The testis-specific apoptosis related gene TTL.6 underwent adaptive evolution in the lineage leading to humans

The TTL.6 gene is a member of the tubulin-tyrosine ligase (TTL) family involved in apoptosis and preferentially expressed in the testis. We sequenced the coding region and part of the introns of TTL.6 in world wide human populations and five representativ

Rapid evolution, genetic variations, and functional association of the human spermatogenesis-related gene NYD-SP12

NYD-SP12 is a recently identified spermatogenesis-related gene with a pivotal role in human testis development. In this study, we analyzed between-species divergence and within-species variation of NYD-SP12 in seven representative primate species, four wo

Cloning, characterization and promoter analysis of common carp hairy/Enhancer-of-split-related gene, her6

Some members of hairy/Enhancer-of-split-related gene (HES) family have important effects on axial mesoderm segmentation and the establishment and maintenance of the somite fringe. In fishes. the her6 gene, a member of the HES family, is the homologue Of heS1 in mammals and chicken. In this study, the her6 gene and its full-length cDNA from the common carp (Cyprinus carpio) were isolated and characterized. The genomic sequence of common carp her6 is approximately 1.7 kb. with four exons and three introns, and the full-length cDNA of 1314 bp encodes a Putative polypeptide of 271 amino acids. To analyse the promoter sequence of common carp her6. sequences of various lengths upstream from the transcription initiation site of her6 were fused to enhanced green fluorescent. protein gene (eGFP) and introduced into zebrafish embryos by microinjection to generate transgenic embryos. Our results show that the upstream sequence of 500 bp can direct highly efficient and tissue-specific expression of eGFP in zebrafish embryos. whereas a fragment of 200 bp containing the TATA box and a partial suppressor of hairless paired site sequence (SPS) is not sufficient to drive eGFP expression in zebrafish embryos.

Expression of a twist-related gene, Bbtwist, during the development of a lancelet species and its relation to cephalochordate anterior structures

Mesoderm formation plays a crucial role in the establishment of the chordate body plan. In this regard, lancelet embryos develop structures such as the anteriorly extended notochord and the lateral divertecula in their anterior body. To elucidate the developmental basis of these structures, we examined the expression pattern of a lancelet twist-related gene, Bbtwist, from the late gastrula to larval stages. In late-gastrula embryos, the transcripts of Bbtwist were detected in the presumptive first pair of somites and the middorsal wall of the primitive gut. The expression of Bbtwist was then upregulated in the lateral wall of somites and the notochord. At the late-neurula stage, it was also expressed in the anterior wall of the primitive gut, as well as in the evaginating lateral diverticula. No signal was detected in the left lateral diverticulum when it was separated from the gut, while in the right one, the gene was expressed later during the formation of the head coelom in knife-shaped larvae, and in the anterior part of the notochord in the same larvae. In 36-h larvae, only faint expression was detected in the differentiating notochordal and paraxial mesoderm in the caudal region. These expression patterns suggest that Bbtwist is involved in early differentiation of mesodermal subsets as seen in Drosophila and vertebrates. The expression in the anterior notochord may be related to its anterior expansion. The expression in the anterior wall of the primitive gut and its derivative, the lateral diverticula, suggests that lancelets share the capability to produce a mesodermal population from the tip of the primitive gut with nonchordate deuterostome embryos. (C) 1998 Academic Press.

A conserved family of doublesex-related genes from fishes

The sex-determining gene Mab-3 of C. elegans and the doublesex gene of Drosophila each contain a common DM domain and share a similar role. Human doublesex-related gene DMRT1 also encodes a conserved DM-related DNA-binding domain. We present here the amplification of a broad range of DM domain sequences from three fish species using degenerate PCR. Our results reveal unexpected complexity of the DM domain gene family in vertebrates. (C) 2002 Wiley-Liss, Inc.

