Development of a rapid, sensitive and specific diagnostic assay for fish Aquareovirus based on RT-PCR

A rapid, sensitive and highly specific detection method for Aquareovirus based on reverse-transcription polymerase chain reaction (RT-PCR) was developed. Based on multiple sequence alignment of the cloned sequences of a local isolates, the Threadfin reovirus (TFV) and Guppy reovirus (GPV) with Grass carp reovirus (GCRV), a pair of degenerate primers was selected carefully and synthesized. Using this primer combination, only one specific product, approximately 450 bp in length was obtained when RT-PCR was carried out using the genomic double-stranded RNA (dsRNA) of TFV, GPV and GCRV. Similar results were also obtained when Chum salmon reovirus (CSRV) and Striped bass reovirus (SBRV) dsRNA were used as templates. No products were observed when nucleic acids other than the dsRNA of the aquareoviruses described above were used as RT-PCR templates. This technique could detect not only TFV but also GPV and GCRV in low titer virus-infected cell cultured cells. Furthermore, this method has also been shown to be able to diagnose GPV-infected guppy (Poecilia reticulata) that exhibit clinical symptoms as well as GPV-carrier guppy. Collectively, these results showed that the RT-PCR amplification method using specific degenerate primers described below is very useful for rapid and accurate detection of a variety of aquareovirus strains isolated from different host species and origin. (C) 2004 Elsevier B.V. All rights reserved.

Genome segment S8 of grass carp hemorrhage virus encodes a virion protein

The complete nucleotide sequence of the genome segment S8 of grass carp hemorrhage virus (GCHV) was determined from cDNA corresponding to the viral genomic RNA. It is 1,287 nucleotides in length and contains a large open reading frame that could encode a protein of 409 amino acids with a predicted molecular mass of 44 kD. The S8 was expressed using the pET fusion protein vector and detected by Western blotting analysis using the chicken egg IgY against intact GCHV particles, indicating that S8 encodes a virion protein. Amino acid sequence comparisons revealed that the protein encoded by S8 is closely related to protein alpha2 of mammalian reovirus, suggesting that the deduced protein of S8 is an inner capsid protein. Copyright (C) 2001 S. Karger AG, Basel.

A zebrafish forebrain-specific zinc finger gene can induce ectopic dlx2 and dlx6 expression

Identifcation of the earliest forebrain-specific markers should facilitate the elucidation of molecular events underlying vertebrate forebrain determination and specification. Here we report the sequence and characterization of fez (forebrain embryonic zinc finger), a gene that is specifically expressed in the embryonic forebrain of zebrafish. Fez encodes a putative nuclear zinc finger protein that is highly conserved in Drosophila, zebrafish, Xenopus, mouse, and human. In zebrafish, the expression of fez becomes detectable at the anterior edge of the presumptive neuroectoderm by 70% epiboly. During the segmentation period, its expression is completely restricted to the rostral region of the prospective forebrain. At approximately 24 h postfertilization, fez expression is mostly confined to the telencephalon and the anterior-ventral region of the diencephalon. Although fez expression is present in one-eyed pinhead (oep) and cyclops (cyc) zebrfish mutants, the pattern is altered. Forced expression of fez induces ectopic expression of dlx2 and dlx6, two genes involved in brain development. Knockdown of fez function using a morpholino-based antisense oligo inhibited dlx2 expression in the ventral forebrain. Our studies indicate that fez is one of the earliest markers specific for the anterior neuroectoderm and it may play a role in forebrain development by regulating Dlx gene expression. (C) 2001 Academic Press.

Molecular and evolutionary analyses of formyl peptide receptors suggest the absence of VNO-specific FPRs in primates

Formyl peptide receptors (FPRs) were observed to expand in rodents and were recently suggested as candidate vomeronasal chemosensory receptors. Since vomeronasal chemosensory receptors usually underwent positive selection and evolved concordantly with the vomeronasal organ (VNO) morphology, we surveyed FPRs in primates in which VNO morphology is greatly diverse and thus it would provide us a clearer view of VNO-FPRs evolution. By screening available primate genome sequences, we obtained the FPR repertoires in representative primate species. As a result, we did not find FPR family size expansion in primates. Further analyses showed no evolutionary force variance between primates with or without VNO structure, which indicated that there was no functional divergence among primates FPRs. Our results suggest that primates lack the VNO-specific FPRs and the FPR expansion is not a common phenomenon in mammals outside rodent lineage, regardless of VNO complexity.

