Cloning and Sequencing of the Gene Encoding the Soluble Fumarate Reductase from Saccharomyces cerevisiae

A gene of the soluble fumarate reductase (FRDS) that binds FADnon-covalently was cloned by polymerase chain reaction (PCR)using degenerate oligonucleotides designed from partial aminoacid sequences of highly purified enzyme. The nucleotide sequenceof a 0.99-kb amplified product was found to be nearly identicalto a partial sequence of an open reading frame (ORF) previouslyreported (EMBL database accession number S-30830). Accordingto the sequence in the EMBL database, we cloned 1.7-kb fragmentcontaining entire sequence of this ORF by PCR and found thatthis fragment contained a perfect match to the 0.99-kb sequenceamplified with the degenerate primers. From these results, weconcluded that this ORF is the FRDS gene. The amino acid sequencesof the regions involved in the non-covalent binding of FAD andthe active site, which are conserved among the flavoproteinsubunits of membrane-bound fumarate reductase and succinatedehydrogenase, were found in FRDS. However, unlike the membrane-boundenzymes, FRDS did not contain the histidine residue that covalentlybinds the isoalloxazine ring of FAD at or near the correspondingposition. FRDS showed high homology to the product of S. cerevisiaeOSM1 gene which was reported to be required for growth in hypertonicmedia.     

番茄烟粉虱传双生病毒PCR检测

From the conserved regions of the reported nucleotide sequences of whitefly-transmitted geminiviruses (WTGV), a pair of degenerate primers was designed to anneal to the conserved sequence.The tomato samples infected geminivirus-like from Guangdong were detected by PCR. The results showed that a 356bp specific fragment was amplified from the samples. The specific fragment was cloned and sequenced, and the sequence was compared with all nucleotide sequences in GenBank by Blast of NCBI. The result showed that the fragment belonged to Geminiviridae DNA. So the degenerate primers may be used to detect the WTGV from tomato in Guangdong. Moreover, both of the homology of the fragment between WTGV from tomato in Guangdong and the reported WTGV in the world and WTGV from tomato in Guangxi were under 82%. These results implied that the WTGV from tomato in Guangdong differed from the above-mentioned WTGV.     

花生RGA 片段的克隆及初步分析

目的:通过同源克隆获得了花生闽花6号的RGA片段,为其抗性的研究及抗性育种提供了参考资料。方法:试验分为两组:其一通过利用抗性基因的NBS保守区所设计的简并引物对花生品种闽花六号进行了RGA片段扩增,其二结合已登录的花生RGA片段序列经过多元比对后设计简并引物进行RGA片段的扩增及序列分析;分析比较两组克隆方法的效果。结果:测序分析表明:前者20条随机测序序列中没有一条与已知RGA片段序列相似;后者20条随机测序序列中有18条为RGA片段序列,其登录号为GenBankEU639668-EU639685。结论:前一种方法克隆扩增RGA基因片段的效率很低,而后一种方法克隆扩增效果更好,这为闽花6号花生的遗传改良提供了理论基础。     

Polymorphisms flanking the mariner integration sites in the rice gall midge (Orseolia oryzae Wood-Mason) genome are biotype-specific.

In an effort to study genome diversity within and between the Indian biotypes of the Asian rice gall midge, Orseolia oryzae, a major insect pest of rice, we made use of mariner transposable element integration site polymorphisms. Using degenerate primers, the design of which is based on mariner sequences, we amplified a ca. 450 bp mariner sequence from the rice gall midge. The mariner sequence showed homology with that of a mariner element isolated from the Hessian fly, Mayetiola destructor, a major dipteran pest of wheat. Southern hybridization, using this mariner fragment as a probe, revealed that the mariner elements are moderately to highly repetitive in the rice gall midge genome. Based on the sequence information of this 450-bp PCR-amplified fragment, outward-directed primers were designed and used in an inverse PCR (iPCR) to amplify the DNA flanking the conserved regions. To study the regions flanking the mariner integration sites, we employed a novel PCR-based approach: a combination of sequence specific amplification polymorphism (SSAP) and amplified fragment length polymorphism (AFLP). The outward-directed mariner-specific primer was used in combination with adapter-specific primers with 1-3 selective nucleotides at their 3' ends. The amplification products were resolved on an agarose gel, Southern-transferred onto nylon membranes, and probed with the iPCR fragment. Results revealed biotype-specific polymorphisms in the regions flanking the mariner integration sites, suggesting that mariner elements in the rice gall midge may be fixed in a biotype-specific manner. The implications of these results are discussed in the context of biotype differentiation.     

