寡氧单胞菌中CRISPR位点的分析

对寡氧单胞菌基因组中的CRISPR位点进行生物信息学分析。CRISPRdb数据库中公布的和NCBI上下载的共26株寡氧单胞菌的基因组序列,分析其CRISPR位点的分布情况、重复序列、间隔序列以及间隔序列和噬菌体序列数量之间的关系。共发现15个确定的CRISPR结构和132个可疑的CRISPR,不同菌株CRISPR结构中的重复序列具有较强的保守性。间隔序列的靶向基因主要来自细菌的基因组,说明寡氧单胞菌CRISPR的的进化与其他细菌基因有关。此外,间隔序列与前噬菌体数量之间的负相关关系,说明CRISPR能阻止噬菌体的入侵。寡氧单胞菌CRISPR位点的分析为进一步研究耐药性及基因组稳定性奠定了基础。     

基于ITS和trnL-trnF序列的草木樨种群遗传多样性研究

以白花草木樨(Melilotus alba)和黄花草木樨(Melilotus officinalis)18个地理种群植物为材料,用ITS序列和trnL-trnF序列研究了2种草木樨不同种群间的遗传多样性。结果表明:(1)trnL-trnF序列对位后长度为459bp,其中包括6个变异位点,6个简约信息位点,G+C含量为33.1%;ITS序列对位后长度为714bp,其中包括5个变异位点,3个简约信息位点,G+C含量为48.9%。(2)在基于trnL-trnF序列构建的系统发育树中,2种草木樨能够形成单系分支,说明trnL-trnF序列在草木樨中的鉴别能力较强。(3)单倍型多样性以及核苷酸多样性分析表明,黄花草木樨的遗传多样性高于白花草木樨。     

Comparative sequence analysis of the SALT OVERLY SENSITIVE1 orthologous region in Thellungiella halophila and Arabidopsis thaliana

To provide a framework for studies to understand the contribution of SALT OVERLY SENSITIVE1 (SOS1) to salt tolerance in Thellungiella halophila, we sequenced and annotated a 193-kb T. halophila BAC containing a putative SOS1 locus (ThSOS1) and compared the sequence to the orthologous 146-kb region of the genome of its salt-sensitive relative, Arabidopsis thaliana. Overall, the two sequences were colinear, but three major expansion/contraction regions in T. halophila were found to contain five Long Terminal Repeat retrotransposons, MuDR DNA transposons and intergenic sequences that contribute to the 47.8-kb size variation in this region of the genome. Twenty-seven genes were annotated in the T. halophila BAC including the putative ThSOS1 locus. ThSOS1 shares gene structure and sequence with A. thaliana SOS1 including 11 predicted transmembrane domains and a cyclic nucleotide-binding domain; however, different patterns of Simple Sequence Repeats were found within a 540-bp region upstream of SOS1 in the two species.     

Cloning and nucleotide sequence of the Salmonella typhimurium LT2 gnd gene and its homology with the corresponding sequence of Escherichia coli K12

Summary The complete nucleotide sequence of the Salmonella strain LT2 gnd gene for 6-phosphogluconate dehydrogenase was determined. The gene contains 1404 bases and encodes a 468 amino acid polypeptide, which is the same as for Escherichia coli K12. The DNA sequence shows 14.8% difference between the two and the amino acid sequence 3.6% difference. Changes are mostly in the third codon base and most of the amino acid changes are conservative.     

Identification of the DNA damage-responsive elements of therhp51+ gene, arecA andRAD51 homolog from the fission yeastSchizosaccharomyces pombe

TheSchizosaccharomyces pombe rhp51 ⁺ gene encodes a recombinational repair protein that shares significant sequence identities with the bacterial RecA and theSaccharomyces cerevisiae RAD51 protein. Levels ofrhp51 ⁺ mRNA increase following several types of DNA damage or inhibition of DNA synthesis. Anrhp51::ura4 fusion gene was used to identify the cis-acting promoter elements involved in regulatingrhp51 ⁺ expression in response to DNA damage. Two elements, designated DRE1 and DRE2 (fordamage-responsiveelement), match a decamer consensus URS (upstream repressing sequence) found in the promoters of many other DNA repair and metabolism genes fromS. cerevisiae. However, our results show that DRE1 and DRE2 each function as a UAS (upstream activating sequence) rather than a URS and are also required for DNA-damage inducibility of the gene. A 20-bp fragment located downstream of both DRE1 and DRE2 is responsible for URS function. The DRE1 and DRE2 elements cross-competed for binding to two proteins of 45 and 59 kDa. DNase I footprint analysis suggests that DRE1 and DRE2 bind to the same DNA-binding proteins. These results suggest that the DRE-binding proteins may play an important role in the DNA-damage inducibility ofrhp51 ⁺ expression.     

The complete amino acid sequence of ribosomal protein S8 fromThermus thermophilus

Protein S8 fromThermus thermophilus consists of 138 amino acids ofM, 15,840. Its primary structure was established using peptide sequences from two different digests. Protein S8 fromT. thermophilus shares a high percentage of identity with protein S8 fromThermus aquaticus. There are some consensus sequences between proteins S8 from eubacteria, archebacteria, chloroplasts, and cyanelles.     

