A novel cryptic plasmid pBMB175 from <Emphasis Type="Italic">Bacillus thuringiensis </Emphasis>subsp. <Emphasis Type="Italic">tenebrionis </Emphasis>YBT-1765

A new cryptic plasmid pBMB175 from Bacillus thuringiensis subsp. tenebrionis YBT-1765 was isolated and characterized. Sequence analysis showed that pBMB175 (14,841 bp and 31% GC content) contained at least eighteen putative open reading frames (ORFs), among which nine ORFs displayed the homology with the hypothetical proteins in rolling-circle replication plasmid pGI3. Deletion analysis revealed that the pBMB175 minireplicon located in a novel 1,151 bp fragment. This fragment contains ORF7 coding sequence, which encodes a protein (Rep175, 149 amino acids [aa]) indispensable for plasmid replication. Rep175 has no significant homology with known function proteins. Furthermore, a putative double-strand origin (dso), having no DNA similarity with characterized dso of other replicon so far, was identified in this minireplicon fragment. These features showed that pBMB175 could be placed into a new plasmid family.     

Complete Sequence of Plasmid pMP1 from the Marine Environmental Vibrio vulnificus and Location of Its Replication Origin

A novel cryptic plasmid, pMP1, from an environmental Vibrio vulnificus MP-4 isolated from Mai Po Nature Reserve in Hong Kong, has been characterized. The 7.6-kb plasmid had guanine–cytosine content of 40.03% and encoded four open reading frames (ORFs) with >100 amino acids. The predicted protein of ORF1 contained 478 amino acids showing 29% identity and 50% similarity over 309 amino acids to the integrase of Vibrio cholerae phage VP2. ORF2 encoded a putative protein of 596 amino acids, which were 23% identity and 42% similarity over 455 amino acids to the tail tape measure protein TP901 of Chromohalobacter salexigens phage. ORF3 and ORF4 encoded putative proteins of 103 and 287 amino acids, respectively, but showed no homologies to any known proteins. Further experiments indicated that a 3.2-kb fragment from EcoRI digestion could self-replicate. Analysis indicated that a sequence upstream of ORF4 had the features characteristic of theta-type replicons: AT-rich region, six potential direct repeats (iterons) spaced approximately two DNA helical turn apart (about 23 bp), two copies of 9 bp dnaA boxes, three Dam methylation sites, and five inverted repeats. Complementation experiments confirmed that the protein encoded by ORF4 was required for plasmid replication. We propose that ORF4 encode a new type of Rep protein and pMP1 is a new type of theta plasmid.     

Conservation of IS66 homologue of octopine Ti plasmid DNA in Rhizobium fredii plasmid DNA

Summary DNA sequences homologous to the T-DNA region of the octopine Ti plasmid from Agrobacterium tumefaciens are found in various fast-growing Rhizobium fredii strains. The largest fragment (BamHI fragment 2) at the right-boundary region of the core T-DNA hybridizes to more than one plasmid present in R. fredii. However, one smaller fragment (EcoRI fragment 19a) adjacent to the core T-DNA shows homology only with the plasmid carrying the symbiotic nitrogen-fixation genes (pSym). Hybridization data obtained with digested R. fredii USDA193 pSym DNA suggests that the homology is mainly with two HindIII fragments, 1.7 kb and 8.8 kb in size, of the plasmid. The 1.7 kb HindIII fragment also hybridizes to two regions of the virulence plasmid of A. tumefaciens, pAL1819, a deletion plasmid derived from the octopine Ti plasmid, pTiAch5. Hybridization studies with an insertion element IS66 from A. tumefaciens indicate that the 1.7 kb HindIII fragment of R. fredii plasmid, homologous to the T-DNA and the virulence region of Ti plasmid, is itself an IS66 homologue.     