Conserved residues of liquefying alpha-amylases are concentrated in the vicinity of active site

Three dimensional structure of three liquefying type Bacillus alpha-amylases were modeled based on sequence analyses and refined structure of Aspergillus oryzae enzyme. The models suggest that the overall folding motif of alpha-amylases is conserved. The active site, substrate binding and stabilizing calcium binding residues are conserved and concentrated in a cleft between two domains. They constitute the core of alpha-amylases to which other, less conserved regions are attached. The bacterial enzymes have a loop of about 45 residues near the active site and Ca2+ binding region. The loop may be important for the liquefying function of these enzymes.     

花生RGA片段的克隆及初步分析

目的：通过同源克隆获得了花生闽花6号的RGA片段,为其抗性的研究及抗性育种提供了参考资料。方法：试验分为两组：其一通过利用抗性基因的NBS保守区所设计的简并引物对花生品种闽花六号进行了RGA片段扩增,其二结合已登录的花生RGA片段序列经过多元比对后设计简并引物进行RGA片段的扩增及序列分析;分析比较两组克隆方法的效果。结果：测序分析表明：前者20条随机测序序列中没有一条与已知RGA片段序列相似;后者20条随机测序序列中有18条为RGA片段序列,其登录号为GenBankEU639668-EU639685。结论：前一种方法克隆扩增RGA基因片段的效率很低,而后一种方法克隆扩增效果更好,这为闽花6号花生的遗传改良提供了理论基础。     

盐胁迫下朝鲜碱茅的甜菜碱醛脱氢酶活性变化及其基因保守区的克隆

文章探讨了朝鲜碱茅甜菜碱醛脱氢酶活性在盐胁迫下的变化,用简并引物扩增了甜菜碱醛脱氢酶基因保守区序列的结果表明,该保守区段长438 bp,推测编码145个氨基酸,包括醛脱氢酶高度保守序列V[T/S]LELGGKSP和其后29位与酶功能有关的Cys。此序列Genbank登录号为EF095710。     

来源于蓝藻、铜绿微囊藻的尿苷二磷酸葡萄糖脱氢酶基因的克隆和鉴定

以已知的尿苷二磷酸葡萄糖脱氢酶基因的保守区为基础,自行设计一对简并引物,该对引物从形成水华的蓝藻(Synechocystis PCC6803)铜绿微囊藻FACHB 905株(Microcystis aeruginosa FACHB 905)的基因组DNA中扩增到一个476 bp的DNA片段.通过TAIL-PCR和连接介导的PCR两种方法分离该片段的侧翼序列,最后得到大小约2.5 kb的DNA片段.序列分析揭示其中有一个编码462个氨基酸的开放阅读框,我们将此开放阅读框对应的蛋白命名为Mud.该Mud蛋白的氨基酸序列与蓝藻(73%相同,87%相似)和细菌(Bacillus subtilis)(51%相同,67%相似)的尿苷二磷酸葡萄糖脱氢酶氨基酸序列表现高度的同源性.将该mud基因克隆于p-GEX-4T-1融合表达载体并在大肠杆菌中表达GST-Mud融合蛋白,经过酶活力测定发现,GST-Mud蛋白具有一定的尿苷二磷酸葡萄糖脱氢酶活性.用抗GST-Mud蛋白的多抗对M.aeruginosa FACHB 905的胞质蛋白组分进行Western印迹分析,结果显示一条分子量大小约49 kD的专一条带,这个分子量与从基因推断出的蛋白分子量大小基本一致.综上所述,我们认为从微囊藻克隆到的Mud蛋白基因是尿苷二磷酸葡萄糖脱氢酶基因,该酶在其他生物如植物和细菌中参与多糖合成,是多糖合成的关键酶之一,而在藻类中对尿苷二磷酸葡萄糖脱氢酶开展研究却是首次报道.     