Chromosomal localization and molecular marker development of the lipopolysaccharide and beta-1,3-glucan binding protein gene in the Zhikong scallop Chlamys farreri (Jones et Preston) (Pectinoida, Pectinidae)

Zhikong scallop Chlamys farreri(Jones et Preston) is an economically important species in China. Understanding its immune system would be of great help in controlling diseases. In the present study, an important immunity-related gene, the Lipopolysaccharide and Beta-1,3-glucan Binding Protein (LGBP) gene, was located on C. farreri chromosomes by mapping several lgbp-containing BAC clones through fluorescence in situ hybridization (FISH). Through the localization of various BAC clones, it was shown that only one locus of this gene existed in the genome of C. farreri, and that this was located on the long arm of a pair of homologous chromosomes. Molecular markers, consisting of eight single nucleotide polymorphism (SNPs) markers and one insertion-deletion (indel), were developed from the LGBP gene. Indel marker testing in an F1 family revealed slightly distorted segregation (p = 0.0472). These markers can be used to map the LGBP gene to the linkage map and assign the linkage group to the corresponding chromosome. Segregation distortion of the indel marker indicated genes with deleterious alleles might exist in the surrounding region of the LGBP gene.

运用减法杂交、差异筛选与信使RNA差别显示聚合酶链式反应克隆冬小麦春化相关基因

对于某些一年生或二年生高等植物，春化作用是诱导其成花的一个重要的环境因子。冬小麦春化进程中存在着一个核酸代谢的关键期，利用分子生物学技术分离特异表达的基因是研究春化诱导成花机理的一个突破口。 利用TRIzol试剂快速提取冬小麦燕大1817(Triticum aestivum L. cv Yanda 1817)未春化、春化4d、春化20d、5d脱春化的胚芽中的总RNA，去除污染的DNA后，将引物P_1(5'TTTTTTTTTTTCA3')、P_2(5'TTTTTTTTTTCC3')与10个碱基的随机引物OPF_1-OPF_(20)、OPG_1-OPG_(20)组成80个引物对，对不同来源的RNA进行差别显示，共显示了大约10,000种mRNA，结果发现了两个仅在春化20d这一关键期表达而在未春化、春化4d、5d脱春化时不表达的春化相关基因(VRG)VRG49与VRG54。Northern分析进一步表明这两个基因仅与春化20d的冬小麦RNA有杂交信号。将VRG49与VRG54亚克隆于pGEM-4Z载体上，利用T_7测序系统获得了VRG49和VRG54的DNA序列，它们的长度分别为307bp与169bp。 春化21d的冬小麦京冬1号(T. aestivum L. cv Jingdong No. 1)胚芽的mRNA在逆转酶作用下反转录成sscDNA杂交，将过量的未春化、脱春化的mRNA与sscDNA杂交，运用磁珠法分离出未杂交上的sscDNA，以特异的sscDNA为模板，用DNA聚合酷I合成了dscDNA。通过对dscDNA内部EcoRI位点的甲基化、末端补平、EcoRI接头的安装、连接进入λgt10载体的EcoRI位置，以及运用包装系统进行体外包装，建立了库容为4 * 10~6pfu的富集低温诱导的冬小麦cDNA噬菌体文库。用来源于未春化、春化21d、脱春化的冬小麦mRNA合成3种cDNA探针，对噬菌斑进行原位杂交，结果筛选出了3个春化相关基因(VRG)VRG79、VRG111和VRG231。Dot blotting与Northern分析表明VRG79仅在冬小麦春化关键期21d表达。运用PCR方法从λgt10DNA中扩增出VRG79片断并亚克隆于PUC18载体上，通过T_7测序系统获得了VGR79的序列，其包括349个碱基。 通过Internet将VRG49、VRG54、VRG79与GenBank、EMBL、DDBJ、PBD中的序列进行同源性分析，结果发现这些基因至少是在植物中新发现的基因，对这些基因推测的一些功能也进行了讨论。

冬小麦春化作用相关基因Verc203的功能鉴定

本文应用反义基因技术对减法杂交和差异筛选所获得的春化相关基因CDNA克隆( Verc203)的功能进行鉴定。将Vcrc203插入PB1221质粒的CaMV35S启动子与GLIS基因之间构成表达载体，采用花粉管途径转基因法导入冬小麦胚，得到300粒种子。将上述处理的冬小麦种子萌发后在2-4℃处理28天，转入自然条件下栽培。以幼叶为材料提取基因组DNA，以Gus基因片断为探针，用点杂交法筛选获得转基因植株．转基因植株在自然条件下生长三个月未见抽穗， 而同样栽培条件下的对照（未经转基因处理的冬小麦，经相同条件春化处理）则全部抽穗。以Vcrc203和Gus基因的部分序列为引物，在转基因材料的基因组DNA中扩增出Vcrc203反义基因序列，Gus活性检测表明Gus基因在转基因小麦的茎尖和叶片中得到表达。上述结果表明Vcrc203的反义基因已整合到冬小麦基因组内，并得到高效表达。转基因植株的发育受到严重影响，显示Vcrc203基因可能参与了冬小麦春化作用对植物发育进程的调控．

