The region essential for efficient autonomous replication of pSa in Escherichia coli

Summary Comparative analyses were made between plasmid pSa17, a deletion derivative of pSa that is capable of replicating efficiently in Escherichia coli and plasmid pSa3, a derivative that is defective for replication. By comparing the restriction maps of these two derivatives, the regions essential for replication and for stable maintenance of the plasmid were determined. A 2.5 kb DNA segment bearing the origin of DNA replication of pSa17 was sequenced. A 36 kDa RepA protein was encoded in the region essential for replication. Downstream of the RepA coding region was a characteristic sequence including six 17 bp direct repeats, the possible binding sites of RepA protein, followed by AT-rich and GC-rich sequences. Furthermore, an 8 bp incomplete copy of the 17 bp repeat was found in the promoter region of the repA gene. Based on the hypothesis that RepA protein binds to this partial sequence as well as to intact 17 bp sequences, an autoregulatory system for the synthesis of RepA protein may be operative. Another open reading frame (ORF) was found in the region required for the stability of the plasmid. The putative protein encoded in this ORF showed significant homology to several site-specific recombination proteins. A possible role of this putative protein in stable maintenance of the plasmid is discussed.     

The marker SCK13<Subscript>603</Subscript> associated with resistance to ascochyta blight in chickpea is located in a region of a putative retrotransposon

The sequence characterized amplified region (SCAR) marker SCK13(603), associated with ascochyta blight resistance in a chickpea recombinant inbred line (RIL) population, was used as anchored sequence for genome walking. The PCRs performed in the walking steps to walk in the same direction produced eight bands in 5' direction and five bands in 3' direction with a length ranking from 530 to 2,871 bp. The assembly of the bands sequences along with the sequence of SCK13(603) resulted in 7,815 bp contig. Blastn analyses showed stretches of DNA sequence mainly distributed from the nucleotides 1,500 to 4,500 significantly similar to Medicago truncatula genomic DNA. Three open reading frames (ORFs) were identified and blastp analysis of predicted amino acids sequences revealed that ORF1, ORF2 and ORF3 had significant similarity to a CCHC zinc finger protein, to an integrase, and to a precursor of the glucoamylase s1/s2, respectively, from M. truncatula. The high homology of the putative proteins derived from ORF1 and ORF2 with retrotransposon proteins and the prediction of the existence of conserved domains usually present in retrotransposon proteins indicate that the marker SCK13(603) is located in a region of a putative retrotransposon. The information generated in this study has contributed to increase the knowledge of this important region for blight resistance in chickpea.     

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.     

哈氏弧菌dam基因的克隆与分析

利用兼并PCR的方法克隆得到哈氏弧菌T4的DNA腺嘌呤甲基化酶(dam)基因,序列分析表明该基因编码279个氨基酸,与其它已知弧菌的Dam具有较高的同源性,其中与副溶血弧菌Dam的相同性达95%。功能检验表明所克隆的dam基因在大肠杆菌中具有DNA腺嘌呤甲基化酶活性,能够甲基化大肠杆菌染色体DNA GATC序列中的腺嘌呤。运用染色体步移法获得dam基因上游的3251 bp DNA,发现该区域含有3个基因,其与dam在染色体上的相对排列顺序为:莽草酸激酶-脱氢奎尼酸合成酶-damX-dam。对dam上游DNA序列研究发现位于翻译起点ATG上游的78bp、112bp和477bpDNA片段皆具有启动子活性,但前者的活性明显高于后二者。     

