水稻组蛋白H_3基因的分离和组织结构分析

用噬菌斑原位杂交法,从一个水稻新品系的EMBL,基因文库中筛选到了两个组蛋白H,基因克隆,并制作了它们的限制性内切酶图谱,分析了它们的组织结构。其中λRH_3-1克隆包含2个组蛋白H_3基因区(分别为0.5kb和0.8kb的BamHI-EcoRI片段,两基因区距离5.8kb)。而λRH_3-2克隆只带有1个结构特异的H_3基因区(5.2kb的EcoRI-HindⅢ片段)。本文还就水稻组蛋白H_3基因在整个基因组中的组织结构特点进行了讨论。     

cosmid克隆筛选的体内同源重组法——一个含有小鼠t复合体连锁DNA顺序的cosmid克隆的分离

一个从cosmid分子克隆库中筛选特别基因顺序的遗传学方法——体内同源重组(invlvo homologous recombination)法。即使探针DNA与分子克隆库中带有与探针同源顺序的克隆发生体内重组,然后以遗传学方法进行筛选。cosmid分子克隆库构建在rec宿主细胞内,经体内包装(in vivo Packaging)成λ噬菌体颗粒,把该噬菌体颗粒转入带有探针DNA的rec~+细胞内,探针是已被克隆在与cosmid载体没有同源顺序的质粒(如PUC8或PUC9)内的。经过一段时间(1—3小时),待重组发生后,把cosmid进行体内包装。此时探针DNA连同质粒已整合入cosmid基因组内,因此它带有原为两个载体所分别带有的双重抗性——Amp~r(氨苄青霉素,PUC8或PUC9)和Kan~r(卡那霉素,cosmid)。这种双重抗性菌落可在含有这2种抗菌素的培养平皿上选出,该重组cosmid借助于λ切除酶的作用将已被整合的探针质粒重新切除,再经体内包装后,该cosmid被还原并纯化,然后可用一含有Xgal的培皿识别和选出。本文用此法以有关DNA探针从cosmid分子克隆库中分离得到含有与小鼠t复合体连锁的基因组顺序的克隆,并对该克隆作了物理图谱分析。     

人胃癌细胞株BGC-823 DNA二轮转化细胞基因组文库的构建及其转化基因的克隆

以人胃癌细胞株BGC-823 DNA二轮转化的鼠成纤维细胞为材料,用λ噬菌体EMBL_3,作克隆载体,构建了转化细胞的基因组文库。用~(32)P标记的人Alu重复顺序和原癌基因c-Ha-ras为探针,对100万基因文库噬斑进行原位杂交筛选,找出了两个可以同时与上述探针杂交的阳性克隆。经对两个阳性克隆DNA进行分子杂交表明,它们均含有来自人胃癌细胞株BGC-823的转化基因Ha-ras,进一步运用质粒载体pBR322对这一转化基因进行次级克隆,并进行了限制性内切酶图谱的初探,从而为研究胃癌转化基因的结构、DNA序列及与原癌基因的异同奠定了基础。     

抗乙型脑炎病毒单抗重链可变区基因的筛选和鉴别

为了制备抗乙型脑炎病毒的人-鼠嵌合抗体,以分泌抗乙型脑炎病毒单抗的51-8杂交瘤细胞株为材料,分离这一单抗的重链可变区基因。杂交瘤细胞的大分子量DNA经Bam HI部分酶切后,以λEMBL-3为载体,构建了总数为2×10~7pfu的基因文库。以抗乙脑病毒单抗重链可变区cDNA为探针,从360000个噬菌斑中筛选出9个阳性斑,经点杂交及Southern杂交证明它们都含有重链可变区基因的片段。进一步以J_(11)探针(含J_3、J_4和重链增强子)对其中4个重组体进行鉴别。经EcoRI酶切后,有3个重组体含有与肝细胞和Sp2/0细胞相同的3.8kb片段,而第4个重组体(λ8a4)没有这一片段,却有一个4.5kb片段,这是在肝细胞和Sp2/0细胞中不存在的。从而证明前3个重组体的插入片段是未经重排的重链可变区基因片段,而λ8a4中的插入片段含有经过重排的功能性可变区基因。这一4.5kb片段不能与含有J_1—J_2的探针杂交,却同时含有V_H、J,或/和J_4和增强子。进一步证明,这是一个功能性的可变区基因。因此,将这一4.5kb片段分离出来之后,在pUC19中亚克隆并作酶切图。为构建抗乙脑病毒的人-鼠嵌合重链基因奠定了基础。     