来自易变山羊草抗禾谷孢囊线虫基因的克隆与序列分析

禾谷孢囊线虫（Heterodera avenae）是严重危害禾谷类作物的病原线虫之一,它广泛分布于澳大利亚、欧洲、北美、印度和中国等世界主要小麦产区,使作物严重减产,造成巨大的经济损失。目前最有效的防治措施之一是将外源抗性基因导入栽培小麦(Triticum aestivum L.)，培育抗禾谷孢囊线虫的新品种。但迄今为止抗禾谷孢囊线虫基因克隆研究的相关报道却很少。 本实验根据此前从抗禾谷孢囊线虫材料E-10扩增得到的与来自节节麦（Aegilops tauschii）的抗禾谷孢囊线虫基因Cre3高度同源的序列Rccn4,设计出三条嵌套引物,采用SON-PCR(single oligonucleotide nested PCR)方法,从E-10基因组DNA中得到一个长为1264 bp的扩增产物(命名为Rccn-L),测序比对结果显示,这一序列将Rccn4的3’端延伸了1209 bp,与抗禾谷孢囊线虫Cre3基因核苷酸同源性为86﹪，核苷酸编码区长1026 bp，含一个不完整的开放阅读框,一个终止密码子，没有起始密码子和内含子结构,编码一个342个氨基酸残基的蛋白质。该蛋白质等电点为5.19，分子量为38112.6Da。从序列的第113位开始到第332位是NBS-LRR类抗病性基因LRR区,呈现XXLXXLXXL重复。LRR编码区内亮氨酸残基的含量达17﹪，与抗禾谷孢囊线虫Cre3基因LRR编码区的核苷酸和氨基酸同源性分别为89﹪和78﹪。本实验首次将SON-PCR成功地运用于植物基因克隆，为植物基因克隆提供了又一有效方法。 此外,还根据Cre3基因及其他的NBS-LRR类植物抗性基因的NBS和LRR区保守序列设计了两对特异性引物，从禾谷孢囊线虫抗性材料易变山羊草基因组DNA中扩增到两个相应的目标条带。测序分析结果表明，它们的长度分别为532bp和1175bp，构成了一个有32bp的共同序列的NBS-LRR编码区。其序列总长为1675bp（命名为RCCN），含有一个不完整的开放阅读框，没有起始密码子、终止密码子和内含子结构。其中编码序列为1673bp，可编码一个557个氨基酸的蛋白质，等电点（pI）为5.39，分子量为63537.5Da。与Cre3的核苷酸和氨基酸同源性分别为87.8﹪和77﹪。RCCN氨基酸序列中含有已知抗病基因NBS区域的几个保守模体：kinase2区的ILDD、kinase3的（ⅰ）ESKILVTTRSK，（ⅱ）KGSPLAARTVGG，（ⅲ）RRCFAYCS及EGF。RCCN NBS区与Cre3 NBS区的核苷酸和氨基酸的同源性分别为96.4﹪和94﹪。从氨基酸序列的274位到548位为LRR保守区，呈现不规则的aXXLXXLXXL（其中a代表I，V，L，F或M）重复，其中亮氨酸的含量为15.6﹪。该区域与Cre3的LRR区的核苷酸和氨基酸同源性分别为80.8﹪和74﹪。推测该序列可能为一个抗禾谷孢囊线虫的新基因。 本文对抗禾谷孢囊线虫基因的克隆研究，为进一步克隆基因全序列，探索其结构与功能，和研究该基因表达与调控提供了关键信息。同时也为通过基因工程途径将抗性基因向优良小麦品种高效、定向转移，最终培育出小麦抗禾谷孢囊线虫新品种奠定了基础。 Cereal cyst nematode (CCN) is a damaging pathogen of broad acre cereal crops in Australia, Europe, North America, India and China. It affects wheat, barley, oat and triticale and causes yield loss of up to 80%. At present, Transferring resistance genes against CCN into wheat cultivars and breeding varieties are considered one of the most effective methods for controlling the CCN. However, there are very limited reports concerning the cloning studies of resistance genes against the cereal cyst nematode. According to the sequence of Rccn4 which had high similarity to the nucleotide binding site (NBS) coding region of cereal cyst nematode resistance gene, Cre3， We designed three 3’ nested primers. Using single oligonucleotide nested PCR (SON-PCR) we successfully amplified one band, Rccn-L, of 1264bp from E-10 which is the wheat-Ae.variabilis translocation line containing the cereal cyst nematode resistance gene of Ae.variabilis. We found that this band of interesting is the 3’ flanking sequence of 1209bp in size of Rccn4. The coding region was 1026bp, which contained an incomplete open reading frame and a terminator codon, without initiation codon and intron, encoding a peptide of 342 amino acid residues, and shared 86﹪nucleotide sequence identity with Cre3. This peptide had a conserved LRR domain, containing the imperfect repeats,XXLXXLXXL, which contains 17﹪ leucine residues and shares, respectively, 89﹪ nucleotide sequence and 78﹪ amino acid sequence identity with the LRR sequence of Cre3 locus. This research firstly used SON-PCR in the research of plant genome successfully, which indicated that SON-PCR is another method of cloning plant gene. At the same time, According to the conversed motif of NBS and LRR region of cereal cyst nematode resistance gene Cre3 from wild wheat (Triticum tauschlii L.) and the known NBS-LRR group resistance genes, we designed two pairs of specific primers for NBS and LRR region respectively. One band of approximately 530bp was amplified using the specific primers for conversed NBS region and one band of approximately 1200bp was amplified with the specific primers for conversed LRR region. After sequencing, we found that these two sequences included 32bp common nucleotide sequence and have 1675 bp in total, which was registered as RCCN in the Genbank. RCCN contained a NBS-LRR domain and an incomplete open reading frame without initiation codon, terminator codon and inxon. Its exon encodes a peptide of 557 amino acid residues. The molecular weight of the protein from the amino acid was 63.537 KDa. The amino acid sequence of RCCN contained conserved motif: ILDD, ESKILVTTRSK, KGSPLAARTVGG, RRCFAYCS, EGF，LRR. RCCN shares 87.8﹪ nucleotide sequence and 77﹪ amino acid sequence identity with cereal cyst nematode gene Cre3. It might be a novel cereal cyst nematode resistance gene. These research results of cloning the resistance genes against cereal cyst nematode bring a great promise for transferring resistance genes into wheat cultivars and breeding new wheat varieties against cereal cyst nematode by gene engineering. And these results also lay the hard foundation for the expressing researches of these genes.

