Characterization of type II protein secretion (xcp) genes in the plant growth-stimulatingPseudomonas putida, strain WCS358

InPseudomonas aeruginosa, the products of thexcp genes are required for the secretion of exoproteins across the outer membrane. Despite structural conservation of the Xcp components, secretion of exoproteins via the Xcp pathway is generally not found in heterologous organisms. To study the specificity of this protein secretion pathway, thexcp genes of another fluorescent pseudomonad, the plant growth-promotingPseudomonas putida strain WCS358, were cloned and characterized. Nucleotide sequence analysis revealed the presence of at least five genes, i.e.,xcpP, Q, R, S, andT, with homology toxcp genes ofP. aeruginosa. Unlike the genetic organization inP. aeruginosa, where thexcp cluster consists of two divergently transcribed operons, thexcp genes inP. putida are all oriented in the same direction, and probably comprise a single operon. Upstream ofxcpP inP. putida, an additional open reading frame, with no homolog inP. aeruginosa, was identified, which possibly encodes a lipoprotein. Mutational inactivation ofxcp genes inP. putida did not affect secretion, indicating that no proteins are secreted via the Xcp system under the growth conditions tested, and that an alternative secretion system is operative. To obtain some insight into the secretory pathway involved, the amino acid sequence of the N-terminus of the major extracellular protein was determined. The protein could be identified as flagellin. Mutations in thexcpQ andR genes ofP. aeruginosa could not be complemented by introduction of the correspondingxcp genes ofP. putida. However, expression of a hybrid XcpR protein, composed of the N-terminal one-third ofP. aeruginosa XcpR and the C-terminal two-thirds ofP. putida XcpR, did restore protein secretion in aP. aeruginosa xcpR mutant.     

Measuring the toxic effects of high gene dosage on yeast cells

 A novel method, which is rapid, reliable and quantitative, is presented for measuring the toxic effects on yeast cells of high dosage of any given gene. It is based on the possibility of monitoring the presence in cells of a plasmid carrying the ADE2 gene from Saccharomyces cerevisiae by direct observation of colonies, the construction of this particular plasmid being easily made by marked homologous recombination in yeast. Four yeast regulatory genes tested were found to result in various degrees of toxicity at high dosage. Possible implications of the measurement of gene toxicity for eukaryotic cell regulatory mechanisms and for the use of novel general approaches to gene selection, such as the gene-gene interference method, are discussed. Received: 18 March 1996 / Accepted: 23 August 1996     

Isolation and characterization of two new negative regulatory mutants for nitrate assimilation inChlamydomonas reinhardtii obtained by insertional mutagenesis

Plasmid DNA carrying either the nitrate reductase (NR) gene or the argininosuccinate lyase gene as selectable markers and the correspondingChlamydomonas reinhardtii mutants as recipient strains have been used to isolate regulatory mutants for nitrate assimilation by insertional mutagenesis. Identification of putative regulatory mutants was based on their chlorate sensitivity in the presence of ammonium. Among 8975 transformants, two mutants, N1 and T1, were obtained. Genetic characterization of these mutants indicated that they carry recessive mutations at two different loci, namedNrg1 andNrg2. The mutation in N1 was shown to be linked to the plasmid insertion. Two copies of the nitrate reductase plasmid, one of them truncated, were inserted in the N1 genome in inverse orientation. In addition to the chlorate sensitivity phenotype in the presence of ammonium, these mutants expressed NR, nitrite reductase and nitrate transport activities in ammonium-nitrate media. Kinetic constants for ammonium (¹⁴C-methylammonium) transport, as well as enzymatic activities related to the ammonium-regulated metabolic pathway for xanthine utilization, were not affected in these strains. The data strongly suggest thatNrg1 andNrg2 are regulatory genes which specifically mediate the negative control exerted by ammonium on the nitrate assimilation pathway inC. reinhardtii.     

Transformation of the extremely thermoacidophilic archaeon Sulfolobus solfataricus via a self-spreading vector

Abstract The comparative chromosomal locations of polymeric β-fructosidase SUC genes have been determined by Southern blot hybridization with the SUC2 probe in 91 different strains of Saccharomyces cerevisiae . Most of the strains exhibited a single SUC2 gene, but in some strains two or three SUC genes were found. All Suc⁻ strains carried a silent suc2⁰ sequence. The accumulation of SUC genes was observed in populations derived from sources containing sucrose and seems to be absent in strains from sources promoting the MEL gene.     

昆虫分子生物学的一些研究进展：生物钟的基因

昆虫分子生物学的一些研究进展：生物钟的基因翟启慧（中国科学院动物研究所北京１０００８０）生物的许多行为和生理现象有周期性波动,称为生物节律或生物钟。长期以来,这是一个十分吸引人却又难以理解的问题。虽然有大量文献描述生物钟的现象,但对其机理却一无所知。...     

