Expression profiling of <Emphasis Type="Italic">Streptomyces peucetius</Emphasis> metabolic genes using DNA microarray analysis

Doxorubicin (DXR) and daunorubicin (DNR) are anthracycline antibiotics produced by Streptomyces peucetius and widely used as cancer chemotherapeutic agents. To improve their productivity, regulation of DXR/DNR synthesis genes as well as central metabolic pathway genes must be understood more clearly. So far, studies have focused on DXR/DNR gene regulation. To investigate the correlation between the central metabolic pathway genes and DXR/DNR productivity, we selected 265 genes involved in glycolysis, fermentation, the citric acid cycle, butanoate metabolism, etc., and searched for their sequences in the S. peucetius genome by comparing gene sequences to those of Streptomyces coelicolor. The homologous genes were amplified by PCR and arrayed on glass microarray slides. Gene expression was monitored under two different growth media conditions, R2YE and NDYE. Genes involved in the production of malonyl-CoA and propionyl-CoA, the main precursors for doxorubicin synthesis, were mainly upregulated in NDYE media. Genes related to acetyl-CoA and the urea cycle were also upregulated. These changes in gene expression were confirmed by real-time RT-PCR.     

Characterization of <Emphasis Type="Italic">chsA,</Emphasis> a new gene controlling the chemotactic response in <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp7

We report, here, the characterization of a mutant strain of Azospirillum brasilense Sp7 impaired in surface motility and chemotactic response. Presence of flagella in the mutant strain was confirmed by western blot analysis, using antisera raised against the polar and lateral flagellins, and by electron microscopy. Genetic complementation and nucleotide sequencing led to the identification of a new gene, named chsA. The deduced translation product, ChsA protein, contained a PAS sensory domain and an EAL domain. As ChsA displayed characteristic signaling protein architecture, it is thought that this protein is a component of the signaling pathway controlling chemotaxis in Azospirillum.     

Regulation of nodulin gene expression

The expression of plant genes specifically induced during rhizobial infection and the early stages of nodule ontogeny (early nodulin genes) and those induced in the mature, nitrogen-fixing nodule (late nodulin genes) is differentially regulated and tissue/cell specific. We have been interested in the signal transduction pathway responsible for symbiotic, temporal and spatial control of expression of an early (Enod2) and a late (Leghemoglobin;lb) nodulin gene from the stem-nodulated legumeSesbania rostrata, and in identifying thecis-acting elements andtrans-acting factors involved in this process (De Bruijn and Schell, 1992). By introducing chimericS. rostrata lb promoter-gus reporter gene fusions into transgenicLotus corniculatus plants, we have been able to show that thelb promoter directs an infected-cell-specific expression pattern inLotus nodules. We have been able to delimit thecis-acting element responsible for nodule-infected-cell-expression to a 78 pb region of thelb promoter (NICE Element) and have analyzed this element in detail by site-specific mutagenesis. We have studied the interaction of the NICE element, and further upstreamcis-acting elements, withtrans-acting factors of both plant- and rhizobial origin. We have obtained evidence for the involvement of rhizobial proteins in infected-cell-specific plant gene expression (Welters et al., 1993). We have purified one of the bacterial binding proteins from theS. rostrata symbiontAzorhizobium caulinodans (AcBBP1), and cloned and mutated the corresponding gene, in order to examine its symbiotic phenotype. We have also found that theS. rostrata Enod2 gene is rapidly induced by physiologically significant concentrations of cytokinins, suggesting the role of cytokinin as a potential secondary signal involved in nodulation (Dehio and De Bruijn, 1992). We are examining whether the observed cytokinin induction, as well as the nodule-specific expression pattern, are modulated by theSrEnod2 promoter.     