苏云金芽孢杆菌chiA,chiB全基因的克隆、表达及其序列分析

以苏云金芽孢杆菌科默尔亚种15A3菌株基因组DNA为模版,用touchdown PCR方法扩增几丁质酶ChiA和ChiB的全基因序列(GenBank登录号:EF103273和DQ512474)。将PCR产物连接pUCm-T克隆载体,获得重组质粒pUCm-chiA和pUCm-chiB,分别转化E.coliXL-Blue。克隆的几丁质酶基因可以利用本身的启动子异源表达各自的蛋白,不需要几丁质作为诱导物。表达的几丁质酶能够分泌到胞外。证明15A3菌株可组成型表达2种几丁质酶。经核苷酸及氨基酸序列分析证明,chiA基因全长1426bp,含有343bp的上游非编码区和1083bp的ORF,编码360个氨基酸。推测成熟蛋白分子量为36kD,只有一个几丁质酶催化域。chiB基因全长2279bp,含有248bp的上游非编码区和2031bp的ORF,编码676个氨基酸。推测成熟蛋白分子量约为70.6kD,具有三个功能域。核苷酸序列分析显示chiA和chiB的启动子所处的位置及转录起始碱基都不相同,-35区相同,而-10区有两个碱基不同,SD序列也不完全一致。     

Identification of replication origins in the genome of the methanogenic archaeon,<Emphasis Type="Italic"> Methanocaldococcus jannaschii</Emphasis>

Methanocaldococcus jannaschii has been notorious as an archaeon in which the replication origins are difficult to identify. Although extensive efforts have been exerted on this issue, the locations of replication origins still remain elusive 7 years after the publication of its complete genome sequence in 1996. Ambiguous results were obtained in identifying the replication origins of M. jannaschii based on all theoretical and experimental approaches. In the genome of M. jannaschii, we found that an ORF (MJ0774), annotated as a hypothetical protein, is a homologue of the Cdc6 protein. The position of the gene is at a global minimum of the x component of the Z curve, i.e., RY disparity curve, which has been used to identify replication origins in other Archaea. In addition, an intergenic region (694,540–695,226 bp) that is between the cdc6 gene and an adjacent ORF shows almost all the characteristics of known replication origins, i.e., it is highly rich in AT composition (80%) and contains multiple copies of repeat elements and AT stretches. Therefore, these lines of evidence strongly suggest that the identified region is a replication origin, which is designated as oriC1. The analysis of the y component of the Z curve, i.e., MK disparity curve, suggests the presence of another replication origin corresponding to one of the peaks in the MK disparity curve at around 1,388 kb of the genome.Communicated by G. Antranikian     

Characterization of a Theta Plasmid Replicon with Homology to All Four Large Plasmids ofBacillus megateriumQM B1551

A replicon from one of an array of seven indigenous compatible plasmids ofBacillus megateriumQM B1551 has been cloned and sequenced. The replicon hybridized with all four of the large plasmids (165, 108, 71, and 47 kb) of strain QM B1551. The cloned 2374-bpHindIII fragment was sequenced and contained two upstream palindromes and a large (>419-amino-acid) open reading frame (ORF) truncated at the 3′ end. Unlike most plasmid origins, a region of four tandem 12-bp direct repeats was located within the ORF. The direct repeats alone were incompatible with the replicon, suggesting that they are iterons and that the plasmid probably replicates by theta replication. The ORF product was shown to act intrans.A small region with similarity to theB. subtilischromosomal origin membrane binding region was detected as were possible binding sites for DnaA and IHF proteins. Deletion analysis showed the minimal replicon to be a 1675-bp fragment containing the incomplete ORF plus 536 bp upstream. The predicted ORF protein of >48 kDa was basic and rich in glutamate + glutamine (16%). There was no significant amino acid similarity to any gene, nor were there any obvious motifs present in the ORF. The data suggest that this is a theta replicon with an expressedrepgene required for replication. The replicon contains its iterons within the gene and has no homology to reported replicons. It is the first characterization of aB. megateriumreplicon.     

Cloning, expression, and nucleotide sequence of the Lactobacillus helveticus 481 gene encoding the bacteriocin helveticin J.