Cloning, sequencing and function ofsanA, a gene involved in nikkomycin biosynthesis ofStreptomyces ansochromogenes

Several genetically stable mutants blocked in nikkomycin biosynthesis were obtained after the slightly germinated spores ofStreptomyces ansochromogenes, a nikkomycin producer, were treated with ultra violet radiation. One of the mutants is the same in morpholotical differentiation as the wild type strain and is designated as NBB19. A DNA library was constructed using plasmid pIJ702 as cloning vector, NBB19 as cloning recipient. A 6 kb DNA fragment which can genetically complement NBB19 was cloned when screening the library for antifungal activity. Sequence analysis showed that the 3 kbBgl II -Sal I fragment contains one complete ORF (ORF1) and one partial ORF (ORF2). ORF1 is designated assanA. sanA is 1 365 bp, encoding a protein consisting of 454 amino acid residues. Database searching indicated thatsanA is homologous to the hypothetical methyltransferase inPyrococcus horikoshii with 25% identities and 41% positives. Disruptant ofsanA lost the ability to synthesize nikkomycin. It indicated thatsanA is a novel gene which is essential for nikkomycin biosynthesis.     

Nucleotide sequence of diatom plasmids: identification of open reading frames with similarity to site-specific recombinases

We have determined the nucleotide sequence of two small circular DNA plasmids, pCf1 and pCf2 [22], from the marine diatom Cylindrotheca fusiformis. pCf1 is 4273 bp, and pCf2 is 4079 bp in size. In each plasmid, all of the major open reading frames (ORFs) are encoded on the same DNA strand. Two ORFs are similar, comparing the two plasmids. ORF218 (pCf1) and ORF217 (pCf2) share 80% amino acid identity and ORF482 (pCf1) and ORF484 (pCf2) share 54% amino acid identity. ORF218/217 shows significant similarity (28–31% amino acid identity) to the Tn3 class of resolvases. Resolvases are most commonly found in bacterial transposons. However, two other features found in the Tn3 class of transposon are missing in the plasmids; an ORF encoding a transposase and terminal inverted repeat sequences. This, and data mapping the portions of the plasmids that hybridize to genomic chloroplast DNA, suggest that the plasmids do not contain active transposons. By analogy with the R46 plasmid from Enterobacter [5, 6], another potential role for the resolvases encoded by pCf1 and pCf2 is the conversion of multimeric forms of the plasmid to monomers. The similarity of ORF218/217 to resolvases documents the first identification of a potential coding function in an algal plasmid.     

Cloning, sequencing and expression of the acylneuraminate lyase gene from Clostridium perfringens A99

The acylneuraminate lyase gene from Clostridium perfringens A99 was cloned on a 3.3 kb HindIII DNA fragment identified by screening the chromosomal DNA of this species by hybridization with an oligonucleotide probe that had been deduced from the N-terminal amino acid sequence of the purified protein, and another probe directed against a region that is conserved in the acylneuraminate lyase gene of Escherichia coli and in the putative gene of Clostridium tertium. After cloning, three of the recombinant clones expressed lyase activity above the background of the endogenous enzyme of the E. coli host. The sequenced part of the cloned fragment contains the complete acylneuraminate lyase gene (ORF2) of 864 bp that encodes 288 amino acids with a calculated molecular weight of 32.3 kDa. The lyase structural gene follows a non-coding region with an inverted repeat and a ribosome binding site. Upstream from this regulatory region another open reading frame (ORF1) was detected. The 3′-terminus of the lyase structural gene is followed by a further ORF (ORF3). A high homology was found between the amino acid sequences of the sialate lyases from Clostridium perfringens and Haemophilus influenzae (75% identical amino acids) or Trichomonas vaginalis (69% identical amino acids), respectively, whereas the similarity to the gene from E. coli is low (38% identical amino acids). Based on our new sequence data, the ‘large’ sialidase gene and the lyase gene of C. perfringens are not arranged next to each other on the chromosome of this species. This revised version was published online in November 2006 with corrections to the Cover Date.     

