains isolated from sickened patients were typable by cpe-genotypes, but surveys of C. perfringens environmental strains or strains from feces of healthy people suggested that this assay might not be useful for some cpe-carrying type A isolates. In the current study, a pulsed-field gel electrophoresis Southern blot assay showed that four cpe-genotype untypable isolates carried their cpe gene on a plasmid of ～65 kb. Complete sequence analysis of the ～65 kb variant cpe-carrying plasmid revealed no intact IS elements and a disrupted cytosine methyltransferase (dcm) gene. More importantly, this plasmid contains a conjugative transfer region, a variant cpe gene and variant iota toxin genes. The toxin genes encoded by this plasmid are expressed based upon the results of RT-PCR assays. The ～65 kb plasmid is closely related to the pCPF4969 cpe plasmid of type A isolates. MLST analyses indicated these isolates belong to a unique cluster of C. perfringens. Overall, these isolates carrying a variant functional cpe gene and iota toxin genes represent unique type E strains.     

Linear plasmids pWR1A and pWR1B of the yeast Wingea robertsiae are associated with a killer phenotype

Wingea robertsiae CBS6693 (synonym Debaryomyces robertsiae) was previously reported to harbor two cryptic linear plasmids, designated pWR1A (8.3 kb) and pWR1B (14.6 kb). Reexamination of a putative plasmid encoded killer phenotype involved UV-curing as well as a highly sensitive toxin assay. Killer activities of concentrated culture supernatants prepared from both, a plasmid carrying and a cured plasmid-free strain, were examined in liquid media. Supernatants collected from plasmid carrying strains subjected to cultures of the plasmid-free derivative had clear concentration-dependent inhibitory effects, whereas plasmid harboring cells were not affected. Incubation at 65 degrees C for 10 min totally destroyed the toxin. Since supernatants prepared from the plasmid-free strain did not possess such killer activity and the presence of the plasmids confered resistance, toxin as well as immunity functions appear plasmid encoded. Beyond this, chitin affinity chromatography and Western blot analysis proved plasmid specific expression and secretion of a protein displaying similarities to the alpha-subunit of the Kluyveromyces lactis killer toxin. The assay applied in this study will most probably allow disclosure of other hidden killer phenomena, which may have escaped detection by conventionally applied plate assays.     

Enterotoxins and toxic shock syndrome toxin in Staphylococcus aureus recovered from human nasal carriers and manually handled foods: epidemiological and genetic findings

A set of 269 Staphylococcus aureus isolates recovered from nasal carriers and manually handled foods in a region of Spain was analyzed for pyrogenic toxin production and toxin genes. Fifty-seven isolates producing at least one of four enterotoxins (SEA, SEB, SEC, SED), 10 isolates producing only toxic shock syndrome toxin (TSST-1), and 10 isolates producing both toxin types were found. The 77 toxigenic isolates were discriminated into 36 SmaI genomic and 13 EcoRI plasmid profiles. A strong relationship between toxin profiles with both SmaI genomic and EcoRI plasmid profiles was revealed. SmaI genomic profiles showing six or less mismatching fragments and similarity coefficient > or =0.7 were included in a lineage. Eight lineages were differentiated; six of them grouped both human and food isolates and two of these also included outbreak-implicated isolates. Two lineages, represented by TSST-SEA and TSST-1, on the one hand, and SEC and SEC-SED isolates, on the other hand, were the most frequent, but only the second was outbreak-related. When SmaI genomic and EcoRI plasmid profiles were hybridized with tst, sea, seb, and sec toxin probes, it was observed that each probe mapped on a different SmaI fragment from isolates falling into the same lineage. All of the probes only mapped on genomic fragments, but sed also mapped on three plasmid fragments. When sej and ser probes were included, they mapped together with sed on the chromosome and on the plasmids. Two plasmids (ca. 33 and 36 kb) carried the expected sed-sej-ser genes, while the other (ca. 53.5 kb) carried sed-sej and ser-like genes. The latter plasmid and the chromosomal location of sed-sej-ser are new findings from this study.     

Cloning, nucleotide sequence and expression of a hemolysin gene of Clostridium septicum

Abstract A genomic library of Clostridium septicum NCTC547 strain was made in Escherichia coli by means of λgt10. The DNA insert of a hemolysin-positive (Hly⁺) λ-clone was transferred into pUC19. The resulting plasmid, pCS21, confers a Hly⁺ phenotype on E. coli . Crude lysates of E. coli (pCS21) possessed a strong lytic activity on human erythrocytes and also a lethal effect on mice, characteristic of an α toxin. Nucleotide sequence analysis revealed that the insert DNA (5.2 kb) in pCS21 included at least one open reading frame of 1380 bp. The coding frame for hemolysin was predicted to be 1329 bp in size and to encode a protein of 49.8 kDa. It coincided with the molecular mass (48 kDa) of the α toxin secreted by C. septicum . Taken together, the data indicated that plasmid pCS21 indeed encoded an α toxin gene of C. septicum .     

酿酒酵母两种不同嗜杀菌的比较研究

以酿酒酵母两种不同类型的嗜杀菌株ＳＫ４（Ｋ１型）和ＥＲＲ１（Ｋ２型）为材料,分析了不同嗜杀酵母的嗜杀特性,两株嗜杀酵母具有相互杀死作用,其嗜杀活性与菌体生长有关。ＳＫ４和ＥＲＲ１的嗜杀质粒的比较表明：Ｍ１－ｄｓＲＮＡ质粒和Ｍ２－ｄｓＲＮＡ质粒分子量分别为１．７ｋｂ和１．５ｋｂ,两株菌的Ｌ－ｄｓＲＮＡ质粒均为４．０ｋｂ。用高温和紫外线处理嗜杀酵母,嗜杀活性随之消失,消除菌中的Ｍ－ｄｓＲＮＡ质粒也相应