Lipopolysaccharides of Escherichia coli K12 Strains That Express Cloned Genes for the Ogawa and Inaba Antigens of Vibrio cholerae O1: Identification of O-Antigenic Factors

Structural and serological studies were performed with the lipopolysaccharide (LPS) expressed by Escherichia coli K12 strains No. 30 and No. 64, into which cosmid clones derived from Vibrio cholerae O1 NIH 41 (Ogawa) and NIH 35A3 (Inaba) had been introduced, respectively. The two recombinant strains, No. 30 (Ogawa) and No. 64 (Inaba), produced LPS that included, in common, the O-polysaccharide chain composed of an α(1 → 2)-linked N-(3-deoxy-L -glycero-tetronyl)-D -perosamine (4-amino-4,6-dideoxy-D -manno-pyranose) homopolymer attached to the core oligosaccharide of the LPS of E. coli K12. Structural analysis revealed the presence of N-(3-deoxy-L -glycero-tetronyl)-2-O-methyl-D -perosamine at the non-reducing terminus of the O-polysaccharide chain of LPS from No. 30 (Ogawa) but not from No. 64 (Inaba). Serological analysis revealed that No. 30 (Ogawa) and No. 64 (Inaba) LPS were found to share the group antigen factor A of V. cholerae O1. They were distinguished by presence of the Ogawa antigen factor B [co-existing with relatively small amounts of the Inaba antigen factor (c)] in the former LPS and the Inaba antigen factor C in the latter LPS. It appears, therefore, that No. 30 (Ogawa) and No. 64 (Inaba) have O-antigenic structures that are fully consistent with the AB(c) structure for the Ogawa and the AC structure for the Inaba O-forms of V. cholerae O1, respectively. Thus, the present study clearly confirmed our previous finding that the Ogawa antigenic factor B is substantially related to the 2-O-methyl group at the non-reducing terminus of the α(1 → 2)-linked N-(3-deoxy-L -glycero-tetronyl)-D -perosamine homopolymer that forms the O-polysaccharide chain of LPS of V. cholerae O1 (Ogawa).     

Detection and Identification of Yersinia pestis by Polymerase Chain Reaction (PCR) Using Multiplex Primers

A PCR method for detection of Yersinia pestis-virulence determinants by the use of multiplex primers was developed. Four pairs of oligonucleotide primers were designed from each gene of three kinds of virulent plasmids and a chromosomal DNA; 60-Md plasmid-located gene (caf1) encoding Y. pestis-specific capsular antigen fraction 1, a Y. pestis-specific region of a yopM gene encoded on 42-Md virulent plasmid, a plasminogen activator gene (pla) encoded on Y. pestis-specific 7-Md plasmid and an invasin protein gene (inv) encoded on chromosomal DNA. This multiplex-primer system was specific for the detection of Y. pestis among pathogenic Yersinia species and other enterobacteriaceae having antigens common to Y. pestis. Since this method is simple and safe, it will be useful to identify and confirm Y. pestis in cases of emergency and for the surveillance of epidemics.     

细胞分裂素生物合成基因在调节一组烟草病理相关蛋白基因表达中的作用

对一组病理相关蛋白基因在烟草 ( N icotiana tabacum cv. Wisconsin 38)中的表达情况进行了研究 ,包括 :碱性几丁质酶、β- 1 ,3-葡萄糖苷酶、渗透蛋白及伸展蛋白。RNA杂交实验表明在正常烟草植株中上述 4个基因具有发育和器官专一性的表达。在含有细胞分裂素生物合成基因的转基因烟草丛生芽中 ,这 4个基因的表达受过量合成的内源细胞分裂素和载体效应的共同调节 ,细胞分裂素降低这些基因的表达 ,而载体效应则促进它们的表达。热激处理也明显降低这 4种基因的表达水平。上述结果表明这些病理相关蛋白基因具有复杂的调控系统     

Structural characterization of genes corresponding to cotton fiber mRNA, E6: reduced E6 protein in transgenic plants by antisense gene

Two genes, each corresponding to fiber mRNA E6, were isolated from cotton cultivars Coker 312 (Gossypium hirsutum L.) and Sea Island (G. barbadense L.). E6 is one of the predominant fiber-specific mRNAs present during early fiber development. The distinguishing feature of the nucleotide-derived E6 protein is the presence of a motif where a dimer, Ser-Gly, is repeated several times. Two of the Sea Island genes contained a pentameric motif, Ser-Gly, while one of the Coker genes had one and the other had four motifs each. cDNA clones containing one or five Ser-Gly motifs were also identified. Thus, it appears that the strict conservation of this motif may not be critical to E6 protein function. Sequence characterizations of the genes and cDNAs showed that multiple members of the E6 family are transcribed in fiber and may result in proteins 238 to 246 amino acids long. The 3 ends of the genes and cDNAs showed considerable heterology among them. Transgenic plants containing antisense genes were generated to decipher E6 function. Transgenic cotton with reduced E6 protein levels in the range of 60 to 98% were identified. However, no discernible phenotypic changes in fiber development or properties were apparent. This result leads to the conclusion that E6 is not critical to the normal development or structural integrity of cotton fibers.