Tomato leaf curl geminivirus associated with cantaloupe yellow leaf disease in Thailand

Geminivirus associated with yellow leaf disease of cantaloupe plants was detected using polymerase chain reaction (PCR) with geminivirus-specific degenerate primers which anneal within the AC1 ORF (replication initiator protein gene) and the AV1 ORF (coat protein gene). A DNA fragment of 1.2 kbp was amplified, cloned and sequenced. The 32-base stem loop region was found in the amplified fragment. This included the conserved nonanucleotide sequence TAATATTAC present in all geminiviruses. The nucleotide sequence of the intergenic region (IR) was compared with 28 whitefly-transmitted geminiviruses. The geminivirus associated with yellow leaf disease of cantaloupe plants showed 96.2% sequence identity with DNA A of tomato leaf curl geminivirus from India (ToLCV-In2). These data suggest that cantaloupe yellow leaf disease was caused by ToLCV.     

Characterization and mapping of NBS-LRR resistance gene analogs in apricot (Prunus armeniaca L.)

Genomic DNA sequences sharing homology with the NBS-LRR (nucleotide binding site-leucine-rich repeat) resistance genes were isolated and cloned from apricot (Prunus armeniaca L.) using a PCR approach with degenerate primers designed from conserved regions of the NBS domain. Restriction digestion and sequence analyses of the amplified fragments led to the identification of 43 unique amino acid sequences grouped into six families of resistance gene analogs (RGAs). All of the RGAs identified belong to the Toll-Interleukin receptor (TIR) group of the plant disease resistance genes (R-genes). RGA-specific primers based on non-conserved regions of the NBS domain were developed from the consensus sequences of each RGA family. These primers were used to develop amplified fragment length polymorphism (AFLP)-RGA markers by means of an AFLP-modified procedure where one standard primer is substituted by an RGA-specific primer. Using this method, 27 polymorphic markers, six of which shared homology with the TIR class of the NBS-LRR R-genes, were obtained from 17 different primer combinations. Of these 27 markers, 16 mapped in an apricot genetic map previously constructed from the self-pollination of the cultivar Lito. The development of AFLP-RGA markers may prove to be useful for marker-assisted selection and map-based cloning of R-genes in apricot.     

Molecular cloning, overproduction and characterization of the Bacillus cereus IMP dehydrogenase

The gene of IMP dehydrogenase of Bacillus cereus ts-4, a temperature-sensitive mutant of B. cereus JCM 2152, was subcloned and its sequence was analyzed. A B. cereus ts-4 DNA fragment of 2,065 bp containing the entire impdh gene and flanking regions was sequenced. The fragment contained an open reading frame of 1,527 bp encoding 509 amino acids with a calculated molecular mass of 55,390 Da. The impdh sequence of JCM 2152 was also analyzed by TA cloning using PCR products amplified with primers from B. cereus ts-4 impdh gene. The gene amplified by PCR was expressed in Escherichia coli using a pET17 x b expression plasmid. The N-terminal amino acid sequence of the overproduced enzyme was identified as Met-Trp-Glu-Ser-Lys-Phe-Val-Lys-Glu-Gly-Leu-Thr-Phe-AspAsp-Val-Leu -Leu-Val- Pro. The overproduced enzyme was eluted at a molecular mass of about 225 kDa by gel filtration. The molecular mass of the subunit was estimated to be 56 kDa by SDS-PAGE. The overproduced enzyme was active against IMP, IDP, and ITP, and showed the highest activity at pH 9.5. These properties of the recombinant enzyme were almost identical to those of IMP dehydrogenase of B. cereus.     

米根霉菌ldhL基因的克隆及其在大肠杆菌中的表达

以米根霉菌基因组DNA为模板,根据GenBank上已公布的米根霉L-乳酸脱氢酶基因(ldhL)序列设计特异性引物,PCR扩增得到963 bp的DNA片段,经序列分析后将其亚克隆到原核表达载体pET30a上,构建成重组质粒pET30a-ldhL.将pET30a-ldhL转化到BL21感受态细菌中,经IPTG诱导表达后进行SDS-PAGE分析,可见约43 kD的与预期大小一致的目的蛋白条带,结果表明ldhL基因在大肠杆菌中进行了表达,经酶活分析产物的酶活力为98 U/mL,证明了表达产物具有预期的酶活性,这为进一步研究利用乳清为发酵原料高产L-乳酸的米根霉基因工程菌株奠定了基础.     