Lactobacillus helveticus 481 produces a 37-kDa bacteriocin called helveticin J. Libraries of chromosomal DNA from L. helveticus were prepared in lambda gt11 and probed for phage-producing fusion proteins that could react with polyclonal helveticin J antibody. Two recombinant phage, HJ1 and HJ4, containing homologous inserts of 350 and 600 bp, respectively, produced proteins that reacted with antibody. These two phage clones specifically hybridized to L. helveticus 481 total genomic DNA but not to DNA from strains that did not produce helveticin J or strains producing unrelated bacteriocins. HJ1 and HJ4 lysogens produced beta-galactosidase fusion proteins that shared similar epitopes with each other and helveticin J. The intact helveticin J gene (hlv) was isolated by screening a library of L. helveticus chromosomal DNA in lambda EMBL3 with the insert DNA from phage HJ4 as a probe. The DNA sequence of a contiguous 3,364-bp region was determined. Two complete open reading frames (ORF), designated ORF2 and ORF3, were identified within the sequenced fragment. The 3' end of another open reading frame, ORF1, was located upstream of ORF2. A noncoding region and a putative promoter were located between ORF1 and ORF2. ORF2 could encode an 11,808-Da protein. The L. helveticus DNA inserts of the HJ1 and HJ4 clones reside within ORF3, which begins 30 bp downstream from the termination codon of ORF2. ORF3 could encode a 37,511-Da protein. Downstream from ORF3, the 5' end of another ORF (ORF4) was found. A Bg/II fragment containing ORF2 and ORF3 was cloned into pGK12, and the recombinant plasmid, pTRK135, was transformed into Lactobacillus acidophilus via electroporation. Transformants carrying pTRK135 produced a bacteriocin that was heat labile and exhibited an acitivity spectrum that was the same as that of helveticin J.     

Molecular cloning,characterization and expression analysis of <Emphasis Type="Italic">Cm</Emphasis>APX

The RT PCR and RACE methods were used to obtain the cDNA sequence of an APX gene of muskmelon after the leaves were induced with powdery mildew. The cDNA length of the APX gene is 1,047 bp with a 750 bp ORF encoded a 249 amino acid and the molecular weight of APX protein is 27.3 kDa. The analysis showed that the CmAPX genomic DNA contained 10 extrons and 9 introns. The identity of the amino acid sequence deduced from the cDNA with the APX family of other homologous members was about 74–97%. A Full-length of ORF was sub-cloned into prokaryotic expression vector pET24a. The recombinant proteins had high expression level in E. coli. Analysis of expression at mRNA level showed that CmAPX exhibited highly tissue-specific patterns of expression. The mRNA level and enzyme activities assays showed that CmAPX might play an important role in the pathogenesis of powdery mildew.     

Nucleotide sequence of IS110, an insertion sequence of Streptomyces coelicolor A3(2).

The nucleotide sequences of the Streptomyces transposable element IS110 and its insertion site in the DNA of a derivative of the temperate phage luminal diameter C31 were determined. The element is inserted about 460 bp from the right-hand end of luminal diameter C31 DNA, in a region of apparently non-coding DNA. The target site (in a run of seven C residues) is within an 11 bp sequence homologous with one end of IS110. The inserted element is flanked by runs of 11 and 15 C residues which form part of more extensive regions of homology between the left and right junction regions. Imperfect inverted repeats (10 matches out of 15 bp) are present near (but not at) the ends of IS110. The whole IS110 element contains about 1550 bp of which 71% are G-C bp. One major potentially protein-coding region (ORF 1215) was detected, of 1215 bp, the product of which, a presumptively soluble protein of MR 43,563, was not overtly related to any entry in a protein sequence database. A smaller open reading frame (ORF 330) was tentatively identified in the opposite strand of the ORF 1215 region.     

丹参中病程相关蛋白基因SmSTH-2的生物信息学分析

对丹参cDNA文库的表达序列标签(EST)序列进行BLAST分析显示,其中一条序列与病程相关蛋白基因STH-2有较高的同源性。该序列全长691bp,包含1个长483bp的开放阅读框(ORF),编码160个氨基酸,命名为SmSTH-2。生物信息学分析显示:SmSTH-2所编码蛋白的分子质量为17990Da,等电点为5.15,富含谷氨酸、赖氨酸、甘氨酸、丝氨酸,无信号肽,属于稳定类蛋白。与NCBI注册的其他6种植物来源的病程相关蛋白基因编码的氨基酸序列的同源性在42%～46%之间。实时荧光定量PCR的方法检测丹参不同组织部位中SmSTH-2表达和病原菌对该基因诱导表达的影响的结果表明:SmSTH-2在植物的根、茎、叶中均有不同程度的表达,其表达丰度为根>叶>茎;丹参叶片接种黄瓜细菌性角斑病病原菌后,4d内可诱导该基因的表达量持续增加。用PCR方法从基因组水平克隆到SmSTH-2的DNA序列,测序表明SmSTH-2的编码序列在DNA水平上含有一个71bp的内含子,DNA序列注册号为EF621486.     