小麦抗禾谷孢囊线虫（ Heterodera avenae）相关基因的研究

禾谷孢囊线虫严重影响禾谷类作物的产量，在小麦中由禾谷孢囊线虫引起的产量损失可达30-100%。尤其在澳大利亚、欧洲、印度和中东危害严重，目前禾谷孢囊线虫已成为危害我国作物的主要病源。控制禾谷孢囊线虫的方法主要有：作物轮作、杀线虫剂、寄主抗性等等，其中基因工程方法培育抗线虫小麦品种被认为是最经济有效的方法。分离抗禾谷类孢囊线虫基因对揭示抗性基因结构与功能及其表达调控具有重要意义。 尽管小麦是重要的粮食作物，在小麦中已发现的抗禾谷孢囊线虫的基因很少，而比其近缘属如节节麦、易变山羊草、偏凸山羊草中含有丰富的抗源。目前已鉴定出禾谷孢囊线虫抗性位点Cre，并发现了9个禾谷孢囊线虫抗性基因(Cre1,2, 3, 4, 5, 6, 7, 8, and R) ，其中只有Cre1和Cre8直接从普通小麦中获得。从节节麦中获得的Cre3基因能最有效的控制线虫数量，其次是Cre1和Cre8。这些基因的克隆对于了解禾谷孢囊线虫抗性机制及进一步的育种应用都是非常关键的。然而，目前为止仅有Cre3基因通过图位克隆的方法从节节麦中被分离得到。该基因已被克隆得到的多数线虫抗性基因一样均属于核苷酸结合位点区（NBS）－亮氨酸重复序列区（LRR）基因家族。目前，已有很多抗性基因被分离，这些已知的NBS-LRR类抗性基因的保守序列为应用PCR的方法克隆新的抗性基因提供了可能。 因此本课题的目的是采用保守区同源克隆、3′RACE 和5′RACE 等方法从抗禾谷孢囊线虫小麦-易变山羊草小片段易位系E10 中克隆小麦抗禾谷孢囊线虫基因全序列，进而通过半定量PCR 和荧光定量PCR 研究该基因的表达模式。同时通过mRNA 差别显示技术和任意引物PCR（RAP-PCR）技术分离克隆植物禾谷孢囊线虫抗性基因及其相关基因，为阐明植物抗病性分子机制以及改良作物抗病性和作物育种提供基础，为通过分子标记辅助育种和基因工程方法实现高效、定向转移抗病基因到优良小麦品种奠定了重要的理论和物质基础。主要研究结果： 1． 本实验根据此前从抗禾谷孢囊线虫材料E-10 扩增得到的与来自节节麦的抗禾谷孢囊线虫Cre3 基因及其他的NBS-LRR 类抗性基因的NBS 和LRR 保守区序列设计了两对特异性引物，从E10 中扩增到532bp 和1175bp 的两个目标条带，它们有一个32bp 的共同序列，连接构成总长为1675bp 的NBS-LRR 编码区（命名为RCCN）。根据RCCN设计引物，利用NBS-LRR区序列设计引物，通过5′RACE 和3′RACE 技术采用3′-Full RACE Core Set(TaKaRa)和5'-Full RACE Kit (TaKaRa)试剂盒，反转录后通过嵌套引物GSP1 和GSP2 分别进行两轮基因特异性扩增，分别将NBS_LRR 区向5′端和3′端延伸了1173bp 和449bp，并包含了起始密码子和终止密码子。根据拼接的得到的序列重新设计引物扩增进行全基因扩增的结果与上面获得的一致。拼接后得到全长2775 bp 的基因序列（记作CreZ, GenBank 号：EU327996）。CreZ 基因包括完整的开放阅读框，全长2775 bp，编码924个氨基酸。序列分析表明它与已知的禾谷孢囊线虫抗性基因Cre3的一致性很高，并且它与已经报到的NBS-LRR 类疾病抗性基因有着相同的保守结构域。推测CreZ基因可能是一个新的NBS-LRR 类禾谷孢囊线虫抗性基因，该基因的获得为通过基因工程途径培育抗禾谷孢囊线虫小麦新品种奠定了基础，并为抗禾谷孢囊线虫基因的调控表达研究提供了参考。 2． 通过半定量PCR和SYBR Green荧光定量PCR技术对CreZ基因的相对表达模式进行了研究。以α-tubulin 2作为参照，采用半定量PCR 分析CreZ 基因在不同接种时期1d, 5d, 10, 15d 的E-10的根和叶的的表达情况。在内参扩增一致的条件下，CreZ 在E-10的根部随着侵染时间的增加表达量有明显的增加，在没有侵染的E-10的根部其表达量没有明显变化，而在叶中没有检测表达，说明该基因只在抗性材料的根部表达。SYBR Green定量PCR分析接种前后E10根部基因CreZ基因的表达水平为检测CreZ基因的表达建立了一套灵敏、可靠的SYBRGreen I 荧光定量PCR 检测方法。接种禾谷孢囊线虫后E10根内CreZ基因的相对表达水平显著高于接种前。随接种时间的延长持续增加，最终CreZ基因的相对表达量达到未接种的对照植株的10.95倍。小麦禾谷孢囊线虫抗性基因CreZ的表达量与胁迫呈正相关，表明其与小麦的的禾谷孢囊线虫抗性密切相关，推测CreZ基因可能是一个新的禾谷孢囊线虫候选抗性基因。 3． 针对小麦基因组庞大、重复序列较多，禾谷孢囊线虫抗性基因及其相关基因的片断难以有效克隆的问题，通过mRNA 差别显示技术及RAP-PCR 技术分离克隆植物禾谷孢囊线虫抗性及其相关基因。试验最终得到154 条差异表达条带，将回收得到的差异条带的二次PCR 扩增产物经纯化后点到带正电的尼龙膜上，进行反向Northern 杂交筛选，最终筛选得到102 个阳性差异点。将其中81 个进行测序，并将序列提交到Genbank 中的dbEST 数据库，分别获得登录号(FE192210 -FE192265，FE193048- FE193074 )。序列比对分析发现，其中26 个序列与已知功能的基因序列同源；有28 条EST 序列在已有核酸数据库中未找到同源已知基因和EST，属新的ESTs 序列；另外27 个EST 序列与已知核酸数据库中的ESTs 具有一定相似性，但功能未知。其所得ESTs 序列补充了Genbank ESTs 数据库，为今后进一步开展抗禾谷类孢囊线虫基因研究工作打下了基础。结合本试验功能基因的相关信息，对小麦接种禾谷孢囊线虫后产生的抗性机制进行了探讨。接种禾谷孢囊线虫后植物在mRNA 水平上的应答是相当复杂的，同时植物的抗病机制是一个复杂的过程，涉及到多个代谢途径的相互作用。 