桃ACC合酶基因的分离及其差异表达

利用5′/3′RACE PCR技术,从桃(Prunus persica (L.) Batsch)果实中克隆了植物乙烯生物合成的关键酶--ACC合酶的全长cDNA pacs,对pacs基因进行全序列测定表明,该基因全长1 848个碱基,编码区为1 449个碱基,5′端有177个碱基的非编码区序列,3′端有219个碱基的非编码区序列(不包括终止密码子TAA).pacs基因编码区共编码483个氨基酸,蛋白质大小为54 kD,等电点为6.43.pacs与番茄(S19677)、梅(AB031026)、番木瓜(U68216)、苹果(AB034993)等其他植物ACC合酶cDNA氨基酸序列同源性分别为65%、70%、75%、90%,并存在与这些ACC合酶氨基酸的活性位点保守序列SLSKDMGFPGFR.RT-PCR结合杂交分析表明,pacs和我们以前克隆的桃ACC合酶cDNA pacs12(AF467782)在叶片和花中基因表达模式基本一致,伤处理和IAA均能诱导叶片pacs 和pacs12基因的表达,但pacs在伤处理叶片的表达水平比pacs12高;pacs 和pacs12基因在果实表达有所不同,pacs在绿熟和成熟果实中均有表达,而pacs12在绿熟果实中基本检测不到,在成熟果实中才有表达,两者在果实中的表达水平比伤处理和IAA处理叶片和花中要低.     

Cloning and sequencing of the cDNA for S-acyl fatty acid synthase thioesterase from the uropygial gland of mallard duck

In vitro translation of poly(A)+ RNA from the uropygial glands of mallard ducks (Anas platyrhynchos) generated a 29-kDa protein which cross-reacted with rabbit antibodies prepared against S-acyl fatty acid synthase thioesterase (Kolattukudy, P. E., Rogers, L., and Flurkey, W. (1985) J. Biol. Chem., 260, 10789-10793). A poly(A)+ RNA fraction enriched in this thioesterase mRNA, isolated by sucrose density gradient centrifugation, was used to prepare cDNA which was cloned in Escherichia coli using the plasmid pUC9. Using hybrid-selected translation and colony hybridization, 17 clones were selected which contained the cDNA for S-acyl fatty acid synthase thioesterase. Northern blot analysis showed that the mature mRNA for this thioesterase contained 1350 nucleotides whereas the cloned cDNA inserts contained 1150-1200 base pairs. Five of the 6 clones tested for 5'-sequence had identical sequences, and the three tested for 3'-end showed the same sequence with poly(A) tails. Two clones, pTE1 and pTE3, representing nearly the full length of mRNA, were selected for sequencing. Maxam-Gilbert and Sanger dideoxy chain termination methods were used on the cloned cDNA and on restriction fragments subcloned in M13 in order to determine the complete nucleotide sequence of the cloned cDNA. The nucleotide sequence showed an open reading frame coding for a peptide of 28.8 kDa. Two peptides isolated from the tryptic digest of the thioesterase purified from the gland showed amino acid sequences which matched with two segments of the sequence deduced from the nucleotide sequence. Another segment containing a serine residue showed an amino acid sequence homologous to the active serine-containing segment of the thioesterase domain of fatty acid synthase. Thus, the clones represent cDNA for S-acyl fatty acid synthase thioesterase. The present results constitute the first case of a complete sequence of a thioesterase.     

Expression cloning of human globoside synthase cDNAs. Identification of beta 3Gal-T3 as UDP-N-acetylgalactosamine:globotriaosylceramide beta 1,3-N-acetylgalactosaminyltransferase

By using a eukaryocytic cell expression cloning system, we have isolated cDNAs of the globoside synthase (beta1, 3-N-acetylgalactosaminyltransferase) gene. Mouse fibroblast L cells transfected with SV40 large T antigen and previously cloned Gb3/CD77 synthase cDNAs were co-transfected with a cDNA library prepared from mRNA from human kidney together with Forssman synthase cDNA, and Forssman antigen-positive cells were panned using an anti-Forssman monoclonal antibody. The isolated cDNAs contained a single open reading frame predicting a type II membrane protein with 351 amino acids. Surprisingly, the cDNA clones turned out to be identical with previously reported beta3Gal-T3, which had been cloned by sequence homology with other galactosyltransferases. Substrate specificity analysis with extracts from cDNA-transfected L cells confirmed that the gene product was actually beta1, 3-N-acetylgalactosaminyltransferase that specifically catalyzes the transfer of N-acetylgalactosamine onto globotriaosylceramide. Results of TLC immunostaining of neutral glycolipids from the cDNA-transfected cells also supported the identity of the newly synthesized component as globoside. The results show that glycosyltransferases apparently belonging to a single glycosyltransferase family do not necessarily catalyze reactions utilizing the same acceptor or even the same sugar donor. The globoside synthase gene was expressed in many tissues, such as heart, brain, testis, etc. We propose the designation beta3GalNAc-T1 for the cloned globoside synthase gene.     