猪Ⅱ型圆环病毒豫A株的全基因组克隆与序列分析

参照国外发表的猪Ⅱ型圆环病毒(porcine circovirus type 2,PCV-2)全基因组序列,设计一对PCV-2特异性引物,用该室分离的PCV-2豫A株感染PK-15细胞,从中提取PCV-2复制型基因组DNA,并以之为模板进行PCR扩增.回收PCR产物,构建重组测序质粒T-PCV-2.测序结果表明,猪Ⅱ型圆环病毒豫A株的全基因组为1767bp,与GenBank收录的PCV-2国外分离株核苷酸的同源性可高达97%.序列分析表明,复制型豫A株的基因组包含10个读码框架,其中ORF1、ORF2是其两个最主要的读码框架,分别编码314、234个氨基酸.豫A株和PCV-1间的ORF1、ORF2的氨基酸序列同源性分别为85%、66%,与其它PCV-2毒株间的ORF1氨基酸同源性均在98%以上,而ORF2的氨基酸同源性为92%～97%.     

A yeast nuclear gene, MRS1, involved in mitochondrial RNA splicing: nucleotide sequence and mutational analysis of two overlapping open reading frames on opposite strands.

We have cloned a 1.6-kb fragment of yeast nuclear DNA, which complements pet- mutant MK3 (mrs1). This mutant was shown to be defective in mitochondrial RNA splicing: the excision of intron 3 from the mitochondrial COB pre-RNA is blocked. The DNA sequence of the nuclear DNA fragment revealed two open reading frames (ORF1 with 1092 bp; ORF2 with 735 bp) on opposite strands, which overlap by 656 bp. As shown by in vitro mutagenesis, ORF1, but not ORF2, is responsible for complementation of the splice defect. Hence, ORF1 represents the nuclear MRS1 gene. Disruption of the gene (both ORFs) in the chromosomal DNA of the respiratory competent yeast strain DBY747 (long form COB gene) leads to a stable pet- phenotype and to the accumulation of the same mitochondrial RNA precursors as in strain MK3. The amino acid sequence of the putative ORF1 product does not exhibit any homology with other known proteins, except for a small region of homology with the gene product of another nuclear yeast gene involved in mitochondrial RNA splicing, CBP2. The function of the MRS1 (ORF1) gene in mitochondrial RNA splicing and the significance of the overlapping ORFs in this gene are discussed.     

The linear mitochondrial plasmid pClK1 of the phytopathogenic fungus Claviceps purpurea may code for a DNA polymerase and an RNA polymerase

Summary Plasmid pClK1, a linear mitochondrial plasmid of Claviceps purpurea, was completely sequenced. The sequence contains two long open reading frames (ORF1, 3291 bp; ORF2, 2910 bp), and at least four smaller ORFs. The potential polypeptide derived from ORF1 shows homology to the family B type DNA polymerases. The product of ORF2 has significant homology to the mitochondrial RNA polymerase of yeast and RNA polymerases from bacteriophages. ORF1 and ORF2 show homology to URF3 and URF1 of the maize plasmids S1 and S2, respectively. No homology to any published protein sequence was found for the smaller ORFs. The origin of the terminal protein attached to the 5 ends of pClK1 remains open; several alternatives for its origin are discussed. The sequence data as a whole confirm the virus-like character of pClK1 already postulated from structural properties. Thus pClK1 together with S plasmids of maize and several other linear plasmids make up a distinct class of DNA species of plants and fungi probably derived from a common virus-like ancestor.     

Identification of a gene conferring resistance to zinc and cadmium ions in the yeast Saccharomyces cerevisiae

Summary A DNA fragment conferring resistance to zinc and cadmium ions in the yeast Saccharomyces cerevisiae was isolated from a library of yeast genomic DNA. Its nucleotide sequence revealed the presence of a single open reading frame (ORF; 1326 bp) having the potential to encode a protein of 442 amino acid residues (molecular mass of 48.3 kDa). A frameshift mutation introduced within the ORF abolished resistance to heavy metal ions, indicating the ORF is required for resistance. Therefore, we termed it the ZRC1 (zinc resistance conferring) gene. The deduced amino acid sequence of the gene product predicts a rather hydrophobic protein with six possible membrane-spanning regions. While multiple copies of the ZRC1 gene enable yeast cells to grow in the presence of 40 mM Zn²⁺, a level at which wild-type cells cannot survive, the disruption of the chromosomal ZRC1 locus, though not a lethal event, makes cells more sensitive to zinc ions than are wild-type cells.     