The new class 11 transposon Tn163 is plasmid-borne in two unrelated Rhizobium leguminosarum biovar viciae strains

Tn163 is a transposable element identified in Rhizobium leguminosarum bv. viciae by its high insertion rate into positive selection vectors. The 4.6 kb element was found in only one further R. leguminosarum bv. viciae strain out of 70 strains investigated. Both unrelated R. leguminosarum bv. viciae strains contained one copy of the transposable element, which was localized in plasmids native to these strains. DNA sequence analysis revealed three large open reading frames (ORFs) and 38 bp terminal inverted repeats. ORF1 encodes a putative protein of 990 amino acids displaying strong homologies to transposases of class 11 transposons. ORF2, transcribed in the opposite direction, codes for a protein of 213 amino acids which is highly homologous to DNA invertases and resolvases of class II transposons. Homology of ORF1 and ORF2 and the genetic structure of the element indicate that Tn163 can be classified as a class II transposon. It is the first example of a native transposon in the genus Rhizobium. ORF3, which was found not to be involved in the transposition process, encodes a putative protein (256 amino acids) of unknown function. During transposition Tn163 produced direct repeats of 5 bp, which is typical for transposons of the Tn3 family. However, one out of the ten insertion sites sequenced showed a 6 by duplication of the target DNA; all duplicated sequences were A/T rich. Insertion of Tn163 into the sacB gene revealed two hot spots. Chromosomes of different R. leguminosarum bv. viciae strains were found to be highly refractory to the insertion of Tn163.     

Characterization of a second gene involved in bacterio-opsin gene expression in a halophilic archaebacterium.

Southern blot analysis and nucleotide sequencing of DNA from three bacterio-opsin-deficient mutants of the archaebacterium Halobacterium halobium (M86, W105, and W109) revealed that they each contain an alteration in a region 2,000 to 3,800 base pairs (bp) upstream of the bacterio-opsin gene (bop). Nucleotide sequence analysis of this region, which is also located downstream of the previously characterized brp gene, revealed that it contains an open reading frame (ORF) of 2,022 bp. This 2,022-bp ORF has a start codon which overlaps the stop codon of the brp gene and is read in the same direction. The ORF could encode an acidic protein of 73,334 daltons (674 amino acids) with a predicted secondary structure typical of a soluble protein. Bop mutant M86 contains a 1,883-bp deletion extending from bp 351 of the ORF, to 197 bp beyond the stop codon. Mutant W105 has an ISH2 element integrated at bp 1239 of the ORF, and mutant W109 has an ISH26 element integrated at bp 1889. Our results suggest that the ORF is a gene (designated bat for bacterio-opsin activator gene) involved in bop gene expression.     

Identification of a replication protein and repeats essential for DNA replication of the temperate lactococcal bacteriophage TP901-1

DNA replication of the temperate lactococcal bacteriophage TP901-1 was shown to involve the gene product encoded by orf13 and the repeats located within the gene. Sequence analysis of 1,500 bp of the early transcribed region of the phage genome revealed a single-stranded DNA binding protein analogue (ORF12) and the putative replication protein (ORF13). The putative origin of replication was identified as series of repeats within orf13 and was shown to confer a TP901-1 resistance phenotype when present in trans. Site-specific mutations were introduced into the replication protein and into the repeats. The mutations were introduced into the TP901-1 prophage by homologous recombination by using a vector with a temperature-sensitive replicon. Subsequent analysis of induced phages showed that the protein encoded by orf13 and the repeats within orf13 were essential for phage TP901-1 amplification. In addition, analyses of internal phage DNA replication showed that the ORF13 protein and the repeats are essential for phage TP901-1 DNA replication in vivo. These results show that orf13 encodes a replication protein and that the repeats within the gene are the origin of replication.