Cloning,expression, purification,and characterization of recombinant flagellin isolated from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Pseudomonas aeruginosa as an opportunistic pathogen causes lethal infections in immunocompromised individuals. This bacterium possesses a polar flagellum made up of flagellin subunits. Flagella have important roles in motility, chemotaxis, and establishment of P. aeruginosa in acute phase of infections. Isolation, cloning, and expression of flagellin were aimed at in this study. Flagellin gene (fliC) of P. aeruginosa strain 8821M was isolated by PCR and cloned into a pET expression vector. The recombinant flagellin (46 kDa) was overexpressed as inclusion bodies (IBs). IBs were solubilized in guanidine hydrochloride (GuHCl) followed by affinity-purification and renatured using Ni²⁺-Sepharose resin. Recombinant flagellins reacted with the serum from a rabbit previously immunized with native flagellin. In addition, polyclonal antiserum raised against the recombinant flagellin was shown to significantly inhibit the cell motility of P. aeruginosa strain 8821M in vitro.     

Nup211, the fission yeast homolog of Mlp1/Tpr,is involved in mRNA export

Synthetic lethal mutants have been previously isolated in fission yeast Schizosaccharomyces pombe, which genetically interact with spmex67, in order to identify the genes involved in mRNA export. The nup211 gene was isolated by complementation of the growth defect in one of the synthetic lethal mutants, SLMex2, under synthetic lethal condition. We showed that Nup211, fission yeast homolog of Mlpl/Mlp2/Tpr, is essential for vegetative growth and Nup211-GFP proteins expressed at endogenous level are localized mainly in nuclear periphery. The accumulation of poly(A)⁺ RNA in the nucleus is exhibited when expression of nup211 is repressed or over-expressed. These results suggest that the Nup211 protein plays a pivotal role of mRNA export in fission yeast.     

RNAi介导珠子参PjCAS基因沉默对皂苷合成的影响

该研究利用Gateway技术构建珠子参环阿屯醇合成酶基因(Panax japonicus cycloartenol synthase,PjCAS)的RNAi表达载体,利用农杆菌介导转化在珠子参细胞中成功实现了PjCAS 的RNA干扰;采用实时荧光定量PCR分析珠子参皂苷生物合成途径中关键酶基因的表达情况,同时检测转基因细胞中皂苷和植物甾醇含量的变化,探讨PjCAS基因对珠子参皂苷合成的调控作用。结果表明：(1)成功获得PjCAS基因的RNAi片段,并成功构建了PjCAS基因RNAi载体pHellsgate PjCAS。(2)经农杆菌遗传转化,获得6株实现PjCAS基因RNA干扰的转基因阳性细胞系。(3)与普通细胞系相比,转基因细胞系中PjCAS基因的表达量大约下降了85%,同时与珠子参皂苷合成直接相关的关键酶基因PjDS、PjAS表达量最高分别上调了90%和150%。(4)转基因细胞系中6种单体皂苷的含量均显著高于对照组,其中达玛烷型单体皂苷Re、Rb1、Rd和齐墩果烷型单体皂苷R0、IV、IVa的平均含量比普通珠子参细胞系分别提高了28%、49%、40%、36%、59%、50%。说明珠子参皂苷含量的变化受PjCAS基因的间接调控。(5)6株转基因细胞系中植物甾醇含量较对照显著降低了53%~73%。研究发现,沉默PjCAS基因可促进珠子参皂苷合成的关键酶基因PjDS、PjAS显著上调表达,并提高转PjCAS基因细胞系中单体皂苷的含量,从而促进了珠子参皂苷合成量的显著增加,证明通过抑制植物甾醇合成通路关键基因PjCAS的表达可以有效降低植物甾醇合成支路的代谢通量,使更多的代谢流朝着珠子参皂苷合成方向流动,最终促进了珠子参皂苷的生物合成。     