Molecular cloning of the nahG gene encoding salicylate hydroxylase from Pseudomonas fluorescens

A gene encoding the salicylate hydroxylase was cloned from the genomic DNA of Pseudomonas fluorescens SME11. The DNA fragment containing the nahG gene for the salicylate hydroxylase was mapped with restriction endonucleases and sequenced. The DNA fragment contained an ORF of 1,305 bp encoding a polypeptide of 434 amino acid residues. The nucleotide and amino acid sequences of the salicylate hydroxylase revealed several conserved regions with those of the enzyme encoded in P. putida PpG7: The homology of the nucleotide sequence is 83% and that of amino acid sequence is 72%. We found large conserved regions of the amino acid sequence at FAD and NADH binding regions. The FAD binding site is located at the amino terminal region and a lysine residue functions as a NADH-binding site.     

Isolation and identification by sequence homology of a putative cytosine methyltransferase from Arabidopsis thaliana.

A plant cytosine methyltransferase cDNA was isolated using degenerate oligonucleotides, based on homology between prokaryote and mouse methyltransferases, and PCR to amplify a short fragment of a methyltransferase gene. A fragment of the predicted size was amplified from genomic DNA from Arabidopsis thaliana. Overlapping cDNA clones, some with homology to the PCR amplified fragment, were identified and sequenced. The assembled nucleic acid sequence is 4720 bp and encodes a protein of 1534 amino acids which has significant homology to prokaryote and mammalian cytosine methyltransferases. Like mammalian methylases, this enzyme has a C terminal methyltransferase domain linked to a second larger domain. The Arabidopsis methylase has eight of the ten conserved sequence motifs found in prokaryote cytosine-5 methyltransferases and shows 50% homology to the murine enzyme in the methyltransferase domain. The amino terminal domain is only 24% homologous to the murine enzyme and lacks the zinc binding region that has been found in methyltransferases from both mouse and man. In contrast to mouse where a single methyltransferase gene has been identified, a small multigene family with homology to the region amplified in PCR has been identified in Arabidopsis thaliana.     

矛尾鱼Hoxa—11基因克隆及5′端序列分析

Hoxa-11基因调节鱼类鳍和四足动物肢的发育,在脊椎动物进化过程中起着重要的作用,利用人和鼠的Hoxa-11基因保守序列设计了两个兼并引物,通过PCR扩增到了矛尾鱼的Hoxa-11基因,经克隆和DNA序列分析,该片段为2065bp,包括绝大部分外显子Ⅰ,内含子和部分外显子Ⅱ,编码204个氨基酸,其氨基酸序列与人、鼠、鸡、蛙和斑马鱼的同源性分别为66.0%、67.6%、74.4%、72.8%和59.7%。外显子Ⅰ的长度从矛尾鱼到蛙、鸡、鼠和人呈现逐步上升趋势,人比矛尾鱼增长了16%,进一步分析,外显子Ⅰ可分为4个区域;两个高度保守区域,1个中度保守区域和1个可变区域,外显子Ⅰ的长度变化主要是由于可变区域内丙氨酸同聚物以及两侧富含甘氨酸和丝氨酸序列的累积。矛尾鱼只有1个由两个丙氨酸组成的同类物,蛙有1个由5个连续丙氨酸组成的同聚物,而鸡、鼠和人有3个丙氨酸同聚物,其中最大的同聚物由7个连续丙氨酸组成,而且在同聚物两侧出现了富含甘氨酸和丝氨酸序列。这表明可变区域可能与脊椎动物进化和鳍-肢转换过程中新功能的获得有关。同源异型盒所在的外显子Ⅱ区和剪接位点是高度保守的。内含子的长度变化较大,但在其内部也发现了两个高度保守的35bp和16bp的DNA片段,这两个片段在人、鼠、鸡、蛙和矛尾鱼中是完全相同的,这些序列的高度保守性提示其功能上的重要性。     