The cereal cyst nematode (CCN), Heterodera avenae Woll, causes severe yieldreductions in cereal crops. The losses caused by CCN can be up to 30-100% in somewheat fields. At present, cereal cyst nematode has become the major disease sourcein China and it also damaged heavily in Australia, Europe, India and Middle East.The damage caused by CCN can be mitigated through several methods, includingcrop rotation, nematicide application, cultural practice, host resistance, and others.Of these methods, incorporating resistance genes into wheat cultivars and breedingresistant lines is considered to be the most cost-effective control measure forreducing nematode populations. Although wheat is an economically important crop around the world, far fewergenes resistant to CCN were found in wheat than were detected in its relatives, suchas Aegilops taucchi, Aegilops variabilis and Aegilops ventricosa. Cloning these genesis essential for understanding the mechanism of this resistance and for furtherapplication in breeding. Because of the huge genome and high repeat sequencescontent, the efficient methods to clone genes from cereal crops, are still lacking. A resistance locus, Cre, has been identified and 9 genes resistant to CCN (designatedCre1, 2, 3, 4, 5, 6, 7, 8, and R) have been described, in which Cre1 and Cre8 werederived directly from common wheat. The Cre3 locus, which was derived from Ae.tauschii, has the greatest impact on reducing the number of female cysts, followed byCre1 and Cre8. Cloning these genes is essential for understanding the mechanism ofthis resistance and for further application in breeding. However, to this point, only Cre3, a NBS-LRR disease resistance gene, has been obtained through mappingcloning in Ae. tauschii. The majority of nematode resistance genes cloned so far belong to a super familywhich contains highly conserved nucleotide-binding sites (NBS) and leucine-richrepeat (LRR) domains. To date, many NBS-LRR resistance genes have been isolated.The conserved sequences of these recognized NBS-LRR resistance genes provide thepossibility to isolate novel resistance genes using a PCR-based strategy. The aim of the present study was to clone the resistance gene of CCN fromWheat/Aegilops variabilis small fragment chromosome translocation line E10 whichis resistant to CCN and investigate the espression profiles of this gene withsemi-quantitative PCR and real-time PCR. Another purpose of this study is cloningthe relational resistance gene for CCN by mRNA differential display PCR andRAP-PCR. These works will offer a foundation for disease defence of crop andbreeding and directional transferring resistance gene into wheat with geneengineering. Primary results as following: 1．