Plasmid RFLP profiling and DNA homology inThermus isolated from hot springs of different geographical areas

Thirty-four strains belonging to various species of the genus Thermus (T. aquaticus, "T. thermophilus," "T. brockianus," T. scotoductus, and genomic species 2) isolated from hot springs of different geographical areas were examined for plasmid content and restriction fragment length polymorphism (RFLP) of plasmid DNAs. The four strains of the numerical taxonomy cluster E of genomic species 2 did not harbor plasmid DNA. Overall examination of the HindIII-RFLP profiling of plasmid DNA showed considerable variability between and within genomic species, with the exception of presumed clonal isolates. In spite of this heterogeneity, HindIII plasmid digests within a numerical taxonomic cluster gave a subset of restriction fragments of similar or identical length. Strains belonging to genomic species 2 or unclassified isolates from S. Pedro do Sul that harbored plasmid DNA (7 of the 14 strains studied) exhibited strong DNA homology between plasmid regions. No homologous sequences to these plasmid regions were found in chromosomal DNA from strains isolated from S. Pedro do Sul in which no plasmids were detected. The strains belonging to T. scotoductus formed two plasmid DNA homology groups, as estimated by probing with a plasmid fragment that coincided with the two numerical taxonomy clusters proposed previously. Among the other species, homology of plasmid regions was also found between some strains. Strong homology was also found between plasmid regions from some strains of different taxonomic groups, isolated from the same and from different sources, suggesting that these sequences are highly conserved in plasmids present in Thermus. For plasmid-containing strains, results of plasmid RFLP profiling/DNA homology appear promising for the typing of Thermus at the level of biotypes or of individual strains, namely, for monitoring the diversity and frequency of isolates from a particular hot spring. Received: 24 October 1994 / Accepted: 6 March 1995     

Molecular cloning, sequence analysis, and characterization of a new cell wall hydrolase, CwIL, of Bacillus licheniformis

We have cloned a DNA fragment containing the gene for a cell wall hydrolase from Bacillus licheniformis FD0120 into Escherichia coli. Sequencing of the fragment showed the presence of an open reading frame (ORF; designated as cwlL), which is different from the B. licheniformis cell wall hydrolase gene cwlM, and encodes a polypeptide of 360 amino acids with a molecular mass of 38 994. The enzyme purified from the E. coli clone is an N-acetylmuramoyl-l-alanine amidase, which has a M_r value of 41 kDa as determined by SDS-polyacrylamide gel electrophoresis, and is able to digest B. licheniformis, B. subtilis and Micrococcus luteus cell walls. The nucleotide and deduced amino acid sequences of cwlL are very similar to those of ORF3 in the putative operon xpaL1-xpaL2-ORF3 in B. licheniformis MC14. Moreover, the amino acid sequence homology of CwlL with the B. subtilis amidase CwlA indicates two evolutionarily distinguishable regions in CwlL. The sequence homology of CwlL with other cell wall hydrolases and the regulation of cwlL are discussed.     

The gene for indole-3-acetyl-L-aspartic acid hydrolase from Enterobacter agglomerans : molecular cloning, nucleotide sequence, and expression in Escherichia coli

A 5.5-kb DNA fragment containing the indole-3-acetyl-aspartic acid (IAA-asp) hydrolase gene (iaaspH) was isolated from Enterobacter agglomerans strain GK12 using a hybridization probe based on the N-terminal amino acid sequence of the protein. The DNA sequence of a 2.4-kb region of this fragment was determined and revealed a 1311-nucleotide ORF large enough to encode the 45-kDa IAA-asp hydrolase. A 1.5-kb DNA fragment containing iaaspH was subcloned into the Escherichia coli expression plasmid pTTQ8 to yield plasmid pJCC2. Extracts of IPTG-induced E. coli cultures containing the pJCC2 recombinant plasmid showed IAA-asp hydrolase levels 5 to 10-fold higher than those in E. agglomerans extracts. Homology searches revealed that the IAA-asp hydrolase was similar to a variety of amidohydrolases. In addition, IAA-asp hydrolase showed 70% sequence identity to a putative thermostable carboxypeptidase of E. coli. Received: 12 March 1998 / Accepted: 30 March 1998     