植物克隆化基因功能分析及蛋白编码基因分离的研究

直到九十年代，以利用拟南芥菜和金鱼草突变体克隆到花器官发育基因为重要标志的植物发育研究取得了重大突破。但由于开花决定过程分子控制机理研究进展却较为缓慢。以mRNA人手利用差异筛选技术获得春化相关基因克隆(verc203，verc17)，Northem blot初步证明这些克隆对春化处理的特异性。根据反义RNA理论和技术对克隆化verc203基因功能进行研究。另外，从纯化蛋白人手，在金针菇中克隆到新的溶血性毒蛋白编码基因，并在细菌中得到表达，这一实验为利用此途径来克隆发育调控蛋白的编码基因进行了有益的技术路线探索。本研究的具体结果如下： 1． 春化相关基因(verc203)对冬小麦花发育调控 根据反义RNA的理论和技术进一步证明verc203在冬小麦开花启动和花发育过程中的可能控制作用。将verc203 DNA序列插入带有CaMV35S启动子和GUS基因的pBI221表达载体， 使之能在植物中高效表达反义RNA，并以转正义基因植株作为对照。通过花粉管途径转基因方法，得到326粒反义质粒冬小麦转化种子和1 98粒正义质粒冬小麦转化种子．所有转化处理种子和正常小麦种子萌发后，经春化处理，与未春化冬小麦植株一起培养直到对照植株达到成熟期。实验观察发现，转反义基因植株的抽穗受到明显的阻抑，开花过程大大推迟。其中表达较强的6株转基因植株甚至晚花五十多天。还发现一些表达较弱的植株尽管晚花20到30天，但花器官发育却受到影响，如，雄蕊缺失，穗发育异常等。Southemblot和PCR实验结果表明外源基因整合到转基因植株基因组。 Northrenblot以及报告基因(GUS)活性等实验证明反义基因在转反义基因植株RNA水平上得到高效表达。同时，GUS基因在蛋白质水平上得到表达。基于上述实验，有理由认为ver203基因可能是控制冬小麦春化诱导的成花启动和花发育过程中的主要基因之一。 2． 金针菇毒蛋白flammutoxin编码基因的克隆及其在细菌中的表达 利用色谱分离技术及SDS．PAGE电泳技术从金针菇(Flammuleina velutips)分离得到一种分子量为31 kDa、对人血红细胞具有溶血活性的蛋白质(flammutoxin)，通过蛋白质序列微量分析技术测得其N-端3 1个氨基酸顺序。利用氨基酸序列推测其编码基因序列信息，通过RT - PCR克隆到一个由973个核苷酸组成的编码基因，5’端93个核苷酸编码肽链序列与测得flammutoxin N-端氨基酸序列完全相同。基因序列同源性分析表明在GenBank (USA)，EMBL Database (Europe)和DDBJ (Japan)基因序列数据库中未找到与之同源的基因序列。这个基因与载体p-半乳糖苷酶部分基因编码的融合蛋白在E coli细胞中有效地得到表达。表达产物的SDS-PAGE，Western blot和溶血活性实验表明它是flammutoxin蛋白原编码基因。并对融合蛋白的溶血活性降低原因作了讨论。