4种薯蓣属植物的psbA-trnH片段序列分析

对4种薯蓣属(Dioscorea)植物——小花盾叶薯蓣(D.sinoparviflora C.T.Ting.)、盾叶薯蓣(D.zingiberensis C.H Wright.)、黄独(D.bulbifera L.)和薯蓣(D.polystachya Turczaninow) 共22个植物类群的叶绿体psbA-trnH基因区进行PCR扩增并测序,获得了该区间的完整序列,小花盾叶薯蓣的psbA-trnH片段全长589~593 bp,盾叶薯蓣全长为575~599 bp,黄独全长为372 bp,薯蓣全长为355~368 bp。将所得序列用Bayesian推断其系统发育关系,结果发现psbA-trnH片段不能够完全区分4种薯蓣属植物种间和种内的各个分类群(不同产地),且不能区分盾叶薯蓣与小花盾叶薯蓣;推测薯蓣的演变过程可能与人工驯化有关,黄独与栽培薯蓣的亲缘关系较近,而与野生薯蓣的亲缘关系较远。     

Use of recombination techniques to examine the structure of the csg locus of Myxococcus xanthus

Summary The technique of chromosome walking was used to isolate approximately 60 kb of DNA from the region containing the complementation group uncoordinated of Drosophila melanogaster, located in that part of the X chromosome which spans the euchromatin-heterochromatin junction. The cloned DNA can be divided into two distinct regions. The first contains sequences that are low copy number or unique and are largely conserved between strains. The second region is characterized by units repeated in tandem arrays and is polymorphic within, and between, strains. Each repetitive unit is separated by a member of an abundant sequence family, part of which is homologous to the ribosomal type 1 insertion sequence of D. melanogaster. The molecular organization of the cloned DNA was compared with that of sequences isolated from regions of intercalary heterochromatin and also with genes which have been characterized from more conventional euchromatic regions.     

Molecular cloning, sequence and regulation of expression of the recA gene of the phototrophic bacterium Rhodobacter sphaeroides

The recA gene of Rhodobacter sphaeroides 2.4.1 has been isolated by complementation of a UV-sensitive RecA^– mutant of Pseudomonas aeruginosa. Its complete nucleotide sequence consists of 1032 bp, encoding a polypeptide of 343 amino acids. The deduced amino acid sequence displayed highest identity to the RecA proteins from Rhizobium mehloti, Rhizobium phaseoli, and Agrobacterium tumefaciens. An Escherichia coli-like SOS consensus region, which functions as a binding site for the LexA repressor molecule was not present in the 215 by upstream region of the R. sphaeroides recA gene. Nevertheless, by using a recA-lacZ fusion, we have shown that expression of the recA gene of R. sphaeroides is inducible by DNA damage. A recA-defective strain of R. sphaeroides was obtained by replacement of the active recA gene by a gene copy inactived in vitro. The resulting recA mutant exhibited increased sensitivity to UV irradiation, and was impaired in its ability to perform homologous recombination as well as to trigger DNA damage-mediated expression. This is the first recA gene from a Gram-negative bacterium that lacks an E. coli-like SOS box but whose expression has been shown to be DNA damage-inducible and auto-regulated.     

Amino acid sequence of myoglobin from the molluscBursatella leachii

The complete amino acid sequence of myoglobin from the triturative stomach of gastropodic molluscBursatella leachii has been determined. It is composed of 146 amino acid residues, is acetylated at the N-terminus, and contains a single histidine residue at position 95 which corresponds to the heme-binding proximal histidine. The E7 distal histidine, which is conserved widely in myoglobins and hemoglobins, is replaced by valine inBursatella myoglobin. The amino acid sequence ofBursatella myoglobin shows strong homology (73–84%) with those ofAplysia andDolabella myoglobins.     

Application of petG-trnP sequence to systematic study of Chinese Cupressus species

Chinese Cupressus L. includes five species. The molecular phylogenetic relationship of the Cupressus species and Chamaecyparis L. were determined by comparing 417–479 bp of chloroplast petG-trnP intergenic spacer sequence. In PAUP^* analysis, Platycladus orientalis was used as the functional out group. By using the maximum likelihood method 1 077 trees were examined and the result showed that one tree had a best score of -Ln=2 232.47. The phylogenetic tree clearly showed that Chamaecyparis nootkatensis was diverged from other Chamaecyparis species. Based on the results, together with evidences from other aspects, we consider that Cupressus funebris and Chamaecyparis nootkatensis should be placed in the genus Cupressus. The use of cpDNA intergenic spacer petG-trnP in Cupressus was also discussed. __________ Translated from Journal of Sichuan University (Natural Science Edition), 2005, 42(5): 1033–1037 [译自: 四川大学学报 (自然科学版) 2005, 42(5): 1033–1037]     

Genetic characterization and sequence analysis of the duplicated nifA/nifB gene region of Rhodobacter capsulatus

