脑组织特异性基因/脑胶质瘤抑瘤基因LRRC4的功能研究进展

LRRC4是一个脑组织相对特异性表达基因,是采用表达序列标签(EST)介导的定位-候选克隆策略结合5′-RACE技术从染色体7q31～32区域克隆出来的一个富亮氨酸重复(LRR)超家族的新成员.LRRC4是神经系统发育与轴突生长的功能基因.LRRC4的表达与胶质瘤的级别进展呈负相关,其表达缺失参与脑胶质瘤进展的晚期事件.LRRC4能够下调一系列神经生长因子或受体(如IGF,EGF,PDGF,CNTF,bFGF,GDNF和BDNF等)的表达,通过调控多种信号转导通路(K-Ras/c-Raf/ERK/MAPK,PI-3K/AKT/NF-κB,p70S6/PKC,STAT3以及JNK2/c-Jun/mp53等)将U251细胞阻滞在G1晚期,抑制脑胶质瘤细胞的增殖和侵袭,而且这种抑制作用依赖于它的LRR结构域.LRRC4不能诱导胶质瘤细胞的凋亡,而是诱导胶质瘤细胞向胶质样细胞分化.     

人类生殖相关新基因的定位和组织表达

基因定位对研究基因之间以及基因与疾病之间的相互关系具有重要意义。应用辐射杂种细胞系技术（RH）对我们克隆的人类新基因HBRP（Human BSP-Related Protein）进行了染色体定位,结果将该基因定位于19q13.2～13.3,同时应用生物信息学方法在人类基因组重叠片段数据库进行该基因的定位,结果相吻合。研究证明,RH技术具有快速、精确、简便等优点,是基因定位研究中一强有力的技术。同时通过RT-PCR方法研究了HBRP基因在人体各组织中的表达分布,结果显示该基因在睾丸、肠、肾、肝、脾、胃、胰腺组织有较高的表达,而在检测的脑、肺、骨骼肌、心肌组织中表达较弱。 Abstract:Gene localization is significant in elucidating the interaction between genes,gene and diseases.Using radiation hybrid (RH) technique,we cloned and localized a novel gene,designated human BSP-related protein (HBRP) on 19q13.2～13.3,in line with its localization in data bank of overlapping fragment of human genome through bioinformatics method.It is suggested RH is rapid,precise,simple and powerful in gene localization.In addition,we detected the expression and distribution of HBRP in human tissues by RT-PCR.The results showed HBRP was highly expressed in intestine,kidney,liver,spleen,stomach and pancreas,whereas lowly in brain,lung,muscle and heart.     

Isolation and Characterization of a Novel Human Radiosusceptibility Gene, NP95

The murine nuclear protein Np95 has been shown to underlie resistance to ionizing radiation and other DNA insults or replication arrests in embryonic stem (ES) cells. Using the databases for expressed sequenced tags and a two-step PCR procedure, we isolated human NP95, the full-length human homologue of the murine Np95 cDNA, which consists of 4,327 bp with a single open reading frame (ORF) encoding a polypeptide of 793 amino acids and 73.3% homology to Np95. The ORF of human NP95 cDNA is identical to the UHRF1 (ubiquitin-like protein containing PHD and RING domain 1). The NP95 gene, assigned to 19p13.3, consists of 18 exons, spanning 60 kb. Several stable transformants from HEK293 and WI-38 cells that had been transfected with the antisense NP95 cDNA were, like the murine Np95-knockout ES cells, more sensitive to X rays, UV light and hydroxyurea than the corresponding parental cells. In HEK293 cells, the lack of NP95 did not affect the activities of topoisomerase IIalpha, whose expression had been demonstrated to be regulated by the inverted CCAAT box binding protein of 90 kDa (ICBP90) that closely resembles NP95 in amino acid sequence and in cDNA but differs greatly in genomic organization. These findings collectively indicate that the human NP95 gene is the functional orthologue of the murine Np95 gene.     

CLAN, a Novel Human CED-4-like Gene

Proteins governing cell death form the basis of many normal processes and contribute to the pathogenesis of many diseases when dysregulated. Here we report the cloning of a novel human CED-4-like gene, CLAN, and several of its alternatively spliced isoforms. These caspase-associated recruitment domain (CARD)-containing proteins are expressed at varying degrees in normal human tissues and may contribute to a number of intracellular processes including apoptosis, cytokine processing, and NF-κB activation. The CARD of the CLAN proteins binds a number of other CARD-containing proteins including caspase-1, BCL10, NOD2, and NAC. Once their physiologic functions are uncovered, CLAN proteins may prove to be valuable therapeutic targets.     

新vip3Aa基因的克隆、表达及其序列分析

克隆了Bt9816C的vip3A基因,并将测序结果提交到GenBank(序列号:AY945939)。该基因是一个新的vip3Aa基因,Bt杀虫晶体蛋白命名委员会将其命名为vip3Aa18。在大肠杆菌BL21中表达了该基因,生物测定结果表明纯化的Vip3Aa18蛋白对棉铃虫和甜菜夜蛾具有很高的杀虫活性。序列分析结果显示Vip3Aa18C端536至667位氨基酸残基间是一个糖类结合域,推测可能参与Vip3Aa18与敏感昆虫中肠受体结合;N端272至292位氨基酸残基间存在一个跨膜螺旋,可能与Vip3Aa18形成穿孔有关。此外,Vip3Aa18还可能具有一个二硫键。这些特殊区域和位点可能与其功能密切相关。     

