A comparative study of tbe regulation of cytochrome P-450 and glutathione transferase gene expression in rat liver

A cDNA clone for the Ya subunit of glutathione transferase from rat liver was constructed in E.coli. The clone hybridized to Ya and Yc subunit messenger RNAs. On the basis of experiments involving cell-free translation and hybridization to the cloned probe, it was shown that prototype inducers of cytochrome P-450 such as phenobarbitone and 3-methylcholanthrene as well as inhibitors such as CoCl2 and 3-amino-l,2,4-triazole enhanced the glutathione transferase (Ya+Yc) messenger RNA contents in rat liver. A comparative study with the induction of cytochrome P-450 (b+e) by phenobarbitone revealed that the drug manifested a striking increase in the nuclear pre-messenger RNAs for the cytochrome at 12 hr, but did not significantly affect the same in the case of glutathione transferase (Ya+Yc). 3-Amino-l, 2,4-tnazole and CoCl- blocked the phenobarbitone mediated increase in cytochrome P-450 (b+e) nuclear pre-messenger RNAs. These compounds did not significantly affect the glutathione transferase (Ya+Yc) nuclear pre-messenger RNA levels. The polysomal, poly (A)- containing messenger RNAs for cytochrome P-450 (b+e) increased by 12–15 fold after phenobarbitone administration, reached a maximum around 16hr and then decreased sharply. In comparison, the increase in the case of glutathione transferase (Ya+Yc) mesenger RNAs was sluggish and steady and a value of 3–4 fold was reached around 24 hr. Run-off transcription rates for cytochrome P-450 (b+e) increased by nearly 15 fold in 4 hr after phenobarbitone administration, whereas the increase for glutathione transferase (Ya+Yc) was only 2.0 fold. At 12 hr after the drug administration, the glutathione transferase (Ya+Yc) transcription rates were near normal. Administration of 3-amino-l,2,4-triazole and CoCl2 blocked the phenobarbitone-mediated increase in the transcription of cytochrome P-450 (b+e) messenger RNAs. These compounds at best had only marginal effects on the transcription of glutathione transferase (Ya+Yc) messenger RNAs. The half-life of cytochrome P-450 (b+e) messenger RNA was estimated to be 3–4 hr, whereas that for glutathione transferase (Ya+Yc) was found to be 8-9 hr. Administration of phenobarbitone enhanced the half-life of glutathione transferase (Ya+Yc) messenger RNA by nearly two fold. It is suggested that while transcription activation may play a primary role in the induction of cytochrome P-450 (b+e), the induction of glutathione transferase (Ya+Yc) may essentially involve stabilization of the messenger RNAs.

Rubisco small subunit, chlorophyll a/b-binding protein and sucrose : fructan-6-fructosyl transferase gene expression and sugar status in single barley leaf cells in situ. Cell type specificity and induction by light

Chungui Lu, Olga A. Koroleva, John F. Farrar, Joe Gallagher, Chris J. Pollock, and A. Deri Tomos (2002). Rubisco small subunit, chlorophyll a/b-binding protein and sucrose : fructan-6-fructosyl transferase gene expression and sugar status in single barley leaf cells in situ. Cell type specificity and induction by light. Plant Physiology, 130 (3) pp.1335-1348 Sponsorship: BBSRC RAE2008

T-cell-specific deletion of a polypeptide N-acetylgalactosaminyl-transferase gene by site-directed recombination.

UDP-N-acetylgalactosamine (GalNAc): polypeptide N-acetylgalactosaminyltransferase (polypeptide GalNAc-T) catalyzes transfer of the monosaccharide GalNAc to serine and threonine residues, thereby initiating O-linked oligosaccharide biosynthesis. Previous studies have suggested the possibility of multiple polypeptide GalNAc-Ts, although attachment of saccharide units to polypeptide or lipid in generating oligosaccharide structures in vertebrates has been dependent upon the activity of single gene products. To address this issue and to determine the relevance of Oglycosylation variation in T-cell ontogeny, we have directed Cre/loxP mutagenic recombination to the polypeptide GalNAc-T locus in gene-targeted mice. Resulting deletion in the catalytic region of polypeptide GalNAc-T occurred to completion on both alleles in thymocytes and was found in peripheral T cells, but not among other cell types. Thymocyte O-linked oligosaccharide formation persisted in the absence of a functional targeted polypeptide GalNAc-T allele as determined by O-glycan-specific lectin binding. T-cell development and colonization of secondary lymphoid organs were also normal. These results indicate a complexity in vertebrate O-glycan biosynthesis that involves multiple polypeptide GalNAc-Ts. We infer the potential for protein-specific O-glycan formation governed by distinct polypeptide GalNAc-Ts.