亚麻荠FUS3转录因子的鉴定及功能分析北大核心CSCD

FUS3转录因子是调控植物种子油脂合成的关键因子。为探讨亚麻荠CsFUS 3基因在脂质合成和积累过程中的作用,该研究对CsFUS 3基因家族进行全基因组鉴定,分析CsFUS 3基因的时空表达模式,并解析CsFUS 3-1和CsFUS 3-2基因在植物油脂合成中的功能,为深入解析CsFUS 3基因在亚麻荠油脂合成中的功能及亚麻荠高油品种选育提供理论基础。结果表明:(1)利用AtFUS3蛋白序列,在亚麻荠基因组数据库中鉴定出2条完整的CsFUS3蛋白序列,分别命名为CsFUS3-1和CsFUS3-2,亚细胞定位发现2个CsFUS3蛋白均位于细胞核。(2)亚麻荠CsFUS3-1和CsFUS3-2蛋白与拟南芥AtFUS3蛋白的亲缘关系最近,具有与拟南芥AtFUS3蛋白相似的理化性质、高级结构以及完整的B3功能域。(3)qRT-PCR结果显示,CsFUS 3-1和CsFUS 3-2基因仅在种子中表达,且随着种子的发育成熟,2个CsFUS 3基因的表达量均呈先增高后降低的变化趋势,并在花后30 d时表达量达到最高。(4)CsFUS3和CsWRI1蛋白互作以及CsFUS 3对OLE和ABI 3基因的转录调控可能是亚麻荠高油性状的关键调控途径。(5)烟草瞬时表达分析表明,与野生型相比,转CsFUS 3-1和CsFUS 3-2基因的烟草叶片总油脂含量分别提高了0.95%和1.12%,表明亚麻荠CsFUS 3基因能够提高烟叶总油脂的合成积累。     

甘薯细胞色素P450单加氧酶基因IbCYP714E1和IbCYP714E2的鉴定及表达分析

CYP714基因在植物赤霉素合成与代谢过程中发挥着重要作用。该研究从甘薯基因组中鉴定出2个CYP714基因,对基因的结构和编码蛋白质的理化性质等进行了生物信息学分析,并利用荧光定量PCR(qRT-PCR)技术分析基因在不同组织和非生物胁迫条件下的表达特征,为解析甘薯CYP714基因的生物学功能提供帮助。结果表明:(1)2个基因为分别编码518个和521个氨基酸的碱性亲水蛋白,被亚细胞定位于细胞质中;(2)2个蛋白质均含有CYP714蛋白亚家族的3个特征结构域,与毛白杨的PtCYP714E2、PtCYP714E4和PtCYP714E5蛋白聚为一类,分别定名为IbCYP714E1和IbCYP714E2;(3)荧光定量PCR分析显示,IbCYP714E1和IbCYP714E2基因的表达部位存在一定差异,IbCYP714E1在柴根、初生根和叶片中表达量较高,而IbCYP714E2基因只在柴根和花上表达量较高,在盐和干旱胁迫下,IbCYP714E1基因表达量均增加,而IbCYP714E2基因只在盐胁迫条件下表达量增加。IbCYP714E1和IbCYP714E2基因可能参与赤霉素的降解和对非生物胁迫的应答。     

Cloning of the lkyB (tolB) gene of Escherichia coli K12 and characterization of its product

Summary The lkyB gene of Escherichia coli K12 has been cloned from the Clarke and Carbon colony bank by selecting a ColE1 plasmid conferring cholic acid resistance to lkyB mutants. The lkyB gene was localized on hybrid plasmid pJC778 by analysis of mutated plasmids generated by Tn5 insertions. Restriction analysis and complementation studies indicated that plasmid pJC778 carried genes nadA, lkyB and sucA which mapped at min 16.5; the lkyB ⁺ allele was dominant over the lkyB207 mutant allele. Analysis of cell envelope proteins from strains carrying plasmids pJC778 (lkyB ⁺), pJC2578 or pJC2579 (lkyB::Tn5), as well as plasmid-coded proteins in a maxicell system, made it likely that the lkyB gene product was a membrane protein of molecular weight 42,000.     

Monitoring dynamic expression of nuclear genes in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> by using a synthetic luciferase reporter gene

For monitoring the expression profile of selected nuclear genes in Chlamydomonas reinhardtii in response to altered environmental parameters or during cell cycle, in the past many RNA or protein samples had to be taken and analyzed by RNA hybridization or protein immunoblotting. Here we report the synthesis of a gene that codes for the luciferase of Renilla reniformis (RLuc) and is adapted to the nuclear codon usage of C. reinhardtii. This crluc gene was expressed alone or as a fusion to the zeocin resistance gene ble under control of different promoter variants. Luciferase activity was monitored in living cells, increased with the promoter strength and paralleled the amount of expressed protein. Under control of the Lhcb-1 promoter the Luc-activity in synchronized cultures was dependent on the dark-light cycle. Additionally, crluc was placed under control of the Chop-2 promoter and activity was measured under different light conditions. Chop-2 promoter activity was found to be most pronouced under low-light and dark conditions, further supporting that channelrhodopsin-2 is most active in dark-adapted cells. We conclude that crluc is a reliable tool for convenient monitoring of nuclear gene expression in C. reinhardtii.     