柔嫩艾美耳球虫新基因的生物信息学分析与克隆表达

为了研究柔嫩艾美耳球虫(Eimeria tenella)的两个主要入侵发育阶段——子孢子和裂殖子的差异基因,根据已报道的子孢子与裂殖子差异的ESTs序列,应用RACE技术克隆获得了子孢子阶段差异表达的新基因全长cDNA序列,命名为ZL583.该基因全长为862 bp,开放阅读框(ORF)为486 bp,编码161个氨基酸,编码蛋白的分子量约为16.9 kD,利用生物信息学分析软件,分析新基因所编码蛋白的性质、定位、结构等特征.利用荧光定量PCR对柔嫩艾美耳球虫不同发育阶段该基因的表达量进行分析显示,子孢子阶段的表达高于其他发育阶段.将ZL583克隆于pET-28a中构建重组质粒,转化大肠杆菌BL21(DE3)后经IPTG诱导表达,获得重组蛋白分子量约23 kD.免疫印迹分析显示该重组蛋白可与兔抗子孢子血清发生特异性反应,表明该蛋白具有较好的反应原性.该结果为进一步研究该基因的生物学功能奠定基础.     

蜂房哈夫尼菌植酸酶基因appA的克隆、表达及酶学性质研究

从蜂房哈夫尼菌(Hafniaalvei)中克隆获得一个植酸酶编码基因appA,该基因全长1335bp,编码444个氨基酸,其中前33个氨基酸为信号肽,成熟蛋白的理论分子量为45.2kD。将基因appA克隆到大肠杆菌E.coli表达载体pET-22b( ),并在大肠杆菌中表达,表达产物具有植酸酶活性。对表达的酶蛋白进行纯化,并初步研究了该酶的酶学性质,结果表明:酶的作用最适pH值为4.5;在pH2.0~10.0范围内,酶活性保留80%以上;酶的作用最适温度为60℃;酶的比活性为356.7U/mg,酶动力学分析表明其Km为0.49mmol/L,Vmax为238U/mg;该酶对胰蛋白酶和胃蛋白酶有一定的抗性。该研究为哈夫尼菌属来源植酸酶的首次报道。     

Identification and Functional Expression of HCx31.9, a Novel Gap Junction Gene

By combining in silico and bench molecular biology methods we have identified a novel human gap junction gene that encodes a protein designated HCx31.9. We have determined its human chromosomal location and gene structure, and we have identified a putative mouse ortholog, mCx30.2. We have observed the presence of HCx31.9 in human cerebral cortex, liver, heart, spleen, lung, and kidney and the presence of mCx30.2 in mouse cerebral cortex, liver and lung. Moreover, preliminary data on the electrophysiological properties of HCx31.9 have been obtained by functional expression in paired Xenopus oocytes and in transfected N2A cells.     

The Pathogenic Properties of a Novel and Conserved Gene Product,KerV, in Proteobacteria

Identification of novel virulence factors is essential for understanding bacterial pathogenesis and designing antibacterial strategies. In this study, we uncover such a factor, termed KerV, in Proteobacteria. Experiments carried out in a variety of eukaryotic host infection models revealed that the virulence of a Pseudomonas aeruginosa kerV null mutant was compromised when it interacted with amoebae, plants, flies, and mice. Bioinformatics analyses indicated that KerV is a hypothetical methyltransferase and is well-conserved across numerous Proteobacteria, including both well-known and emerging pathogens (e.g., virulent Burkholderia, Escherichia, Shigella, Vibrio, Salmonella, Yersinia and Brucella species). Furthermore, among the 197 kerV orthologs analyzed in this study, about 89% reside in a defined genomic neighborhood, which also possesses essential DNA replication and repair genes and detoxification gene. Finally, infection of Drosophila melanogaster with null mutants demonstrated that KerV orthologs are also crucial in Vibrio cholerae and Yersinia pseudotuberculosis pathogenesis. Our findings suggested that KerV has a novel and broad significance as a virulence factor in pathogenic Proteobacteria and it might serve as a new target for antibiotic drug design.     

血吸虫新抗原基因SjMF4的克隆、表达及功能分析

根据东方田鼠天然抗日本血吸虫病的现象 ,首次利用东方田鼠健康血清结合羊抗小鼠IgG3抗体免疫筛选日本血吸虫 (中国大陆株 )成虫cDNA表达型文库 ,获得两个阳性克隆 ,采用RACE技术对其中一cDNA片段进行扩增 ,获一含ORF的基因片段。序列分析表明该基因为一日本血吸虫新基因 ,命名为SjMF4 (SchistosomajaponicumMicrotusfortis 4 ,SjMF4 )。利用ExPASy的ScanProsite软件对此基因编码的蛋白质结构和功能域进行了分析。把该基因克隆到原核表达载体pET 2 8a( ) ,Western印迹显示表达产物具有良好的抗原性。又构建了真核表达质粒pcD NA3 SjMF4重组DNA疫苗 ,小鼠实验表明可诱导一定的保护作用。     

Identification of a Novel Garlic Cellulase Gene

Genes encoding cellulase enzymes have been investigated in various plants due to the importance of cellulase enzymes in industrial applications, especially in the conversion of biomass into biofuels. Although several cellulase genes have been cloned and characterized, little is known about cellulase genes from garlic or enzyme activities of their gene products. In this study, a cellulase gene from garlic was cloned and characterized in gene and protein levels for the first time. The DNA sequence of the garlic cellulase gene showed 81% identity with the sequence of the endo-beta-1,4-glucanase of Pisum sativum. The open reading frame of this gene is 1,506 bp, which corresponds to 501 deduced amino acids. We identified the novel ORF region, which was translated into a 55.2 kDa protein using the protein expression vector, pET28a, in Escherichia coli and we confirmed that this protein has cellulase activity in vitro. Our study demonstrates that garlic is very useful, not only for the culinary and pharmaceutical industries, but also as an excellent natural source of various kinds of important genes and enzymes.