According to the conversed motif of NBS and LRR region of cereal cystnematode resistance gene Cre3 from wild wheat (Triticum tauschlii) and the knownNBS-LRR group resistance genes, we designed two pairs of specific primers for NBSand LRR region respectively. One band of approximately 530bp was amplified usingthe specific primers for conversed NBS region and one band of approximately 1175bpwas amplified with the specific primers for conversed LRR region. After sequencing,we found that these two sequences included 32bp common nucleotide having 1675bpin total, which was registered as RCCN in the Genbank. Based on the conservedregions of known resistance genes, a NBS-LRR type CCN resistance gene analog wasisolated from the CCN resistant line E-10 of the wheat near isogenic lines (NILs), by5′RACE and 3′ RACE.designated as CreZ (GenBank accession number: EU327996) .It contained a comlete ORF of 2775 bp and encoded 924 amino acids. Sequencecomparison indicated that it shared 92% nucleotide and 87% amino acid identitieswith those of the known CCN-resistance gene Cre3 and it had the same characteristic of the conserved motifs as other established NBS-LRR disease resistance genes. 2． Usingα-tubulin 2 as exoteric reference, semi-quantitative PCR and real-timePCR analysis were conducted. The expression profiling of CreZ indicated that it wasspecifically expressed in the roots of resistant plants and its relative expression levelincreased sharply when the plants were inoculated with cereal cyst nematodes. therelative expression level of the 15days-infected E10 is the 10.95 times as that ofuninfected E10,ultimately. It was inferred that the CreZ gene be a novel potentialresistance gene to CCN. 3．We cloned the relational resistance gene for CCN by mRNA differentialdisplay PCR and arbitrarily primed PCR fingerprinting of RNA from wheat whichpossess huge and high repeat sequence content genomes. Total 154 differentialexpression bands were separated and second amplified by PCR. The products werenylon membrane. The 102 positive clones were filtrated by reverse northern dot blotand 81 of those were sent to sequence. The EST sequences were submitted toGenbank (Genbank accession: FE192210 - FE192265, FE193048 - FE193074). Thesequences alignment analysis indicated 26 of them were identical with known genes;28 were not found identical sequence in nucleic acid database; another 27 ests wereidentical with some known ests, but their functions were not clear. These ESTsenriched Genbank ESTs database and offered foundation for further research ofresistance gene of CCN.