Stable transformation of insect cells to coexpress a rapidly selectable marker gene and an inhibitor of apoptosis

Summary We have constructed several plasmid expression vectors to express foreign genes in stably transformed insect cells. Unlike baculovirus-based expression vectors by which genes of interest are expressed transiently before lysis of the virus-infected cells, genes can be expressed continuously over many passages in a stable cell line. Furthermore, the function of a gene or genes expressed in a stable cell line from an insect-specific promoter that is constitutively expressed can be studied in the absence of virus infection and viral gene expression. In this study, we have expressed a novel, selectable marker gene, puromycin acetyltransferase, under the control of the Drosophila melanogaster hsp70 promoter or under the control of the AcMNPV ie-1 promoter which is active in Spodoptera frugiperda cells in the absence of virus infection. In addition, we have constructed expression vectors which coexpress two genes from separate promoters, the pac gene which confers resistance to puromycin and a baculovirus gene which inhibits apoptosis, derived from Orygia pseudotsugata nuclear polyhedrosis virus. Both genes were expressed in stable populations of S. frugiperda cells in the absence of continuous drug selection.     

Identification and germline transformation of the ribosomal protein rp21 gene of Drosophila: complementation analysis with the Minute QIII locus reveals nonidentity

Summary Minute loci represent a class of about 50 different Drosophila genes that appear to be functionally related. These genes may code for components of the protein synthetic apparatus. While one Minute locus has been recently shown to code for a ribosomal protein, it is not yet known whether any of the other Minute loci also code for ribosomal proteins. We have addressed this question by a combined molecular and genetic approach. In this report, a cloned DNA encoding the ribosomal protein rp21 is partially characterized. The rp21 gene maps to the same region (region 80 of chromosome 3L) as the temperature-sensitive Minute QIII gene. Using P-element mediated transformation, the rp21 gene was transformed into the germline of Drosophila. RNA blot experiments revealed that the transformed gene is expressed in transgenic flies. However, genetic complementation analysis indicated that the QIII locus and the rp21 gene are not identical. Implications of these findings for the relationship between Minutes and ribosomal protein genes are discussed.     

Genetic interactions between the Polycomb locus and the Antennapedia and Bithorax complexes of Drosophila

Summary We have studied the embryonic and adult phenotypes of genetic combinations between Polycomb (Pc), Regulator of bithorax (Rg-bx) and the genes of the Bithorax complex (BX-C) and the Antennapedia complex (ANT-C). The products of Pc and Rg-bx genes act antagonistically, their mutant combinations leading to the ectopic expression of genes of the BX-C and ANT-C. The genetic analysis of the Pc locus suggests it is a complex gene. Pc⁺ products behave as members of a regulatory set that negatively control the expression of BX-C and ANT-C genes. Genetic combinations between different doses of Pc, Rg-bx and the genes of the BX-C and ANT-C have phenotypes which may be interpreted as resulting from ectopic derepression of posterior selector genes repressing selector genes of anterior segments. The transformation phenotypes of certain genetic combinations differ in embryos and adults. A model of regulation of the BX-C and the ANT-C genes during the imaginal cell proliferation is presented, in which the specification state is maintained by self-activation of a given selector gene and down modulation of other selector genes in the same cell.     

Evidence for the thiamine biosynthetic pathway in higher-plant plastids and its developmental regulation

Thiamine or vitamin B-1, is an essential constituent of all cells since it is a cofactor for two enzyme complexes involved in the citric acid cycle, pyruvate dehydrogenase and -ketoglutarate dehydrogenase. Thiamine is synthesized by plants, but it is a dietary requirement for humans and other animals. The biosynthetic pathway for thiamine in plants has not been well characterized and none of the enzymes involved have been isolated. Here we report the cloning and characterization of two cDNAs representing members of the maize thi1 gene family encoding an enzyme of the thiamine biosynthetic pathway. This assignment was made based on sequence homology to a yeast thiamine biosynthetic gene and by functional complementation of a yeast strain in which the endogenous gene was inactivated. Using immunoblot analysis, the thi1 gene product was found to be located in a plastid membrane fraction. RNA gel blot analysis of various tissues and developmental stages indicated thi1 expression was differentially regulated in a manner consistent with what is known about thiamine synthesis in plants. This is the first report of cDNAs encoding proteins involved in thiamine biosynthesis for any plant species.     