粘虫核型多角体病毒凋亡抑制基因的定位,序列和启动子结构

以ＡｃＮＰＶ凋亡抑制基因ｐ３５为探针,与ＬｓＮＰＶＤＮＡ的限制性片段和ＬｓＮＰＶＤＮＡＥｃｏＲＶ片段杂交,发现ＥｃｏＲＶ５．５ｋｂ片段有强烈的杂交信号。将此片段亚克隆后,测定了１２４４ｂｐ序列,发现一个完整的ＯＲＦ,推导的３０２个氨基酸与ＡｃＮＰＶｐ３５蛋白有７０．４％的氨基酸同源性,证明所测ＯＲＦ为ＬｓＮＰＶ的ｐ３５基因。结构分析发现其５′端有早期基因启动子元件ＧＣ、ＡＣＧＴ和ＴＡＴＡｂｏｘ。有２２ｂｐ的顺向重复序列,包括由两个重叠的ＴＡＴＡｂｏｘ和上下游两个ＡＣＧＴｍｏｔｉｆ组成的两套启动子元件,这些结构特征与ＡｃＮＰＶ的凋亡抑制基因十分相似。     

Molecular cloning and analysis of Schizosaccharomyces pombe rad9, a gene involved in DNA repair and mutagenesis

Summary The mutant allele rad9-192 renders Schizosaccharomyces pombe cells sensitive to ionizing radiation and UV light. We have isolated from a S. pombe genomic DNA library a unique recombinant plasmid that is capable of restoring wild-type levels of radioresistance to a rad9 192-containing cell population. Plasmid integration studies using the cloned DNA, coupled with mating and tetrad analyses, indicate that this isolated DNA contains the wild-type rad9 gene. We inactivated the repair function of the cloned fragment by a single insertion of the S. pombe ura4 gene. This nonfunctional fragment was used to create a viable disruption mutant, thus demonstrating that the rad9 gene does not encode an essential cellular function. In addition, the rad9-192 mutant population is as radiosensitive as the disruption mutant, indicating that rad9 gene function is severely if not totally inhibited by the molecular defect responsible for the rad9-192 phenotype. DNA sequence analysis of rad9 reveals an open reading frame of 1,278 bp, interrupted by three introns 53 bp, 57 bp, and 56 by long, respectively, and ending in the termination codon TAG. This gene is capable of encoding a protein of 426 amino acids, with a corresponding calculated molecular weight of 47,464 daltons. No significant homology was detected between the rad9 gene or its deduced protein sequence and sequences previously entered into DNA and protein sequence data banks.     

Complete DNA sequence and gene analysis of the virulence plasmid pCP301 of Shigella flexneri 2a

The complete nucleotide sequence and organization of the large virulence plasmid pCP301 (termed by us) of Shigella flexneri 2a strain 301 were determined and analyzed. The result showed that the entire DNA sequence of pCP301 is composed of 221618 bp which form a circular plasmid. Sequence analysis identified 272 open reading frames (ORFs), among which, 194 correspond to the proteins described previously, 61 have low identity (<60%) to known proteins and the rest 17 have no regions of significant homology with proteins in database. The genes of pCP301 mainly include the genes associated with bacterial virulence, the genes associated with regulation and the genes relating to plasmid maintenance, stability and DNA metabolism. Insertion sequence (IS) elements are 68 kb in length and account for 30 percent of complete sequence of the plasmid which indicates that gene multiple rearrangements of the pCP301 have taken place in Shigella flexneri evolution history. The research result is helpful for interpreting the pathogenesis of Shigella, as well as the genetics and evolution of the plasmid.