小麦春化相关基因的克隆及其功能分析

一． 小麦相关基因ver203F cDNA全序列克隆与功能分析 　　根据本实验室通过差异筛选技术克隆到的与春化相关的基因cDNA verc203的序列，设计PCR 5’端PCR引物，利用RACE（rapid amplification of cDNA ends）克隆策略，得到春化相关基因ver203基因的同源基因ver203F cDNA的3’端序列，长度为1,197bp。Northern分析表明ver203F全长约为1.5 kb，且其表达具有春化处理的特异性。根据3’RACE克隆的ver203F 3 ’端核苷酸序列设计了3’端PCR引物，利用5’RACE克隆到该基因的5’端片段，经DNASIS核酸分析软件分析将5’45RACE和3’RACE DNA序列拼接合并，得到ver203F全长cDNA，从TdT加尾5’末端到poly A全长为1,561 bp，5’端起始密码子ATG上游非编码区－1～－192共了192bp，终止密码子TGA到poly A的非编码区有253bp，cDNA编码区全长1,119 bp，推测编码373氨基酸残基。国际基因序列数据库检索表明该基因序列（GenBank/EMBL/DDBJ：AB012013）与大麦茉莉酸诱导基因有部分同源性。因上推测该基因在调控开花过程中可能参与茉莉酸介导的信号传导途径，ver203F作用的发挥可能需要其它蛋白的参与，或ver203F本身就是一个受体蛋白。 　　为了研究ver203F基因的功能，将通过3’RACE克隆到的ver203F 3’端序到分别构建正义和反义植物表达载体，通过花粉管通道法、农杆菌介导的叶圆片法以及农杆菌介导的真空转化法分别转化小麦、烟草和拟南芥菜。获得转基因植株后，PCR、DNA Dob Blot、Southern Blot分析以及GUS活性检测证明外源基因已整合到转基因植株中，并得到表达。在获得的小麦、烟草和拟南芥菜转基因植株中，它们开花时间都相应地推迟，表明正常植物体内该基因在控制营养生长向生殖生长的转变中起作用。ver203F可以影响小麦和拟南茶菜花序的发育，首先无论正义还是反义都使得花序的发育受到抑制，在小麦中表现为顶部小穗退化，在拟南芥菜中表现为顶花。其次在转化正义基因的转基因拟南芥菜中，观察到产生的顶花为对称的两朵或以对称的两朵顶花基部为生长点长出丛生花，这种对称花的麦型小麦小穗中小花的表型相类似，说明ver203F基因可能在小麦小花的发育过程中也起着重要作用。 二． 春化相关基因ver17在开花过程中功能的分析 以春化处理冬小麦（京冬1号）幼苗cDNA为材料，通过减法杂交与差异筛选得到春化相关cDNA克隆verc17。为了研究该基因的功能，以包含CaMV 35S启动子的pBI221为载体，将ver17cDNA片段分别从两个方向插入pBI221的BamH I－Sma I, Xba I－BamH I间，构建正义和反义表达质粒：p17S和p17X，通过花粉管通道法转化小麦。对T0和T1两代转基因小麦的观察发现，在转化反义基因p17X的转基因小麦首先表现为抽穗推迟，其次穗的顶部和基部小穗严重退化，另外还发现转化反义基因的小麦败育现象严重（主要是花粉败育），因此推测ver17基困能可具有以下几方面的特点：a．春化诱导型表达;b．促进植物开花;c．促进穗顶端和基部花的发育，减少其退化;d．影响雄蕊的发育。

水稻转录因子编码基因的表达谱和功能分析

水稻是重要的经济作物，也是单子叶植物分子遗传研究的模式植物。对水稻生长发育中基因的表达调控研究将为其遗传改良提供重要线索。转录因子在植物发育过程中对基因表达调控起关键作用，对其表达谱和功能的研究具有重要的理论和应用价值。 本研究一方面在水稻基因组水平上，利用cDNA芯片技术研究了水稻(中花11)种子发育过程中388个转录相关基因的表达谱;另一方面，在分离转录因子基因的基础上，利用分子生物学和遗传学方法研究了有关基因详细、具体的表达特征和功能。 利用cDNA芯片技术鉴定了123个种子优先表达、属于12类不同表达模式的转录相关基因。首次发现了许多主要在种子发育特定阶段表达的转录相关基因，还发现一些种子优先表达的转录相关基因参与了激素和非生物胁迫信号转导，为阐释水稻种子发育中的转录调节和信号网络的分子基础提供了很多有价值的线索。 在相关基因工作方面，分离了一个MADS-box类转录因子编码基因OsMDP（全长），表达模式析以及初步的功能分析揭示该基因作为一个负调控因子参与了BR控制的水稻叶节弯曲、胚芽鞘伸长、主根伸长等过程的信号转导，为水稻根、叶、胚芽鞘的发育、以及BR信号转导机制的研究提供了线索。 另外，从水稻的cDNA文库中分离并初步研究了三个分属BELL1、KNOX和HD-ZIP亚族的同源盒基因（片段），OsHB1主要和水稻种子发育相联系，OsHB2可能参与激素调节的发育或响应，OsHB3和水稻花药早期发育相关、对花药早期发育的调控研究有一定价值。

