水稻MYB cDNA的克隆和表达分析

根据植物MYB类转录因子DNA结合功能域的保守区设计一对简并引物 ,以水稻根、小苗和未成熟种子中的RNA为材料 ,用RT PCR方法扩增出约 180bp的片段。序列分析表明 ,它们与MYB基因的保守区有很好的同源性。以未成熟种子中获得的这一 180bp片段作探针 ,从水稻未成熟种子cDNA文库中分离到 5个新的MYB基因家族成员 ,它们是OsMYB12、13、14、15和5 1。在酵母系统中证实OsMYB13、OsMYB15和Os MYB5 1蛋白具有转录激活功能。Northern印迹分析表明 ,OsMYB5 1主要在未成熟种子中表达 ,在根和小苗中表达水平较低。RT PCR分析表明 ,OsMYB15在根、茎、小穗、叶片和种子中有低水平的表达     

Effect of Tenuazonic Acid on In Vitro Amino Acid Incorporation by Rice Embryo Ribosomes

Deacetylcephalosporin C negative mutants, lacking a certain step in the pathway of deacetylcephalosporin C biosynthesis, were obtained from the deacetylcephalosporin C producing mutant No. 40 of Cephalosporium acremonium by treatment with N-methyl-N′-nitro-N-nitrosoguanidine. Among these mutants, the strain No. 40-20 was found to mainly accumulate a cephalosporin compound other than deacetylcephalosporin C and cephalosporin C. The cephalosporin was isolated as crystals from the culture broth of the mutant No. 40-20, and identified as deacetoxycephalosporin C, possessing a D-a-aminoadipyl side chain at C-7, by physical, chemical and biological methods. The profile of deacetoxycephalosporin C fermentation and the examination of the biochemical reduction of deacetylcephalosporin C led us to the conclusion that deacetoxycephalosporin C would be produced through de novo synthesis by this mutant.     

Cloning,Expression, and Functional Characterization of Secondary Amino Acid Transporters of Lactococcus lactis

Fourteen genes encoding putative secondary amino acid transporters were identified in the genomes of Lactococcus lactis subsp. cremoris strains MG1363 and SK11 and L. lactis subsp. lactis strains IL1403 and KF147, 12 of which were common to all four strains. Amino acid uptake in L. lactis cells overexpressing the genes revealed transporters specific for histidine, lysine, arginine, agmatine, putrescine, aromatic amino acids, acidic amino acids, serine, and branched-chain amino acids. Substrate specificities were demonstrated by inhibition profiles determined in the presence of excesses of the other amino acids. Four knockout mutants, lacking the lysine transporter LysP, the histidine transporter HisP (formerly LysQ), the acidic amino acid transporter AcaP (YlcA), or the aromatic amino acid transporter FywP (YsjA), were constructed. The LysP, HisP, and FywP deletion mutants showed drastically decreased rates of uptake of the corresponding substrates at low concentrations. The same was observed for the AcaP mutant with aspartate but not with glutamate. In rich M17 medium, the deletion of none of the transporters affected growth. In contrast, the deletion of the HisP, AcaP, and FywP transporters did affect growth in a defined medium with free amino acids as the sole amino acid source. HisP was essential at low histidine concentrations, and AcaP was essential in the absence of glutamine. FywP appeared to play a role in retaining intracellularly synthesized aromatic amino acids when these were not added to the medium. Finally, HisP, AcaP, and FywP did not play a role in the excretion of accumulated histidine, glutamate, or phenylalanine, respectively, indicating the involvement of other transporters.     

基于编码序列、基因间序列和氨基酸序列构建的系统发生关系比较

以7种古菌、46种细菌和10种真核生物的基因组为样本,考虑碱基间的短程关联和长程关联作用,得到编码序列的密码对和基因间序列的三联体对中不同位点的二核苷酸频率,据此构建了基于编码序列和基因间序列的系统发生关系。无论是基于编码序列还是基因间序列对信息进行聚类,古菌或真核均被聚在一支上,表明聚类参数的选择是合适的;与基于氨基酸序列构建的系统发生关系进行两两比较,发现大部分硬壁菌的编码序列与基因间序列之间,以及编码序列与氨基酸序列之间的进化都存在较大差异。通过分析认为,只有综合考虑这三类序列的进化信息,才可能得到更自然的系统发生关系。     

Characteristic Amino Acid Sequences of Chloroplast and Cytosol Isozymes of CuZn-Superoxide Dismutase in Spinach, Rice and Horsetail

