High-performance liquid chromatography purification of 26-bp serial analysis of gene expression ditags results in higher yields,longer concatemers,and substantial time savings

In contrast to DNA chips, serial analysis of gene expression (SAGE) is not dependent on genes having been previously identified for their monitoring. Although useful, the method can be technically challenging, and particularly the last steps including concatenation and cloning may result in less than optimal results. We propose that many of the encountered problems can be attributed to the purification of the 26-bp ditags by polyacrylamide gel electrophoresis. Low yields, gel contaminants, potential exposure to degrading enzymes during handling and lengthy separation all disfavor the method. We introduce purification of 26-bp ditags by reverse-phase high-performance liquid chromatography (HPLC) using polystyrene/divinylbenzene columns and tetraethylammonium acetate buffer with acetonitrile as mobile phase. The method is fast and gives excellent results. Ditags purified by HPLC readily ligate to high-molecular-weight concatemers leading to their efficient cloning. The method should substantially facilitate the construction of SAGE libraries.     

鹦鹉热衣原体主要外膜蛋白基因序列的扩增、克隆和原核表达

主要外膜蛋白在鹦鹉热衣原体感染过程中起主要作用。扩增了主要外膜蛋白基因,克隆入pGEM-T和pET32a( ),经PCR筛选和酶切鉴定,进行诱导表达和重组蛋白的纯化与复性研究,为进一步进行鹦鹉热衣原体的诊断试剂和疫苗研究创造了条件。     

Gene expression profiling of human diseases by serial analysis of gene expression

Until recently, the approach to understanding the molecular basis of complex syndromes such as cancer, coronary artery disease, and diabetes was to study the behavior of individual genes. However, it is generally recognized that expression of a number of genes is coordinated both spatially and temporally and that this coordination changes during the development and progression of diseases. Newly developed functional genomic approaches, such as serial analysis of gene expression (SAGE) and DNA microarrays have enabled researchers to determine the expression pattern of thousands of genes simultaneously. One attractive feature of SAGE compared to microarrays is its ability to quantify gene expression without prior sequence information or information about genes that are thought to be expressed. SAGE has been successfully applied to the gene expression profiling of a number of human diseases. In this review, we will first discuss SAGE technique and contrast it to microarray. We will then highlight new biological insights that have emerged from its application to the study of human diseases.     

Amplification and molecular cloning of murine adenosine deaminase gene sequences

A previously isolated mouse Cl-1D derived cell line (B-1/25) overproduces adenosine deaminase (EC 3.5.4.4) by 3200-fold. The present studies were undertaken to determine the molecular basis of this phenomenon. Rabbit reticulocyte lysate and Xenopus oocyte translation studies indicated that the B-1/25 cells also overproduced adenosine deaminase mRNA. Total poly(A+) RNA derived from B-1/25 was used to construct a cDNA library. After prehybridization with excess parental Cl-1D RNA to selectively prehybridize nonamplified sequences, 32P-labeled cDNA probe synthesized from B-1/25 total poly(A+) RNA was used to identify recombinant colonies containing amplified mRNA sequences. Positive clones containing adenosine deaminase gene sequences were identified by blot hybridization analysis and hybridization-selected translation in both rabbit reticulocyte lysate and Xenopus oocyte translation systems. Adenosine deaminase cDNA clones hybridized with three poly(A+) RNA species of 1.5, 1.7, and 5.2 kilobases in length, all of which were overproduced in the B-1/25 cell line. Dot blot hybridization analysis using an adenosine deaminase cDNA clone showed that the elevated adenosine deaminase level in the B-1/25 cells was fully accounted for by an increase in adenosine deaminase gene copy number. The adenosine deaminase cDNA probes and the cell lines with amplified adenosine deaminase genes should prove extremely useful in studying the structure and regulation of the adenosine deaminase gene.     

Accurate and unambiguous tag-to-gene mapping in serial analysis of gene expression

Background  

In this study, we present a robust and reliable computational method for tag-to-gene assignment in serial analysis of gene expression (SAGE). The method relies on current genome information and annotation, incorporation of several new features, and key improvements over alternative methods, all of which are important to determine gene expression levels more accurately. The method provides a complete annotation of potential virtual SAGE tags within a genome, along with an estimation of their confidence for experimental observation that ranks tags that present multiple matches in the genome.     