Genomic organization, nucleotide sequence analysis of the core histone genes cluster in Chlamys farreri and molecular evolution assessment of the H2A and H2B

This work represents the nucleotide sequence of the core histone gene cluster from scallop Chlamys farreri. The tandemly repeated unit of 5671 bp containing a copy of the four core histone genes H4, H2B, H2A and H3 was amplified and identified by the techniques of homology cloning and genomic DNA walking. All the histone genes in the cluster had the structures in their 3' flanking region which related to the evolution of histone gene expression patterns throughout the cell cycle, including two different termination signals, the hairpin structure and at least one AATAAA polyadenylation signal. In their 5' region, the transcription initiation sites with a conserved sequence of 5'-PyATTCPu-3' known as the CAP site were present in all genes except to H2B, generally 37-45 bp upstream of the start code. Canonical TATA and CAAT boxes were identified only in certain histone genes. In the case of the promoters of H2B and H2A genes, there was a 5'-GATCC-3' element, which had been found to be essential to start transcription at the appropriate site. After this element, in the promoter of H2B, there was another sequence, 5'-GGATCGAAACGTTC-3', which was similar to the consensus sequence of 5'-GGAATAAACGTATTC-3' corresponding to the H2B-specific promoter element. The presence of enhancer sequences (5'-TGATATATG-3') was identified from the H4 and H3 genes, matching perfectly with the consensus sequence defined for histone genes. There were several slightly more complex repetitive DNA in the intergene regions. The presence of the series of conserved sequences and reiterated sequences was consistent with the view that mollusc histone gene cluster arose by duplicating of an ancestral precursor histone gene, the birth-and-death evolution model with strong purifying selection enabled the histone cluster less variation and more conserved function. Meanwhile, the H2A and the H2B were demonstrated to be potential good marks for phylogenetic analysis. All the results will be contributed to the characterization of repeating histone gene families in molluscs.

Polydispersity of ethylene sequence length in metallocene ethylene/alpha-olefin copolymers. I. Characterized by thermal-fractionation technique

In this article, the polydispersity of the ethylene sequence length (ESL) in ethylene/alpha-olefin copolymers was studied by atomic force microscopy (AFM) and the thermal-fractionation technique. The crystal morphology observation by AFM showed that morphology changed gradually with decreasing average ESL from complete lamellae over shorter and more curved lamellae to a granular-like morphology, and the mixed morphology was observed after stepwise crystallization from phase-separated melt. This result indicated that the ethylene sequence with different lengths crystallized into a crystalline phase with a different size and stability at the copolymer systems. The thermal-fractionation technique was used to characterize the polydispersity of ESL. Three of the following statistical terms were introduced to describe the distribution of ESL and the lamellar thickness: the arithmetic mean (L) over bar (n), the weight mean (L) over bar (w), and the broadness index I = (L) over bar (w)/(L) over bar (n). It was concluded that the polydispersity of ESL could be quantitatively characterized by the thermal-fractionation technique. The effects of temperature range, temperature-dependent specific heat capacity C-p of copolymer, and the molecular weight on the results of thermal fractionation were discussed,

Isolation, characterization and electron microscopic single particle analysis of the NADH : ubiquinone oxidoreductase (complex I) from the hyperthermophilic eubacterium Aquifex aeolicus

The proton-translocating NADH:ubiquinone oxidoreductase (complex I) has been purified from Aquifex aeolicus, a hyperthermophilic eubacterium of known genome sequence. The purified detergent solubilized enzyme is highly active above 50 degreesC. The specific activity for electron transfer from NADH to decylubiquinone is 29 U/mg at 80 degreesC. The A. aeolicus complex I is completely sensitive to rotenone and 2-n-decyl-quinazoline-4-yl-amine. SDS polyacrylamide gel electrophoresis shows that it may contain up to 14 subunits. N-terminal amino acid sequencing of the bands indicates the presence of a stable subcomplex, which is composed of subunits E, F, and G. The isolated complex is highly stable and active in a temperature range from 50 to 90 degreesC, with a half-life of about 10 h at 80 degreesC. The activity shows a linear Arrhenius plot at 50-85 degreesC with an activation energy at 31.92 J/mol K. Single particle electron microscopy shows that the A. aeolicus complex I has the typical L-shape. However, visual inspection of averaged images reveals many more details in the external arm of the complex than has been observed for complex I from other sources. In addition, the angle (90degrees) between the cytoplasmic peripheral arm and the membrane intrinsic arm of the complex appears to be invariant.