Primary structure of a base non-specific ribonuclease from Rhizopus niveus

The primary structure of a base non-specific ribonuclease from Rhizopus niveus (RNase Rh) was determined by nucleotide sequence analysis of the DNA fragment encoding RNase Rh gene including signal peptide sequence, and amino acid sequence analysis of the peptide obtained from RNase Rh and RNase Rh' (a protease-modified RNase Rh created during the course of purification). The sequence determined was: MKAVLALATLIGSTLASSCSSTA LSCSNSANSDTCCSPEYGLVVLNMQWAPGYGPANAFTLHGLWPDKCSGAYAPSGGCDSN RASSSIASVIKSKDSSLYNSMLTYWPSNQGNNNVFWSHEWSKHGTCVSTYDPDCYDNYE EGEDIVDYFQKAMDLRSQYNVYKAFSSNGITPGGTYTATEMQSAIESYFGAKAKIDCSSG TLSDVALYFYVRGRDTYVITDALSTGSCSGDVEYPTK (the sequence of signal peptide is underlined). The sequence indicates that the homology with the sequence of RNase T2 from A. oryzae with the same base specificity is about 42% and that the sequences around the two histidine residues which are supposed to be involved in the active site are fairly conserved.     

Non-LTR retrotransposons (LINEs) as ubiquitous components of plant genomes

During the course of work aimed at isolating a rice gene from Oryza australiensis by PCR, the oligonucleotide primers used were found to generate a fragment that showed sequence homology to the endonuclease (EN) region of the maize non-LTR retrotransposon (LINE) Cin4. We carried out further PCRs using oligonucleotide primers that hybridized to these sequences, and found that they amplified several fragments, each with homology to the EN regions, from Oryza sativa cv. Nipponbare as well as O. australiensis. We mapped the approximate locations of two rice LINE homologues by screening clones in a YAC library made from a rice (O. sativa) genome, and found that each homologue was present in a low copy number apparently at nonspecific regions on rice chromosomes. We then carried out PCR using degenerate oligonucleotide primers which hybridized to the rice LINE homologues and Cin4 to ascertain whether LINE homologues are present in a variety of members of the plant kingdom, including angiosperms, gymnosperms, bracken, horsetail and liverwort. Cloning and nucleotide sequencing revealed that 53 clones obtained from 27 out of 33 plant species contained LINE homologues. In addition to these homologues, we identified four homologues with EN regions in the Arabidopsis thaliana genome by a computer search of databases. The nucleotide sequences of almost all the LINE homologues were greatly diverged, but the derived amino acid sequences were well conserved, and all contained glutamic acid and tyrosine residues at almost the same relative positions as in the the active site regions of AP (apurinic/apyrimidinic)-endonucleases. The EN regions in the LINE homologues from closely related plant species show a closer phylogenetic relationship, indicating that sequence divergence during vertical transmission has been a major influence upon the evolution of plant LINEs. Received: 13 July 1998 / Accepted: 13 October 1998     

NMR reveals structural rearrangements associated to substrate insertion in nucleotide‐adding enzymes

The protein NP_344798.1 from Streptococcus pneumoniae TIGR4 exhibits a head and base‐interacting neck domain architecture, as observed in class II nucleotide‐adding enzymes. Although it has less than 20% overall sequence identity with any member of this enzyme family, the residues involved in substrate‐recognition and catalysis are highly conserved in NP_344798.1. NMR studies showed binding affinity of NP_344798.1 for nucleotides and revealed μs to ms time scale rate processes involving residues constituting the active site. The results thus obtained indicate that large‐amplitude rearrangements of regular secondary structures facilitate the penetration of the substrate into the occluded nucleotide‐binding site of NP_344798.1 and, by inference based on sequence and structural homology, probably a wide range of other nucleotide‐adding enzymes.     

cDNA cloning of a novel cysteine protease of Plasmodium falciparum

The cDNA for a novel Plasmodium cysteine protease (falcipain-2) has been isolated from a Plasmodium falciparum cDNA library. A 602 bp fragment was amplified from P. falciparum by PCR using degenerate oligonucleotide primers. The primers were designed based upon the amino acids flanking the active site cysteine and asparagine residues that are conserved in the eukaryotic cysteine proteases. This fragment was used to screen a P. falciparum cDNA library and isolated a 2.1 kb clone that encoded a novel cysteine protease. The sequence of the 2.1 kb clone predicted a 56 kDa protein containing a typical signal sequence, a prosequence and a 24.7 kDa mature protease with 37% identity to falcipain-1, a hemoglobin-degrading cysteine protease of P. falciparum. Northern blot analysis detected a 2.1 kb message in trophozoites. Taken together, we have isolated a novel cysteine protease of P. falciparum, which may play an important role at the late stages of the erythrocytic cycle of the parasite.