Two isozymes of CuZn-superoxide dismutase (SOD) were purifiedfrom spinach. One (CuZn-SOD II) was localized in chloroplastsand had the same properties as the enzyme previously reported[Asada et al. (1973) Eur. J. Biochem. 36: 257–266]. Theother isozyme (CuZn-SOD I) was predominantly expressed in seedsand in etiolated seedlings of spinach, but was localized inthe cytosol of the leaves as a minor enzyme. The isozymes havesimilar molecular weights, subunit structures, and metal contents;but their amino acid compositions, absorption spectra, CD spectraand sensitivities to hydrogen peroxide are different. The amino acid sequences of 50 amino-terminal residues of thechloroplast and cytosol isozymes of CuZn-SOD from spinach, riceand horsetail were determined and compared with those of CuZn-SODsfrom other plants. The sequences can be divided into chloroplastand cytosol types, and characteristic sequences can be identifiedin accordance with the observations that the two types of CuZn-SODisozymes from green algae, ferns and angiosperms can be distinguishedimmunologically from each other. Differences in amino acid sequencesamong the cytosol enzymes are greater than those among the chloroplastenzymes, indicating that the rate of mutation of the cytosolCuZn-SOD is higher than that of the chloroplast CuZn-SOD. Theseresults provide further evidence that the divergence of thetwo types of isozyme of CuZn-SOD occurred at a very early stageof its acquisition, and that each type of CuZn-SOD has evolvedindependently. (Received September 1, 1989; Accepted November 6, 1989)     

Amino Acid Sequences of Glycopeptides from Cucumisin

Oxidation of short-chain iso-alkanes (isobutane, isopentane, 2-methylpentane, and 3-methylpentane) was studied with propane-grown resting mycelia of Scedosporium sp. A-4. Isobutane was oxidized to terf-butanol, but both isobutane and tert-butanol were not used for growth. Isopentane was oxidized to 3-methyl-1-butanol, 2-methyl-2-butanol, and 3-methyl-2-butanol but not to 2-methyl-1-butanol. 2-Methylpentane was oxidized to 4-methyl-1-pentanol, 2-methyl-2-pentanol, and 4-methyl-2-pentanol but not to 2-methyl-1-pentanol or 2-methyl-3-pentanol. 3-Methylpentane was not oxidized. Oxidation of branched alcohols was also studied.     

Cloning and Mapping of Telomere-Associated Sequences from Rice

We have isolated three telomere-associated sequences from riceusing cassette-ligation-mediated polymerase chain reaction (PCR).Each of the obtained clones hybridized to the terminal of oneor several rice chromosome arms. The telomeres recognized bythe clones displayed a high level of polymorphism between tworice varieties, Nipponbare (a japonica variety) and Kasalath(an indica variety). Variability in the chromosome termini wasalso detected among individual F₂ progeny plants, which werederived from a cross between the two rice varieties. One clonecontaining telomere-associated sequences was located to oneend of chromosome 5, and another clone to one end of chromosome11. For another clone, non-allelic segregation of polymorphichybridization bands was observed between japonica and indicarice; this clone was mapped to one end of chromosome 12 in japonicaand to one end of chromosome 11 in indica rice. This indicatesan exchange of termini between nonhomologous chromosomes.     

Identification and Amino Acid Sequences of Tryptic Peptides of a Novel Ferredoxin-NADP+ Oxidoreductase from Rice

A 37-kDa protein purified from rice thylakoid membranes hasbeen identified as a ferredoxin-NADP⁺ oxidoreductase based onits catalysis of the reduction of nitro blue tetrazolium viaNADPH and its recognition by antibodies against ferredoxin-NADP⁺oxidoreductase. Amino acid sequences determined from trypticfragments of the enzyme further confirm the identity of theprotein and show the presence of unique sequences at the amino-terminus. (Received May 21, 1996; Accepted September 3, 1996)     

CodonTest: Modeling Amino Acid Substitution Preferences in Coding Sequences

Codon models of evolution have facilitated the interpretation of selective forces operating on genomes. These models, however, assume a single rate of non-synonymous substitution irrespective of the nature of amino acids being exchanged. Recent developments have shown that models which allow for amino acid pairs to have independent rates of substitution offer improved fit over single rate models. However, these approaches have been limited by the necessity for large alignments in their estimation. An alternative approach is to assume that substitution rates between amino acid pairs can be subdivided into rate classes, dependent on the information content of the alignment. However, given the combinatorially large number of such models, an efficient model search strategy is needed. Here we develop a Genetic Algorithm (GA) method for the estimation of such models. A GA is used to assign amino acid substitution pairs to a series of rate classes, where is estimated from the alignment. Other parameters of the phylogenetic Markov model, including substitution rates, character frequencies and branch lengths are estimated using standard maximum likelihood optimization procedures. We apply the GA to empirical alignments and show improved model fit over existing models of codon evolution. Our results suggest that current models are poor approximations of protein evolution and thus gene and organism specific multi-rate models that incorporate amino acid substitution biases are preferred. We further anticipate that the clustering of amino acid substitution rates into classes will be biologically informative, such that genes with similar functions exhibit similar clustering, and hence this clustering will be useful for the evolutionary fingerprinting of genes.     