Complete mitochondrial genome of the sea cucumber Apostichopus japonicus (Echinodermata: Holothuroidea): The first representative from the subclass Aspidochirotacea with the echinoderm ground pattern

Complete mitochondrial genome plays an important role in the accurate revelation of phylogenetic relationships among metazoans. Here we present the complete mitochondrial genome sequence from a sea cucumber Apostichopus japonicus (Echinodermata: Holothuroidea), which is the first representative from the subclass Aspidochirotacea. The mitochondrial genome of A. japonicus is 16,096 bp in length. The heavy strand consists of 31.8% A, 20.2% C, 17.9% G, and 30.1% T bases (AT skew = 0.027: GC skew = 0.062). It contains thirteen protein-coding genes (PCGs), twenty-two transfer RNA genes, and two ribosomal RNA genes. There are a total of 3793 codons in all thirteen mitochondrial PCGs, excluding incomplete termination codons. The most frequently used amino acid is Leu (15.77%), followed by Set (9.73%), Met (8.62%), Phe (7.94%), and Ala (7.28%). Intergenetic regions in the mitochondrial genome of A. japonicus are 839 bp in total, with three relatively large regions of Unassigned Sequences (UAS) greater than 100 bp. The gene order of A. japonicus is identical to that observed in the five studied sea urchins, which confirms that the gene order shared by the two classes (Holothuroidea and Echinoidea) is a ground pattern of echinoderm mitochondrial genomes. Bayesian tree based on the cob gene supports the following relationship: (outgroup, (Crinoids, (Asteroids, Ophiuroids, (Echinoids, Holothuroids)))). (C) 2009 Elsevier B.V. All rights reserved.

Analysis of tandem repeats in the genome of Chinese shrimp Fenneropenaeus chinensis

Through random sequencing, we found a total of 884000 base-pairs (bp) of random genomic sequences in the genome of Chinese shrimp (Fenneropenaeus chinensis). Using bio-soft Tandem Repeat Finder (TRF) software, 2159 tandem repeats were found, in which there were 1714 microsatellites and 445 minisatellites, accounting for 79.4% and 20.6% of repeat sequences, respectively. The cumulative length of repeat sequences was found to be 116685 bp, accounting for 13.2% of the total DNA sequence; the cumulative length of microsatellites occupied 9.78% of the total DNA sequence, and that of minisatellites occupied 3.42%. In decreasing order, the 20 most abundant repeat sequence classes were as follows: AT (557), AC (471), AG (274), AAT (92), A (56), AAG (28), ATC (27), ATAG (27), AGG (18), ACT (15), C (11), AAC (11), ACAT (11), CAGA (10), AGAA (9), AGGG (7), CAAA (7), CGCA (6), ATAA (6), AGAGAA (6). Dinucleotide repeats, not only in the aspect of the number, but also in cumulative length, were the preponderant repeat type. There were few classes and low copy numbers of repeat units of the pentanucleotide repeat type, which included only three classes: AGAGA, GAGGC and AAAGA. The classes and copy numbers of heptanucleotide, eleven-nucleotide and thirteen-nucleotide primer-number-composed repeats were distinctly less than that of repeat types beside them.

Assessment of genetic diversities of selected Laminaria (Laminariales, Phaeophyta) gametophytes by inter-simple sequence repeat analysis

Inter-simple sequence repeat (ISSR) analysis was used to assess genetic diversity among 10 pairs of male and female Laminaria gametophytes. A total of 58 amplification loci was obtained from 10 selected ISSR primers, of which 34 revealed polymorphism among the gametophytes. Genetic distances were calculated with the Dice coefficient ranging from 0.006 to 0.223. A dendrogram based on the unweighted pair-group method arithmetic (UPGMA) average showed that most male and female gametophytes of the same species were clustered together and that 10 pairs of gametophytes were divided into four groups. This was generally consistent with the taxonomic categories. The main group consisted of six pairs of gametophytes, which were selected from Laminaria japonica Aresch. by intensive inbreeding through artificial hybridization. One specific marker was cloned, but was not converted successfully into a sequence characterized amplified region (SCAR) marker. Our results demonstrate the feasibility of applying ISSR markers to evaluate Laminaria germplasm diversities.

