稻瘟净（Blasticidin S）脱氨酶基因的克隆

从一株抗稻瘟净（ＢＳ）的Ａｓｐｅｒｇｉｌｌｕｓ ｔｅｒｒｅｕｓ菌中克隆到一个ｂｌａｓｔｉｅｉｄｉｎＳ脱氨酶基因,命名为ｂｓｒＡＳ。ＤＮＡ序列分析表明ｂｓｒＡＳ不含内含子。编码区长３９０ｂｐ,编码１３０个氨基酸。将ｂｓｒＡＳ转化到稻瘟菌中,能使受体菌表达出ＢＳ脱氨酶的活性,从而产生抗药性。该基因可作为抗药标记基因使用,建立稻瘟菌的基因转化系统。     

Studies on Amino Acid Sequence Determination from N-Terminal of Peptide by Methylthiohydantoin

Methylisothiocyanate reacts with the amino group of amino acid in alkaline solution to give methylthiocarbamyl amino acid. This is converted to the methylthiohydantoin derivative (MTH-amino acid) by subsequent acidification.

Gas chromatographical identification of 20 amino acids were examined by making MTH-amino acids. Among them, ten amino acids (glycine, alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, serine and threonine) were successfully identified. Aspartic acid and glutamic acid could be identified as the methyl esters of the MTH-amino acids.     

Active Center and Mode of Reaction of Blasticidin S Deaminase

Blasticidin S deaminase (EC 3.5.4.23) was purified by affinity chromatography using a column of Sepharose 4B coupled with pyrimidinoblasticidin S as a ligand. The Michaelis constant Km and the maximum velocity F_max varied with pH, which suggests that enzyme ionization is important either for substrate binding or for catalytic activity. The inflection point at pH 8.6 ~ 8.9 in the plot of pKm versus pH was attributed to an enzyme amino group which corresponded to the carboxyl group of substrates, and an inflection at pH 5.3 in the log V_max-pH plot was assigned to the imidazole group of histidine, which appeared to be critical for catalytic deaminohydroxylation. The binding of substrates by the enzyme was inferred to be promoted thermodynamically, and activation of the substrate-enzyme complex was presumed to proceed endothermically. From the results obtained, a hypothetical mode of reaction for blasticidin S deaminase is proposed.     

Molecular Cloning and Nucleotide Sequence of the Porphobilinogen Deaminase Gene,hemC, from Chlorobium vibrioforme

We previously reported the DNA sequence and expression of the Chlorobium vibrioforme glutamyl-tRNA reductase (hemA) gene (Majumdar et al., Arch Microbiol 156:281, 1991). The sequence downstream of the hemA gene indicated homology to Escherichia coli and Bacillus subtilis porphobilinogen deaminase (hemC) gene. The Chlorobium gene was confirmed to be the porphobilinogen deaminase gene, and complete sequence of the structural gene was obtained. A 2.8-kb DNA fragment containing the 1.3-kb hemA gene of Chlorobium was cloned into a hemC auxotroph (Sz16) of Bacillus subtilis, and complementation of the auxotroph to prototrophy was achieved. DNA sequence data showed a single open reading frame of 840 bp coding a protein of 279 amino acid residues. The deduced amino acid sequence of the Chlorobium porphobilinogen deaminase revealed 39% to 46% homology with the corresponding prokaryotic and eukaryotic sequences. Received: 20 September 1996 / Accepted: 26 October 1996     

Adenine Deaminase Activity of the yicP Gene Product of Escherichia coli

During previous work on deriving inosine-producing mutants of Escherichia coli, we observed that an excess of adenine added to the culture medium was quickly converted to hypoxanthine. This phenomenon was still apparent after disruption of the known adenosine deaminase gene (add) on the E. coli chromosome, suggesting that, like Bacillus subtilis, E. coli has an adenine deaminase. As the yicP gene of E. coli shares about 35% identity with the B. subtilis adenine deaminase gene (ade), we cloned yicP from the E. coli genome and developed a strain that overexpressed its product. The enzyme was purified from a cell extract of E. coli harboring a plasmid containing the cloned yicP gene, and had significant adenine deaminase [EC 3.5.4.2] activity. It was deduced to be a homodimer, each subunit having a molecular mass of 60 kDa. The enzyme required manganese ions as a cofactor, and adenine was its only substrate. Its optimum pH was 6.5-7.0 and its optimum temperature was 60°C. The apparent K_m for adenine was 0.8 mM.     