细胞分裂素的测定及其转基因植物的基因表达和调控的研究

细胞分裂素是一类重要的植物激素，它参与调节许多植物的生命活动过程。本文从几个方面研究了细胞分裂素的作用。 在细胞分裂素的活性测定中，通过改进尾穗蔸苋红素合成法建立了一种简便、准确的生物试法，同时还建立了根据物理化学和免疫学原理而测定细胞分裂素的HPLC和ELISA方法，使得细胞分裂素的定量更加准确。经过对上述三种方法的相互验证实验表明，同时采用二种方法可以保证细胞分裂素分析的准确和可靠。 细胞分裂素可以促进黄瓜子叶的扩张。利用离体黄瓜子叶，分析BA诱发其扩张与子叶内源细胞分裂素之间的关系，实验证明，BA能促进玉米素及其核苷的迅速积累，进而诱发子叶的扩张。上述结果还表明，黄瓜子叶可能具有合成细胞分裂素的能力。 荸荠球茎是一种贮藏器官，但实验测定发现其中含有细胞分裂素的生理活性形式——异戊烯基腺嘌呤核苷(iPA)，而且合成它的前体腺嘌呤的含量也十分丰富，考虑到球茎与种子的类似之处，推测它可能做为合成细胞分裂素的一个源，而且其合成途径可能有别于植物其它组织。 农杆菌中的异戊烯基转移酶(ipt)基因是负责细胞分裂素生物合成的关键基因。将ipt基因克隆后对其启动子进行了改造，分别构建了如下三种基因：(1) ipt启动子+ipt编码区和3，区(ipt)，(2)磷酸核酮糖羧化酶小亚基启动子SSU 301+ipt编码区和3，区(SSU -ipt)，(3)豌豆种子特异性启动子viciln+ipt编码区和3，区(vic-ipt)。上述三种基因经农杆菌介导转化烟草，获得了16株再生植株，经Southern杂交证明其中15株的基因组上含有正常整合的ipt基因。Northern杂交表明有13株转基因烟草中的ipt基因能转录出大小正常的ipt mRNA并促进了细胞分裂素的生物合成。 实验表明，转基因烟草中ipt基因的表达受到多种因素的调控。首先启动子决定了ipt基因的表达模式，SSU -ipt基因的表达受光的诱导，黑暗中这种基因的转录完全停止，而vic-ipt基因的表达是种子特异性的，它不在烟草营养生长器官如根、茎、叶和愈伤组织中表达。第二，生长素能降低ipt基因的表达活性。第三，在整体植物的根中，存在某些反式因子，能够控制ipt基因的过量表达，这其中可能涉及到细胞内的蛋白因子、基因的甲基化作用及细胞分裂素的反馈调节等。 vic-ipt基因在烟草种子中的特异性表达导致种子内形成了一个细胞分裂素合成的源(source)。对种子中营养物质积累的研究表明，ipt基因的表达促进了种子干物质的积累，其中作用最明显的是增加种子内蛋白质的合成。转入vic-ipt基因后的烟草种子其萌发率没有显著变化，但幼苗的生长速率明显加快，这表明细胞分裂素能调节植株的生长。 通过Northern杂交检测转基因烟草中基因表达的调控，实验证明，ipt基因的表达明显抑制一组植物病理相关蛋白(PR)基因的转录活性，这组基因编码：几丁质酶，β-1，3一葡萄糖苷酶，伸展蛋白和渗调蛋白。对这些调控作用的生理学意义还有待进一步探索。 上述结果表明，在高等植物中，除了传统上认为根是合成细胞分裂素的部位之外，其它组织和器官也具有合成细胞分裂素的能力，其中合成能力最强的是一些离体组织和贮藏器官。农杆菌中的细胞分裂素生物合成基因(ipt)能够在高等植物的基因组中正常的整合和表达，并受到植物体内生理、发育等多种因素的调控，而与整体植物的正常生理过程协调一致。ipt基因的表达还能够调节植物体的生长和发育，包括种子发育时营养物质的积累、幼苗的生长和某些相关基因的表达。对上述问题的深入研究,必将促进细胞分裂素及其相关生理学和发育学研究的进展。

Effects of Cor15a-IPT Gene Expression on Leaf Senescence in Transgenic Petunia x hybrida and Dendranthema x grandiflorum.