Human Indolethylamine N-Methyltransferase: cDNA Cloning and Expression, Gene Cloning, and Chromosomal Localization

Indolethylamine N-methyltransferase (INMT) catalyzes the N-methylation of tryptamine and structurally related compounds. We recently cloned and characterized the rabbit INMT cDNA and gene as a step toward cloning the cDNA and gene for this enzyme in humans. We have now used a PCR-based approach to clone a human INMT cDNA that had a 792-bp open reading frame that encoded a 263-amino-acid protein 88% identical in sequence to rabbit INMT. Northern blot analysis of 35 tissues showed that a 2.7-kb INMT mRNA species was expressed in most tissues. When the cDNA was expressed in COS-1 cells, the recombinant enzyme catalyzed the methylation of tryptamine with an apparent K_m value of 2.9 mM. The human cDNA was then used to clone the human INMT gene from a human genomic BAC library. The gene was 5471 bp in length, consisted of three exons, and was structurally similar to the rabbit INMT gene as well as genes for nicotinamide N-methyltransferase and phenylethanolamine N-methyltransferase in several species. All INMT exon–intron splice junctions conformed to the “GT-AG” rule, and no canonical TATA or CAAT sequences were present within the 5′-flanking region of the gene. Human INMT mapped to chromosome 7p15.2–p15.3 on the basis of both PCR analysis and fluorescence in situ hybridization. Finally, two possible single nucleotide polymorphisms were identified within exon 3, both of which altered the encoded amino acid. The cloning and expression of a human INMT cDNA, as well as the cloning, structural characterization, and mapping of its gene represent steps toward future studies of the function and regulation of this methyltransferase enzyme in humans.     

Chloroplast and Cytosol Isozymes of Cu,Zn-Superoxide Dismutase: Their Characteristic Amino Acid Sequences

lsozymes of CuZn-superoxide dismutase (SOD) were purified from angiosperms (spinach and rice), fern (horsetail) and green alga (Spirogyra). Occurrence of CuZn-SOD was confirmed by its purification in the group of green algae which shows the phragmoplast type of cell division. Purified CuZn-SODS are divided to chloroplast and cytosol types by their cellular localization and immunological properties. Their amino acid compositions, absorption spectra, CD spectra, and sensitivity to hydrogen peroxide also are distinguished from each other. All organisms including Spirogyra contain both types of isozyme. Thus, the divergence of the two types of CuZn-SOD isozyme occurred immediately after its acquisition by the most evolved green algae.

Amino acid sequences of amino-terminal regions of CuZn-SOD isozyrnes from spinach, rice and horsetail were determined and compared with those of CuZn-SODS from other plants. The chloroplast and cytosol isozymes of CuZn-SOD show each characteristic sequences. Sequence differences among the cytosol CuZn-SODS are greater than those among the chloroplast CuZn-SODS. These observations indicate that each type of isozyme had independently evolved after the acquisition of CuZn-SOD.     

水稻乙醛脱氢酶基因的克隆及其在不育系中的表达

从水稻中分离了乙醛脱氢酶基因 (Osaldh)的全长cDNA ,序列分析显示OsaldhcDNA含有一个完整的编码 5 49个氨基酸的开放阅读框 ,其编码蛋白OSALDH的N端为预期的线粒体前导肽 ,中部具有醛类脱氢酶基因家族的酶催化活性中心 ,OSALDH与玉米、烟草、人类的线粒体乙醛脱氢酶同源性分别高达 87%、77%、5 9%。Northern分析显示 ,幼根中Osaldh的表达水平高于幼苗、幼穗 ,不育品种幼穗中Osaldh的转录水平普遍高于对应的可育恢复系。植物线粒体乙醛脱氢酶的功能及其与雄性不育的关系值得进一步研究     

cDNA Cloning of Squalene Synthase Genes from Mono- and Dicotyledonous Plants, and Expression of the Gene in Rice

cDNA clones encoding squalene synthases were isolated from rice,maize and soybeans. A phylogenetic tree showed that the enzymesof monocots and dicots form distinct subgroups. In rice, squalenesynthase mRNA was detected in tissues containing dividing cellsand its level was repressed by illumination. (Received July 9, 1997; Accepted October 7, 1997)     

大熊猫RPS15 cDNA的克隆及序列分析

本研究运用RT-PCR技术,首次从大熊猫 Ailuropoda melanoleuca的肌肉组织总RNA中成功克隆了核糖体蛋白S15 (RPS15)基因的表达序列,并对其进行了初步分析.结果 表明:大熊猫RPS15基因的表达序列全长为442 bp,开放阅读框(ORF)为438 bp,编码145个氨基酸,该蛋白的分子量为17.0401 KDa, 等电点为10.3,含有2个依赖于cAMP和cGMP的蛋白激酶磷酸化位点, 5个蛋白激酶C磷酸化位点,4个N-酰基化位点及1个RPS19蛋白signature位点.进一步分析发现,大熊猫RPS15基因的表达序列及其编码的氨基酸序列与已报道的部分哺乳动物具有很高的相似性.     

几条水稻MAR序列的克隆及其功能分析

利用水稻基因组数据库和MAR预测技术,快速筛选和克隆到10条水稻MAR序列。将其分别构建到转基因结构的两侧并整合到水稻中,分析所克隆的序列对转基因表达的功能效应。结果表明:其中的3条序列具有MAR序列的功能,能明显地提高外源转基因的表达水平和表达稳定性,转基因的表达水平平均提高约2~19倍,最高可达28.9倍。