Molecular cloning, heterologous expression, and functional characterisation of a malate synthase gene from Streptomyces coelicolor A3(2)

With the rapid generation of genetic information from the Streptomyces coelicolor genome project, deciphering the relevant gene products is critical for understanding the genetics of this model streptomycete. A putative malate synthase gene (aceB) from S. coelicolor A3(2) was identified by homology-based analysis, cloned by polymerase chain reaction, and fully sequenced on both strands. The putative malate synthase from S. coelicolor has an amino acid identity of 77% with the malate synthase of S. clavuligerus, and possesses an open reading frame which codes for a protein of 540 amino acids. In order to establish the identity of this gene, the putative aceB clones were subcloned into the expression vector pET24a, and heterologously expressed in Escherichia coli BL21(DE3). Soluble cell-free extracts containing the recombinant putative malate synthase exhibited a specific activity of 1623 (nmol.mg-1.min-1), which is an increment of 92-fold compared to the non-recombinant control. Thus, the gene product was confirmed to be a malate synthase. Interestingly, the specific activity of S. coelicolor malate synthase was found to be almost 8-fold higher than the specific activity of S. clavuligerus malate synthase under similar expression conditions. Furthermore, the genomic organisation of the three Streptomyces aceB genes cloned thus far is different from that of other bacterial malate synthases, and warrants further investigation.     

The pattern of expression of the Xenopus laevis tadpole alpha-globin genes and the amino acid sequence of the three major tadpole alpha-globin polypeptides

We have isolated cDNA clones derived from three tadpole alpha-globin mRNAs of Xenopus laevis. The entire nucleotide sequence of the three mRNAs has been determined from the cDNA clones and is presented together with the deduced amino acid sequence of the encoded polypeptides. Two of the three polypeptide sequences are 96% homologous whilst the third sequence is highly diverged, with only a 72% homology. The three tadpole alpha-globin genes are all similarly diverged from the two X. laevis adult alpha-globin genes with which they display approximately 50% homology. Analysis of several independent clones from each class of tadpole alpha-globin sequence reveals a very high degree of coding region polymorphism for each of the three corresponding genes. Using the cloned DNA sequences as hybridisation probes, we have analysed the expression of the corresponding genes during larval development. We show that all three genes are activated simultaneously early in development and that thereafter all three are expressed at an approximately equivalent level. A fourth tadpole alpha-globin mRNA sequence, for which we do not have a cDNA clone, accumulates co-ordinately with the three major mRNA sequences but to a much lower concentration. This pattern of gene expression differs significantly from that of the tadpole beta-globin genes of X. laevis, despite the two classes of genes being closely linked in the genome.     

Molecular cloning and analysis of a cDNA coding for chorismate synthase from the higher plant Corydalis sempervirens Pers.

Chorismate synthase catalyzes the last common step in the biosynthesis of the three aromatic amino acids in microorganisms and plants. We have cloned a cDNA for this enzyme from the higher plant Corydalis sempervirens. This is the first chorismate synthase cDNA from a eukaryotic organism. The nucleotide sequence was determined and the identity of the cDNA was confirmed by the amino acid sequence of tryptic peptides obtained from purified chorismate synthase. The homology to the two known bacterial sequences is about 48%. The cDNA contains an open reading frame of 1341 base pairs, encoding a protein of 447 amino acids. This protein with a molecular mass of 48,100 daltons resembles a chorismate synthase precursor targeted for chloroplast import. Multiple sites of polyadenylation were observed in chorismate synthase mRNAs.     

Application for PCR technology to subtractive cDNA cloning: identification of genes expressed specifically in murine plasmacytoma cells.

We describe a simple method for preparing a renewable source of subtractive cDNA which can be used as a hybridization probe or as insert which can be cloned into a variety of convenient vectors. This has been done by ligating a double-stranded oligonucleotide to each end of double-stranded subtractive cDNA, and then using this oligonucleotide sequence to amplify the heterogeneous population of cDNA molecules using the polymerase chain reaction and thermostable Taq DNA polymerase. This method improves the chances for identifying cDNA clones representing low abundance mRNAs that are expressed differentially. Using this approach, we have identified cDNA clones which detect three different low abundance mRNAs that are expressed in mouse plasmacytoma cell lines but not in mouse pre-B or B lymphoma cell lines.     