To prevent leaf senescence of young transplants or excised shoots during storage under dark and cold conditions, the cytokinin biosynthetic gene isopentenyl transferase (ipt) was placed under the control of a cold-inducible promoter cor15a from Arabidopsis thaliana and introduced into Petunia x hybrida 'Marco Polo Odyssey' and Dendranthema x grandiflorum (chrysanthemum) 'Iridon'. Transgenic cor15a-ipt petunia and chrysanthemum plants and excised leaves remained green and healthy during prolonged dark storage (4 weeks at 25 degrees C) after an initial exposure to a brief cold-induction period (4 degrees C for 72 h). However, cor15a-ipt chrysanthemum plants and excised leaves that were not exposed to a cold-induction period, senesced under the same dark storage conditions. Regardless of cold-induction treatment, leaves and plants of non-transformed plants senesced under prolonged dark storage. Analysis of ipt expression indicated a marked increase in gene expression in intact transgenic plants as well as in isolated transgenic leaves exposed to a short cold-induction treatment prior to dark storage. These changes correlated with elevated concentrations of cytokinins in transgenic leaves after cold treatment. Cor15a-ipt transgenic plants showed a normal phenotype when grown at 25 degrees C.

sporamin A ipt嵌合质粒的构建及其对马铃薯的转化

细胞分裂素在植物的生长、发育过程中起着重要的作用，来源于农杆菌Ti质粒上基因4区域的ipt基因特异地编码控制细胞分裂素生物合成中的关键酶－－异戊烯基转移酶。 启动子是一类非常重要的基因表达调控元件，在植物的生长、发育过程中控制着基因的时空及顺序表达。来源于甘薯的Sporamin有两种类型（A和B），它是甘薯块根中的主要贮藏蛋白，并且特异地在块根中表达。我们将sporamin A启动子和ipt基因嵌合构成双元载体pBz213，使之进入农杆菌，然后再转化马铃薯，已经得到了具有卡那霉素抗性的小植株，进一步的检测工作正在进行中。在马铃薯块茎中可望异戊烯基转移酶特异地合成，并且能够增加细胞分裂素的水平。

The gene for albicidin detoxification from Pantoea dispersa encodes an esterase and attenuates pathogenicity of Xanthomonas albilineans to sugarcane

Albicidin phytotoxins are pathogenicity factors in a devastating disease of sugarcane known as leaf scald, caused by Xanthomonas albilineans. A gene (albD) from Pantoea dispersa has been cloned and sequenced and been shown to code for a peptide of 235 amino acids that detoxifies albicidin. The gene shows no significant homology at the DNA or protein level to any known sequence, but the gene product contains a GxSxG motif that is conserved in serine hydrolases. The AlbD protein, purified to homogeneity by means of a glutathione S-transferase gene fusion system, showed strong esterase activity on p-nitrophenyl butyrate and released hydrophilic products during detoxification of albicidins. AlbD hydrolysis of p-nitrophenyl butyrate and detoxification of albicidins required no complex cofactors. Both processes were strongly inhibited by phenylmethylsulfonyl fluoride, a serine enzyme inhibitor. These data strongly suggest that AlbD is an albicidin hydrolase. The enzyme detoxifies albicidins efficiently over a pH range from 5.8 to 8.0, with a broad temperature optimum from 15 to 35°C. Expression of albD in transformed X. albilineans strains abolished the capacity to release albicidin toxins and to incite disease symptoms in sugarcane. The gene is a promising candidate for transfer into sugarcane to confer a form of disease resistance.

Extratos de Ascophyllum nodosum no tratamento de sementes de milho e soja: avaliações fisiológicas e moleculares