黄河裸裂尻鱼肌肉生长抑制素基因克隆及表达分析

肌肉生长抑制素(myostatin,MSTN)属于转化生长因子β(TGF-β)超家族中的一个成员,是骨骼肌生长发育的负调控因子。该文以黄河裸裂尻鱼肌肉总RNA为模板,采用RT-PCT、5’-RACE和3’-RACE法获得MSTN基因全长cDNA序列为2180bp,包含长为1128bp的开放阅读框,编码375个氨基酸。以肌肉总DNA为模板,通过PCR法进一步获得了MSTN基因的2个内含子序列,分析表明,黄河裸裂尻鱼MSTN基因与其他脊椎动物具有相似的基因结构(包括3个外显子和2个内含子)。黄河裸裂尻鱼MSTN具有脊椎动物MSTN的共同序列特征,含有1个蛋白酶水解位点RXXR和8个位于TGF-β功能区域保守的半胱氨酸残基。氨基酸序列同源性分析表明,黄河裸裂尻鱼MSTN序列与其他鲤科鱼类MSTN具有较高的同源性;而与哺乳动物和禽类的MSTN同源性较低。系统发育分析表明,黄河裸裂尻鱼MSTN与其他鲤科鱼类聚于同一进化支。RT-PCR分析表明,该基因在黄河裸裂尻鱼9个被检组织中均有表达,但在心、肾、肠、精巢中表达量较高。Real-TimePCR分析显示,MSTN基因在胚胎中的相对表达量,随胚胎发育阶段的不同而有所差异,暗示MSTN的功能可能并不局限在对肌肉生长发育的负调控作用,可能还有其他功能。     

Amplification and cloning of the Chinese hamster glutamine synthetase gene.

A Chinese hamster ovary cell line, KG1MS which is resistant to 5 mM methionine sulphoximine overproduces glutamine synthetase. Overproduction of this 42 000 mol. wt. polypeptide is not seen in either parental KG1 or revertant KG1MSC4-0 cell lines which are resistant to 3 microM and 8 microM methionine sulphoximine, respectively. Restriction endonuclease analysis of DNA from KG1MS cells produces a pattern of amplified DNA fragments not seen in parental KG1 or revertant KG1MSC4-0 digests. Hybridization of cDNA probes complementary to KG1MS poly(A) mRNA against Southern blots of KG1MS restriction digests identifies a specific subset within these amplified sequences which is not detected by cDNA probes made from parental KG1 poly(A) mRNA. One 8.2-kb BglII fragment of KG1MS DNA identified by cDNA hybridization has been cloned to produce recombinant pGS-1. mRNA hybrid selected by pGS-1 translates to a 42 000 mol. wt. polypeptide which co-migrates in polyacrylamide gels with the over-produced protein in KG1MS cells and purified glutamine synthetase. pGS-1 also hybridizes to several mRNA species abundant in KG1MSC4-M, but not KG1, poly(A) mRNA extracts. The high level of resistance to methionine sulphoximine shown by KG1MS cells is due to amplification of a DNA sequence of at least 50 kb which contains the coding region for the enzyme glutamine synthetase.     

北京棒杆菌转酮酶:基因克隆、序列分析与表达

【目的】转酮酶是非氧化磷酸戊糖途径中的关键酶。从北京棒杆菌(Corynebacterium pekinense PD-67)中克隆转酮酶(transketolase,EC2.2.1.1,TK)基因,并将转酮酶基因在C.pekinense PD-67中进行表达,研究增加转酮酶活性对C.pekinense PD-67生理特性的影响。【方法】分别以C.pekinense野生株AS1.299和突变株PD-67的基因组为模板,用PCR方法扩增tkt的全基因序列和前端控制序列;通过pAK6载体提高tkt基因在C.pekinense PD-67中的拷贝数,从而提高C.pekinense PD-67中转酮酶的活性。【结果】tkt基因核苷酸序列及其编码的氨基酸序列与结构分析结果表明,C.pekinense突变株PD-67与野生株AS1.299相比较,二者调控序列及结构基因核苷酸序列完全一致。与谷氨酸棒杆菌ATCC13032相比较,突变株PD-67的氨基酸序列有5个氨基酸差异,其中4个位于与辅因子硫胺素焦磷酸结合的结构域内。突变株PD-67来源的tkt基因在北京棒杆菌PD-67中得到了表达,重组菌转酮酶比活力比对照菌株提高了2倍。C.pekinense PD-67(pTK3)与对照菌株PD-67(pAK6)相比,生长加快,L-色氨酸的最终积累量也较高。【结论】本工作从C.pekinense1.299和PD-67中克隆到tkt基因,并实现tkt基因的同源表达。适当提高菌株转酮酶活力,有助于菌体生长和色氨酸积累。     

cDNA cloning and analysis of tissue-specific gene expression of rat urocortin II

The corticotropin-releasing hormone (CRH) family of neuropeptides includes CRH (a 41-amino acid hypothalamic peptide) and urocortin. Corticotropin-releasing factor (CRF), a peptide first isolated from mammalian, plays an important role in the regulation of the pituitary-adrenal axis, and in endocrine, autonomic, immune and behavioral responses to stress. In this study, we cloned rat urocortin II (UCN II) cDNA from rat mid-brain by RT-PCR. The rat UCN II clone contained an open reading frame (ORF) 109 amino acids which shared 90% and 63% homology with mouse and human homologues, respectively. The expression of UCN II mRNA is mainly distributed in bone marrow, ovary, uterus, hypophysis, adrenal gland, and skin. In this study, rat recombinant UCN was expressed in E. coli and purified in active form. Furthermore, purified recombinant UCN II protein specifically binds to CRF receptor 2 in rat ROS 17 / 2.8 and GH3 cells by flow-cytometry analysis. UCN II cDNA clone obtained in this study will be useful for further investigation on behavioral responses to stress in rats.