Summary A DNA region showing homology to Klebsiella pneumoniae nifA and nifB is duplicated in Rhodobacter capsulatus. The two copies of this region are called nifA/nifB copy I and nifA/nifB copy II. Deletion mutagenesis demonstrated that either of the two copies is sufficient for growth in nitrogen-free medium. In contrast, a double deletion mutant turned out to be deficient in nitrogen fixation. The complete nucleotide sequence of a 4838 bp fragment containing nifA/nifB copy I was determined. Two open reading frames coding for a 59653 (NifA) and a 49453 (NifB) dalton protein could be detected. Comparison of the amino acid sequences revealed that the R. capsulatus nifA and nifB gene products are more closely related to the NifA and NifB proteins of Rhizobium meliloti and Rhizobium leguminosarum than to those of K. pneumoniae. A rho-independent termination signal and a typical nif promoter region containing a putative NifA binding site and a consensus nif promoter are located within the region between the R. capsulatus nifA and nifB genes. The nifB sequence is followed by an open reading frame (ORF1) coding for a 27721 dalton protein in nifA/nifB copy I. DNA sequence analysis of nifA/nifB copy II showed that both copies differ in the DNA region downstream of nifB and in the noncoding sequence in front of nifA. All other regions compared, i.e. the 5 part of nifA, the intergenic region and the 3 part of nifB, are identical in both copies.     

Cloning and sequencing of the HU-2 gene of Escherichia coli

Summary The Escherichia coli HU-2 gene was cloned using a DNA fragment from the HU-1 gene as a probe. The amino acid sequence of the HU-2 protein deduced from the nucleotide sequence is in good agreement with the published sequence. The nucleotide sequence has a possible promoter and a typical ribosomal binding site upstream of the translation initiation codon (AUG) and a possible rhoindependent terminater site downstream of the termination codon (UAA) of the gene.     

Cloning and nucleotide sequence of the Salmonella typhimurium pepM gene

Summary The pepM gene coding for a methionine-specific aminopeptidase was cloned from Salmonella typhimurium and its nucleotide sequence determined. The gene encoded a 264 amino acid protein that was homologous to a similar protein from Escherichia coli. The sequence of an overproducer mutant allele, pepM100, contained a single base change in the likely –35 region of the pepM promoter that increased its homology to the consensus promoter sequence. A region downstream from the pepM coding sequence contained extensive inverted repeats and was homologous to sequences found elsewhere in both Salmonella and other bacterial species.     

Cloning, sequencing and enhanced expression of the Trichoderma reesei endoxylanase II (pI 9) gene xln2

The Trichoderma reesei xln2 gene coding for the pI 9.0 endoxylanase was isolated from the wild-type strain QM6a. The gene contains one intron of 108 nucleotides and codes for a protein of 223 amino acids in which two putative N-glycosylation target sites were found. Three different T. reesei strains were transformed by targeting a construct composed of the xln2 gene, including its promoter, to the endogenous cbh1 locus. Highest overall production levels of xylanase were obtained using T. reesei ALK02721, a genetically engineered strain, as a host. Integration into the cbh1 locus was not required for enhanced expression under control of the xln2 promoter.     

Cloning and nucleotide sequence of the gene braB coding for the sodium-coupled branched-chain amino acid carrier in Pseudomonas aeruginosa PAO

Summary The gene braB, encoding the Na^–-coupled carrier for branched-chain amino acids in Pseudomonas aeruginosa PAO, was cloned on cosmid pMMB34. The cosmid clones carrying the braB gene were identified as those that restored growth at low leucine concentration and Na^–-dependent leucine transport activity to P. aeruginosa PAO3536 defective in the transport of branched-chain amino acids. Determination of the nucleotide sequence of the DNA fragment shows that the braB gene comprises 1311 bp and encodes a hydrophobic protein of 437 amino acids with a calculated M_r of 45279. The hydropathy profile suggests that there exist in the carrier protein 12 hydrophobic segments long enough to traverse the membrane. The amino acid sequence shows a high degree of homology with thebrnQ product, a branched-chain amino acid carrier of Salmonella typhimurium, while no homology in the nucleotide sequences is found in the braB and brnQ genes.     

Identification of the coding region for the putidaredoxin reductase gene from the plasmid of Pseudomonas putida

The first 12 NH₂-terminal amino acids of the Pseudomonas putida putidaredoxin reductase were shown to be Met-Asn-Ala-Asn-Asp-Asn-Val-Val-Ile-Val-Gly-Thr. Comparison of these data with the DNA sequence of the BamHI-HindIII 197-base fragment derived from the PstI 2.2-kb fragment obtained from the P. putida plasmid showed that the putidaredoxin reductase gene was downstream from the cytochrome P-450 gene and the intergenic region had the 24-nucleotide sequence TAAACACATGGGAGTGCGTGCTAA. The Shine-Dalgarno sequence GGAG was detected in this region. The initiating triplet for the reductase gene was GTG, which normally codes for valine, but in the initiating codon position codes for methionine. From the amino acid sequence and X-ray data comparisons with other flavoproteins, what appears to be the AMP binding region of the FAD can be recognized in the NH₂-terminal portion of the reductase involving residues 5–35.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.