Pseudogenization of the tumor-growth promoter angiogenin in a leaf-eating monkey

Physiological functions of human genes may be studied by gene-knockout experiments in model organisms such as the mouse. This strategy relies on the existence of one-to-one gene orthology between the human and mouse. When lineage-specific gene duplication occurs and paralogous genes share a certain degree of functional redundancy, knockout mice may not provide accurate functional information on human genes. Angiogenin is a small protein that stimulates blood-vessel growth and promotes tumor development. Humans and related primates only have one angiogenin gene, while mice have three paralogous genes. This makes it difficult to generate angiogenin-knockout mice and even more difficult to interpret the genotype-phenotype relation from such animals should they be generated. We here show that in the douc langur (Pygathrix nemaeus), an Asian leaf-eating colobine monkey, the single-copy angiogenin gene has a one-nucleotide deletion in the sixth codon of the mature peptide, generating a premature stop codon. This nucleotide deletion is found in five unrelated individuals sequenced, and therefore is likely to have been fixed in the species. Five colobine species that are closely related to the douc langur have intact angiogenin genes, suggesting that the pseudogenization event was recent and unique to the douc langur lineage. This natural knockout experiment suggests that primate angiogenin is dispensable even in the wild. Further physiological studies of douc largurs may offer additional information on the role of this cancer-related gene in normal physiology of primates, including humans. Our findings also provide a strong case for the importance of evolutionary analysis in biomedical studies of gene functions. (C) 2003 Elsevier Science B.V. All rights reserved.

Early expressed genes showing a dichotomous developing pattern in the lancelet embryo

Lancelets (amphioxus), although showing the most similar anatomical features to vertebrates, never develop a vertebrate-like head but rather several structures specific to this animal. The lancelet anatomical specificity seems to be traceable to early developmental stages, such as the vertebrate dorsal and anterior-posterior determinations. The BMP and Wnt proteins play important roles in establishing the early basis of the dorsal structures and the head in vertebrates. The early behavior of BMP and Wnt may be also related to the specific body structures of lancelets. The expression patterns of a dpp-related gene, Bbbmp2/4, and two wnt-related genes, Bbwnt7 and Bbwnt8, have been studied in comparison with those of brachyury and Hnf-3 beta class genes The temporal expression patterns of these genes are similar to those of vertebrates; Bbbmp2/4 and Bbwnt8 are first expressed in the invaginating primitive gut and the equatorial region. respectively, at the initial gastrula stage. However, spatial expression pattern of Bbbmp2/4 differs significantly from the vertebrate cognates. It is expressed in the mid-dorsal inner layer of gastrulae and widely in the anterior region, in which vertebrates block BMP signaling, The present study suggests that the lancelet embryo may have two distinct developmental domains from the gastrula stage, the domains of which coincide later with the lateral diverticular and the somitocoelomic regions. The embryonic origin of the anterior-specific structures in lancelets corresponds to the anterior domain where Bbbmp2/4 is continuously expressed.

Cloning and characterization of the first amphibian bradykinin gene

More than ten bradykinin-related peptides and their cDNAs; have been identified from amphibians, but their genes are unknown. In present study, four cDNAs encoding one, two, four and six copies of bradykinin-related peptides were cloned from the frog (Odorrana grahami) skin cDNA library, respectively. Three bradykinin-related peptides (bradykinin, Thr6-bradykinin, Leu5Thr6-bradykinin) were deduced from these four cDNA sequences. Based on the cDNA sequence, the gene sequence encoding an amphibian bradykinin-related peptide from O. grahami was determined. It is composed of 7481 base pairs including two exons and two introns. The first exon codes signal peptide and the second exon codes acidic spacer peptide and Thr6-bradykinin. The promoter region of the bradykinin gene contains several putative recognition sites for nuclear factors, such as SRY, GATA-1, LYF-1, DeltaE, CDXA, NKX-2.5, MIF1 and S8. The current work may facilitate to understand the regulation and possible functions of amphibian skin bradykinin-related peptides. (C) 2009 Elsevier Masson SAS. All rights reserved.