Position of the Extra Amino Acid Sequence in the Precursor Arabinose-Binding Protein of Escherichia coli

The extra amino acid sequence in the precursor arabinose-binding protein was shown to be either close to or at the N-terminus.     

Purification and N-Terminal Amino Acid Sequence of Solanapyrone Synthase,A Natural Diels-Alderase from Alternaria solani

The first natural Diels-Alderase, solanapyrone synthase, was purified 1,630-fold from a crude extract. The 41-kDa protein on SDS-polyacrylamide gel electrophoresis was identified as truncated solanapyrone synthase, and its N-terminal amino acid sequence was found to be QETQNLNNFLESNAINP.     

奇异变形杆菌的L-氨基酸脱氢酶在大肠杆菌中的异源表达和鉴定

本研究在大肠杆菌中表达了奇异变形杆菌的L-氨基酸脱氢酶, 并对产物苯丙酮酸进行了鉴定。奇异变形杆菌的L-氨基酸脱氢酶在大肠杆菌中得到了很好的表达, 具有酶活性, 并且发现其活性与细胞膜相关。L-氨基酸脱氢酶与细胞膜的密切相关性, 对酶活来说可能是必不可少的。     

Amino Acid Differences in a 30S Ribosomal Protein from Two Strains of Escherichia coli

The 30S ribosomal proteins of the K-12 and B strains of Escherichia coli differ in at least one protein component. This component, which is allelic in the two strains, has been isolated from both organisms. Amino acid analyses show that the protein from strain B contains between 20 and 28 more amino acids than does the analogue protein from strain K-12.     

陆地棉脂肪酶基因克隆及氨基酸序列分析

根据陆地棉(Gossypium hirsutum L.)EST序列设计一对引物,采用RT-PCR方法从萌发的棉籽中获得了脂肪酶(triacylglycerol acylhydrolases,EC 3.1.1.3)基因,该基因编码483个氨基酸;比对结果显示,棉籽脂肪酶与拟南芥、水稻、蓖麻等脂肪酶相似性较低,具有由Ser-Asp-His组成的三联体催化活性中心,在亲核Ser残基周围有GXSXG保守序列.软件预测显示该脂肪酶为可溶性蛋白,分子量约为55.4 kD,等电点为9.07,不含N端信号肽,亚细胞定位可能是过氧化物酶体或胞质溶胶.     

Blasticidin S Deaminase Gene (BSD): a New Selection Marker Gene for Transformation of Arabidopsis thaliana and Nicotiana tabacum

Arabidopsis thaliana and Nicotiana tahacum were transformed to blasticidin S (BS) resistance with BSD (the BS deaminase gene from Aspergillus terreus) using the Agrohacterium-mediated transformation method. Expression of BSD allowed direct selection of transformants by the fungicide, and both kinds of transgenic plants showed high level of resistance phenotype at 100 ppm of BS sprayed on the leaves. Using Botrytis cinerea, the causal fungus of gray mold disease, it was exemplified that application of BS could control the disease in transgenic tobacco with negligible phytotoxicity.     

Nucleotide Sequence of the Gene for an Alkaline Endoglucanase from an Alkalophilic Bacillus and Its Expression in Escherichia coli and Bacillus subtilis

The gene for an alkaline endoglucanase from the alkalophilic Bacillus sp. KSM-64 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HB101. The nucleotide sequence of a 4.1-kb region of the HindIII insert had two open reading frames, ORF-1 and ORF-2. The protein deduced from ORF-1 was composed of 244 amino acids with an M_r of 27,865. Subcloning analysis proved that the alkaline endoglucanase was encoded by ORF-2 (822 amino acids with an M_r of 91,040). Upstream from ORF-2, there were three consensus like sequences of the sigma A-type promoter of Bacillus subtilis, a putative Shine-Dalgarno sequence (AGGAGGT), and a catabolite repression operator-like sequence (TGTAAGC-GGTTAACC). The HindIII insert was subcloned into a shuttle vector, pHY300PLK, and the encoded alkaline endoglucanase gene was highly expressed both in E. coli and B. subtilis. One of the three promoter-like sequences in ORF-2 could be suitable for high levels of enzyme expression in both host organisms.     