A centromeric satellite sequence in the Pacific oyster (Crassostrea gigas Thunberg) identified by fluorescence in situ hybridization

A highly repetitive satellite sequence was previously identified in the Pacific oyster Crassostrea gigas Thunberg. The sequence has 168 bp per unit, present in tandem repeats, and accounts for 1% to 4% of the genome. We studied the chromosomal location of this satellite sequence by fluorescence in situ hybridization (FISH), A probe was made by polymerase chain reaction and incorporation of digoxigenin-11-dUTP. Hybridization was detected with fluorescein-labeled antidigoxigenin antibodies. FISH signals were located at centromeric regions of 7 pairs of the Pacific oyster chromosomes. No interstitial site was found. Signals were strong and consistent on chromosomes 1, 2, 4, and 7, but weak or variable oil chromosomes 5, 8, and 10. No signal was observed on chromosomes 3, 6, and 9. Our results showed that this sequence is clearly a centromeric satellite, disputing its previous assignment to the telomeric and submetacentric regions of 2 chromosomes. No signal was detected in the American oyster (Crassostrea virginica Gmelin).

Intergeneric conjugation in holomycin-producing marine Streptomyces sp strain M095

Marine Streptomyces are potential candidates for novel natural products and industrial catalysts. In order to set up biosynthesis approach for a holomycin-producing strain M095 isotated from Jiaozhou Bay, China, a genetic transformation system was established using intergeneric conjugation. The plasmid pIJ8600 consists of an origin of replication for Escherichia coli, a phage integrase directing efficient site-specific integration in bacterial chromosome, thiostrepton-induced promoter and an attP sequence. Using E. coli ET12567 (pUZ8002) carrying pIJ8600 as a conjugal donor, while it was mated with strain M095, pIJ8600 was mobilized to the recipient and the transferred DNA was also integrated into the recipient chromosome. The frequency of exconjugants was 1.9 +/- 0.13 x 10(-4) per recipient cell. Analysis of eight exconjugants showed pIJ8600 was stable integrated at a single chromosomal site (attB) of the Streptomyces genome. The DNA sequence of the attB was cloned and shown to be conserved. The results of growth and antimicrobial activity analysis indicated that the integration of pIJ8600 did not seem to affect the biosynthesis of antibiotics or other essential amino acids. To demonstrate the feasibility of above gene transfer system, the allophycocyanin gene (apc) from cyanobacterium Anacystis nidulans UTEX625 was expressed in strain M095, and the results indicated heterologous allophycocyanin could be expressed and folded effectively. (c) 2006 Elsevier GmbH. All rights reserved.

Sequence analysis of Arthrospira maxima based on fosmid library

Arthrospira (Spirulina) (Setchell& Gardner) is an important cyanobacterium not only in its nutritional potential but in its special biological characteristics. An unbiased fosmid library of Arthrospira maxima FACHB438 that contains 4300 clones was constructed. The size distribution of insert fragments is from 15.5 to 48.9 kb and the average size is 37.6 kb. The recombination frequency is 100%. Therefore the library is 29.9 equivalents to the Arthrospira genome size of 5.4 Mb. A total of 719 sample clones were randomly chosen from the library and 602 available sequences, which consisted of 307,547 bases, covering 5.70% of the whole genome. The codon usage of A. maxima was not strongly biased. GC content at the first position of codons (46.9%) was higher than the second (39.8%) and the third (45.5%) positions. GC content of the genome was 43.6%. Of these sequences, 287 (47.7%) showed high similarities to known genes, 63 (10.5%) to hypothetical genes and the remaining 252 (41.8%) had no significant similarities. The assigned genes were classified into 22 categories with respect to different biological roles. Remarkably, the high presence of 25 sequences (4.2%) encoding reverse transcriptase indicates the RT gene may have multiple copies in the A. maxima genome and might play an important role in the evolutionary history and metabolic regulation. In addition, the sequences encoding the ATP-binding cassette transport system and the two-component signal transduction system were the second and third most frequent genes, respectively. These genomic features provide some clues as to the mechanisms by which this organism adapts to the high concentration of bicarbonate and to the high pH environment.