Cloning of the excC and excD genes involved in the release of periplasmic proteins by Escherichia coli K12

Summary Strains of Escherichia coli K12 carrying a tolA, tolB, lky or exc mutation located at min 16.5 on the genetic map released periplasmic proteins into the extracellular medium. Wild-type genes defined by these mutations have been cloned from E. coli genomic bank made with plasmid pBR328. Subcloning experiments and complementation studies showed that lky and exc mutations were located either in the previously described tolA and tolB genes or in the newly characterized excC and excD genes. Using minicells, excC and excD gene products were identified as proteins with a molecular mass of 19 and 21 kDa, respectively.     

Cloning of a cDNA for rape chloroplast 3-isopropylmalate dehydrogenase by genetic complementation in yeast

Both insect and mammalian genes have previously been cloned by genetic complementation in yeast. In the present report, we show that the method can be applied also to plants. Thus, we have cloned a rape cDNA for 3-isopropylmalate dehydrogenase (IMDH) by complementation of a yeast leu2 mutation. The cDNA encodes a 52 kDA protein which has a putative chloroplast transit peptide. The in vitro made protein is imported into chloroplasts, concomitantly with a proteolytic cleavage. We conclude that the rape cDNA encodes a chloroplast IMDH. However, Southern analysis revealed that the corresponding gene is nuclear. In a comparison of IMDH sequences from various species, we found that the rape IMDH is more similar to bacterial than to eukaryotic proteins. This suggests that the rape gene could be of chloroplast origin, but has moved to the nucleus during evolution.     

Specific truncations of an acetolactate synthase gene from Brassica napus efficiently complement ilvB/ilvG mutants of Salmonella typhimurium

Summary The expression of an acetolactate synthase (ALS) gene isolated from the cruciferous plant Brassica napus was investigated in Salmonella typhimurium. Using an expression plasmid containing the highly active trc (trp-lac) promoter, several plant ALS constructs were made containing successive in-frame truncations from the 5 end of the coding region. Functional complementation by these plant ALS constructs of a S. typhimurium mutant devoid of ALS enzymic activity was assayed on minimal medium. Truncations which eliminated a large portion of the transit peptide coding sequence proved to act as efficient ALS genes in the bacterial host. Truncations close to the putative processing site of the plant protein were inactive in the complementation test. A full length copy of the gene, including the entire transit peptide coding region, was also inactive. The efficiency of the complementation, estimated by comparison to the growth rate of wild-type S. typhimurium, was found to correlate with levels of ALS activity in the transformed bacteria. Specific mutations, known to produce herbicide resistance in plants, were introduced into the truncated ALS coding sequence by site-directed mutagenesis. When expressed in bacteria these constructs conferred a herbicide resistance phenotype on the host. The potential of this system for mutagenesis and enzymological studies of plant proteins is discussed.     

Gene expression analysis of the response by Escherichia coli to seawater

Gene expression of Escherichia coli cells exposed to seawater for 20 h was compared to that of exponentially growing cells (mops-glucose 0.2%) using DNA microarray technology. The expression of most (ca. 3000) of the 4228 open reading frames on the microarray remained unchanged; the relative expression of about 320 genes decreased in seawater, whereas that of ca. one fourth (937) increased. Clearly coherent expression patterns were observed for several functional gene groups. Induced genes were numerous in groups specifying the degradation of small molecules (carbon compounds, amino acids and fatty acids), energy metabolism (aerobic and anaerobic respiration, pyruvate dehydrogenase and TCA cycle), chemotaxis and mobility, flagella biosynthesis, surface structures and phage related functions. Repressed genes were clustered in two groups, cell division and nucleotides biosynthesis, indicating a cessation of growth. This revised version was published online in August 2006 with corrections to the Cover Date.