N-Terminal Amino Acid Sequence and Processing Site of Sweet Potato Cytochrome c Oxidase Subunit II

The N-terminal amino acid sequence of sweet potato cytochromec oxidase subunit II polypeptide was determined. Comparisonsbetween the sequence and amino acid sequences deduced from thenucleotide sequences of other higher plant subunit II genesindicate a post-translational clevage of N-terminal extensionpart. ¹Present address: Institute of Low Temperature Science, HokkaidoUniversity, Sapporo, 060 Japan. (Received June 13, 1989; Accepted September 8, 1989)     

龙葵叶绿体的阿特拉津抗性基因psbA的核苷酸序列及有关分析

对克隆在psb135质粒上的来自龙葵阿特拉津抗性生物型的psbA基因进行DNA序列分析,测得长为1384个核苷酸的全序列,包括该基因的全部编码区和5′上游顺序。该基因的核苷酸序列与另一个独立来源的龙葵阿特拉津抗性基因的核苷酸序列完全相同,在由核苷酸推导的氨基酸序列的基础上,比较了分别由龙葵抗阿特拉津和对阿特拉津敏感的psbA基因编码的32kD蛋白质的二级结构,并对其可能的含意进行了讨论。     

Sequence determination of protein S9 from the Escherichia coli ribosome.

The complete amino acid sequence of ribosomal protein S9 of Escherichia coli has been established. The protein was digested with trypsin and Staphylococcus aureus protease and the resulting peptides were separated by ion exchange chromatography on a new Dowex 50W-X7 microcolumn or a small phosphocellulose column. If necessary, they were rechromatographed on purified cellulose thin-layer plates on a preparative scale. The sequences of the peptides were determined by the micro dansyl-Edman technique, whereas the alignments of the tryptic peptides were mainly established from large cyanogen bromide fragments which were sequenced by the automatic Edman degradation process. Protein S9 is 128 amino acids long and has the following composition: Asx7, Thr5, Ser7, Glx16, Pro3, Gly13, Ala10, Val10, Met3, Ile7, Leu9, Tyr5, Phe4, His1, Lys10, Arg18. The molecular weight as calculated from the amino acid composition is 14 569. A total of 92.6 mg of the lyophilized protein was used for the determination of the primary structure of S9. Most of the material was needed to isolate sufficient amounts of the CNBr-fragments for the automatic degradation in the sequenator.     

Ultrastructure of Escherichia coli Depleted of an Amino Acid

Particles accumulating during amino acid starvation of Escherichia coli C600 are described and illustrated.     

Radiation Inhibition of Amino Acid Uptake by Escherichia coli

The inhibition of macromolecular synthesis in Escherichia coli by ionizing radiation has been investigated. The survival of the ability to incorporate arginine, leucine, isoleucine, histidine, uracil, and glucose after various doses of gamma radiation, deuteron and alpha particle bombardment has been measured. All amino acids are incorporated by processes which show the same radiation sensitivity. The sensitivity of uracil corresponds to a volume which is roughly spherical, of radius about 160A, whereas the amino acids possess sensitive regions which are long and thin in character. The uptake of glucose is concerned with a smaller, roughly spherical unit. The possible identification of the radiation-sensitive targets with cellular constituents is discussed. The long thin character observed for amino acids suggests that the sensitive region affected by radiation is an unfolded form of a ribosome, or alternatively a long nucleic acid molecule. For uracil the sensitive region fits with a 70S ribosome, while for glucose a smaller particle would fit the data.     

Purification and N-Terminal Amino Acid Sequence of Dextranicin 24, a Bacteriocin of Leuconostoc sp.

Leuconostoc mesenteroides subsp. dextranicum strain J24 synthesized a bacteriocin named Dextranicin 24 (Dex-24), which inhibited only other Leuconostoc sp. strains. It was purified by a two-step procedure from the fraction of the bacteriocin bound to the producer cells at the end of the growth: desorption form the cells at acidic pH, followed by reserve phase HPLC. The N-terminal sequence of Dex-24 was the following: NH₂₋ K G V L G W L S M A S S A L T G P Q Q . . . Received: 26 January 1996 / Accepted: 28 March 1996     

Cloning and Nucleotide Sequence of the Pullulanase Gene of Thermus thermophilus HB8 and Production of the Enzyme in Escherichia coli

A 3.4-kb SphI fragment carrying the pullulanase gene of Thermus thermophilus HB8 was cloned. Based on the nucleotide sequence of it and the flanking region analyzed by direct sequencing of the inverse PCR product, an expression vector was constructed. The E. coli cells harboring the plasmid produced an about 80-kDa protein having pullulanase activity, the optimum temperature of which was 70°C.