A obtenção de uma lavoura com população adequada de plantas depende da utilização de diferentes práticas agronômicas, estando o sucesso condicionado ao uso de sementes de boa qualidade. No entanto, a semeadura dificilmente é realizada em condições ideais o que resulta em problemas na emergência das plantas. Grande é a procura por alternativas que melhorem a germinação e o desenvolvimento inicial dos cultivos resultando em uniformidade de emergência, garantindo o estande e culminando em produção. O tratamento de sementes com agroquímicos fitossanitários é uma solução parcial no combate de agentes fitopatológicos e pragas, mas poucas são as soluções adotadas para melhorar o desenvolvimento vegetal inicial, que pode levar a desuniformidade e falhas no estande gerando prejuízos econômicos. Os extratos de algas já demonstraram em diversos estudos sua eficiência no desenvolvimento vegetal quando aplicados em plantas. Porém poucos são os estudos voltados para os efeitos dos extratos de algas na germinação e emergência. Assim, o intuito deste trabalho foi testar o extrato comercial de Ascophyllum nodosum, e diferentes fracionamentos do mesmo, no tratamento de sementes de soja e milho. Avaliou-se o efeito de diferentes doses no desenvolvimento das plântulas e as doses de melhor resposta foram utilizadas no tratamento de sementes de soja a fim de associar as respostas obtidas à expressão gênica de 9 genes relacionados ao processo germinativo em 24 e 48h de embebição. Sementes de soja tratadas com o extrato comercial resultaram em plântulas menos desenvolvidas o que pode estar relacionado ao alto teor de sais contidos no produto. O tratamento com as demais frações favoreceu o desenvolvimento das plântulas, principalmente o desenvolvimento radicular. Sementes de milho tratadas não apresentaram desenvolvimento tão satisfatório quanto as sementes de soja tratadas. A análise da expressão gênica relativa demonstrou que o tratamento com frações do extrato comercial é capaz de regular algumas vias do metabolismo hormonal, como a isopentenil transferase e a GA20 oxidase 2, e do catabolismo de reservas, como a acil-CoA oxidase. Em condições ótimas, o tratamento de sementes de soja com frações do extrato comercial de A. nodosum favoreceu o desenvolvimento inicial das plântulas de soja, no entanto não ocasionou grandes alterações no desenvolvimento de milho. Este estudo demonstrou a possibilidade de utilização de frações do extrato de A. nodosum no favorecimento do desenvolvimento inicial de plântulas de soja. Maiores estudos são necessários quanto às respostas em campo e na atenuação de estresses para viabilizar seu uso como um bioestimulante em sementes.

Initiation of transcription of rDNA in rice

Three direct repeats of 320, 340 and 238 nucleotides were detected upstream to the 5′ end of the 18S rRNA gene of an rDNA unit present on a 9.8 kb EcoRT fragment of the rice DNA. The primer extension analysis showed that the site of initiation of transcription is in the 1st repeat at an A, the 623rd nucleotide upstream to the 5′ end of the 18S rRNA gene. Different stretches of the intergenic spacer DNA linked to the Chloramphenicol acetyl transferase gene were transcribed in the intact nuclei of rice embryos. The S1 nuclease protection analysis of the transcripts using [32P]-labelled Chloramphenicol acetyl transferase gene as the probe showed the presence of multiple promoters for rDNA transcription.

水稻Phi类谷胱甘肽转移酶基因家族的功能分化研究

在植物中大多数功能基因是以基因家族的形式存在的，而基因重复则是基因家族的一种重要的进化方式。很多基因往往是由重复事件产生形成不同的拷贝，进而分化形成基因家族。谷胱甘肽转移酶（GSTs）是一类古老、庞大、行使解毒、抗逆、信号转导等多种功能的一个基因家族。本研究以栽培水稻（Oryza sativa ssp. japonica c.v. Nipponbare）为研究材料，以栽培水稻的Phi类GST的5个基因（OsGSTF3、OsGSTF6、OsGSTF14、OsGSTF15、OsGSTF16）为研究对象，分析了它们的系统发生和起源历史、不同组织的差异性表达、编码蛋白质的功能差异等问题，探讨了基因重复后5个基因的功能变化，主要结果如下： 1. OsGSTF3、OsGSTF14、OsGSTF15、OsGSTF16由串联重复产生，而OsGSTF6则由DNA转座产生;它们起源时间早在稻属（Oryza）分化之前。 2. 对水稻不同部位组织的RT-PCR结果表明这5个基因在水稻中的特异性表达组织部位有较大差异：OsGSTF3基因在叶、叶鞘、茎、根4个部位均有大量表达;OsGSTF6基因仅在叶中有表达;OsGSTF14基因在叶鞘、茎2个部位中有表达;OsGSTF15基因在茎、根2个部位中有表达;OsGSTF16则在叶、茎、根3个部位中有表达。 3. 将这5个基因连接原核表达载体PET30a并转化大肠杆菌BL21（DE3），获得了高表达菌株。将表达菌株进行大量表达，表达形式分析显示OsGSTF3蛋白是可溶性表达，而其余4个蛋白以包涵体的形式表达。通过亲和层析获得了纯化的OsGSTF3融合蛋白，OsGSTF3融合蛋白对底物CDNB和NBD-Cl具有高活性，酶动力学分析显示OsGSTF3融合蛋白对GSH与NBD-Cl有较高的亲和力，热力学分析显示该蛋白在40℃以下是热稳定的。通过对包涵体进行洗涤、亲和层析获得了纯化的OsGSTF6、OsGSTF14、OsGSTF15、OsGSTF16的融合蛋白，OsGSTF14融合蛋白对NBD-Cl有微弱活性，OsGSTF15融合蛋白对NBC有较高的活性，而没有检测到OsGSTF6与OsGSTF16融合蛋白的活性。