Nucleotide sequence of a protamine component CII gene of Salmo gairdnerii.

We have isolated, using nick-translated cloned protamine cDNA's as probes, several genomic clones containing protamine gene sequences from a Charon 4A library of Eco R1 digested rainbow trout (Salmo gairdnerii) DNA. One clone was chosen for detailed study and the 2.5 kbp Bam HI-Eco R1 restriction fragment containing the gene was subcloned in the plasmid pBR322. A 920 bp Bg1 II - Bam HI restriction fragment contains a sequence coding for protamine component CII as well as regions 5' and 3' to the mRNA coding portion. Present in the region 5' to the mRNA coding sequence are the promoter associated signals "TATA" box and "CAAT" box. The 5' untranslated region of the mRNA whose length and sequence were not established from the cDNA clones (1) was determined by nuclease mapping and starts within a sequence similar to the "capping signal" found in other genes. The protamine gene for CII contains no introns, a situation common to most histone genes, but, unlike the histone genes does not occur close to other protamine genes in a "cluster".     

Cloning and expression of mouse fatty acid synthase and other specific mRNAs. Developmental and hormonal regulation in 3T3-L1 cells

Mouse liver mRNA enriched in sequence coding for fatty acid synthase by sucrose density gradient centrifugation was used as template for cDNA synthesis. Double-stranded cDNA sequences were inserted into pBR322 and lambda gt10 and cloned. Clones containing putative cDNA sequences for fatty acid synthase were identified by differential hybridization with [32P] cDNAs synthesized from sucrose gradient-purified liver mRNA from mice fasted or fasted and refed a high carbohydrate diet. Thirteen out of 45 differentially expressed clones were found to contain sequences complementary to fatty acid synthase mRNA. Northern blot analysis revealed that, unlike in avian and rat tissues, a single 8.2-kilobase (kb) mRNA codes for fatty acid synthase in mice. In addition to the fatty acid synthase cDNA clones, cDNA clones to two specific mRNAs of 5.1 and 7.2 kb were selected to study nutritional, hormonal, and developmental regulation at the level of mRNA abundance in mouse liver and in 3T3-L1 cells. The induction of fatty acid synthase in the livers of previously fasted mice fed a high carbohydrate diet was controlled pretranslationally by modulation of the fatty acid synthase mRNA content. The level of the two mRNAs with sizes of 5.1 and 7.2 kb were also elevated dramatically in the liver of mice fasted and refed a high carbohydrate diet. A detectable, but very low level of fatty acid synthase mRNA was found in 3T3-L1 preadipocytes. During the differentiation to adipocytes, both the rate of synthesis and relative mRNA level for fatty acid synthase increased in a parallel fashion to a maximum of 17-fold. The levels of 5.1- and 7.2-kb mRNAs, coding for proteins possibly involved in lipogenesis, increased 45- and 25-fold, respectively, during differentiation of 3T3-L1 adipocytes. Treatment of mature 3T3-L1 adipocytes with insulin elicited a 3-fold increase in both rate of synthesis and mRNA content of fatty acid synthase, while treatment with dibutyryl cAMP caused a 60% decrease in fatty acid synthase mRNA and an 80% decrease in the rate of the enzyme synthesis, indicating pretranslational control of fatty acid synthase expression by the lipogenic and lipolytic hormones. Similarly, insulin caused a 2- to 3-fold increase in both 7.2- and 5.1-kb mRNAs and dibutyryl cAMP decreased the levels of 7.2- and 5.1-kb mRNAs to 10 and 20% of control levels, respectively.     

应用限制性显示PCR技术构建细菌poly(A)化mRNA的cDNA文库

针对细菌mRNA poly(A)化位点的高度多态性,利用oligo(dT)与poly(A)特异结合的特性,以oligo(dT)一纤维素纯化mRNA,并以oligo(dT)18为引物逆转录合成cDNA,用限制性内切酶消化cDNA,所得的限制性内切酶片段与通用接头相连,通过10个选择性引物组合进行选择性PCR,使各片段得以扩增并分布于10个亚组中,并进行克隆,成功地克隆了100多个基因片段,已对其中40个进行了测序分析,探讨了限制性显示PCR技术在细菌poly(A)化mRNA cDNA库构建中的应用价值。