Avian influenza virus infection induces differential expression of genes in chicken kidney

The pathogenic process of highly pathogenic avian influenza virus (HPAIV) infection is poorly understood. To explore the differential expression of kidney genes as a result of HPAIV infection, two cDNA libraries were constructed from uninfected and infected kidneys by suppression subtractive hybridization (SSH). Fifteen genes including IFN-stimulated genes (ISG12), lymphocyte antigen 6 complex locus E gene (LY6E), matrix Gla protein gene (MGP), lysozyme gene, haemopoiesis related membrane protein I gene, KIAA1259, MGC68696, G6pe-prov protein gene (G6PC), MGC4504, alcohol dehydrogenase gene (ADH), glutathione S-transferase gene (GST), sodium-dependent high-affinity dicarboxylate transporter gene (SDCT), Synaptotagmin XV (SytXV) and two novel genes were found significantly up-regulated or dramatically suppressed. Differential expression of these genes was further identified by Northern blot. Functional analysis indicated that the regulation of their expression might contribute to the pathogenic process of HPAIV infection. In contrast, the increased expression of three IFN-stimulated genes named ISG12, LY6E, and haemopoiesis related membrane protein 1 gene might reflect host defense responses. Further study showed that ISG12 protein failed to directly interact with NS1 protein of HPAIV which expressed simultaneously in the organs where HPAIV replication occurred, by use of BacterioMatch two-hybrid system. Therefore, our findings may provide new insights into understanding the molecular mechanism underlying the pathophysiological process of HPAIV infection in chicken. (c) 2007 Elsevier Ltd. All rights reserved.

Difterentially expressed genes of Tetrahymena thermophila in response to tributyltin (TBT) identified by suppression subtractive hybridization and real time quantitative PCR

Tributyltin (TBT) is widely used as antifouling paints, agriculture biocides, and plastic stabilizers around the world, resulting in great pollution problem in aquatic environments. However, it has been short of the biomonitor to detect TBT in freshwater. We constructed the suppression subtractive hybridization library of Tetrahymena thermophila exposed to TBT, and screened out 101 Expressed Sequence Tags whose expressions were significantly up- or down-regulated with TBT treatment. From this, a series of genes related to the TBT toxicity were discovered, such as glutathione-S-transferase gene (down-regulated), plasma membrane Ca2+ ATPase isoforms 3 gene (up-regulated) and NgoA (up-regulated). Furthermore, their expressions under different concentrations of TBT treatment (0.5-40 ppb) were detected by real time fluorescent quantitative PCR. The differentially expressed genes of T thermophila in response to TBT were identified, which provide the basic to make Tetrahymena as a sensitive, rapid and convenient TBT biomonitor in freshwater based on rDNA inducible expression system. (c) 2006 Elsevier B.V. All rights reserved.

Identification of differentially expressed genes in Tetrahmena thermophila in response to dichlorodiphenyltrichloroethane (DDT) by suppression subtractive hybridization

The insecticide dichlorodiphenyltrichloroethane (DDT) is persistent in the environment, and continues to cause health problems. Tetrahymena has potential as a model organism for assaying low levels of DDT and for analysing the mechanisms of its toxicity. We constructed the suppression subtractive hybridization library of T thermophila exposed to DDT, and screened out 90 Expressed Sequence Tags whose expressions were significantly up- or downregulated with DDT treatment. From this, a series of important genes related to the DDT metabolism and detoxification were discovered, such as P450 gene, glutathione S-transferase gene and sterol carrier protein 2 gene. Furthermore, their expressions under different concentrations of DDT treatment were detected by real-time fluorescent quantitative PCR. The results show that Tetrahymena is a relevant and useful model organism for detecting DDT in the environment and for discovering biomarkers that can be used to develop specific bio-reporters at the molecular and genomic levels.