体内遗传标记成团肠杆菌固氮质粒pEA9

用卡那霉素抗性（Kan^r)基因对成团肠杆菌固氮质粒pEA9进行活体遗传标记。将来自质粒pEA9的3.0kb片段(nif ENX)克隆到pBR322载体中,再将卡那霉素抗性(Kan^r)基因插入到3.0kb的片段中,构建成供体质粒pST5。将该质粒转化到含有待标记质粒pEA9的E.a.339菌株中,然后在AP培养基中消除供体质粒,筛选得到40个失去了pST5并保持卡那霉素抗性的克隆,分析表明它们不是质粒pEA9和pST5的共整合体,而是卡那霉素抗性基因通过两个质粒在nifENX区域内的DNA间的同源重组整合到了质粒pEA9上。     

果蝇组蛋白去乙酰化酶HDAC1和HDAC3选择性调控形态发生素靶基因转录

在真核细胞中,组蛋白的乙酰化状态对于基因转录的正常进行具有重要的调控作用。组蛋白的乙酰化修饰由组蛋白乙酰转移酶(histone acetyltransferases,HATs)执行,这种修饰是动态的、可逆的,负责去乙酰化修饰的酶是组蛋白去乙酰化酶(histone deacetylases,HDACs),推测HDACs可能通过影响组蛋白的乙酰化状态在基因的转录过程中发挥调控作用。该文以组蛋白去乙酰化酶HDAC1和HDAC3为对象,研究了它们在果蝇翅膀发育过程中对Wg(Wingless)、Hh(Hedgehog)以及Dpp(Decapentaplegic)信号通路下游靶基因转录的调控作用。结果发现,HDAC1功能缺失可导致Dpp下游靶基因Omb(optomotor-blind)和Hh下游靶基因Ptc(patched)的表达上调。Real-time quantitative PCR(RT-q PCR)结果显示,在HDAC1基因敲除的果蝇中,Ptc、Ci(cubitus interruptus)以及Omb的转录水平增加。HDAC3缺失导致Sal(spalt)的表达上调。RT-q PCR结果证实了HDAC3基因敲除果蝇的Sal转录增加,同时发现Vg(vestigial)的转录下降。而过表达HDAC1或HDAC3对下游靶基因的表达则没有影响。综上所述,该研究表明,HDAC1和HDAC3可以选择性地调控形态发生素下游靶基因的转录。     

猪基因组文库的构建及其生长激素基因的分离

本工作以长白猪为材料,分别用Charon28及EMBL3为载体,构建了猪的基因组文库。用牛生长激素基因为探针对基因文库进行筛选,由两个基因文库中各获得一个阳性克隆λPGH1,λPGH2。其后,以质粒pUCl9为载体对λPGHl进行了亚克隆。通过对亚克隆pPGH的酶切图谱及其Southern杂交结果的分折表明,在pPGH中含有完整的猪生长激素基因。     

马铃薯甲虫两个系统性RNA干扰缺失基因cDNA的克隆、序列分析和时空表达

【目的】克隆马铃薯甲虫Leptinotarsa decemlineata(Say)两条Sid-1基因的全长并分析其时空表达。【方法】本研究通过RT-PCR和5'/3'RACE等技术从马铃薯甲虫中克隆全长序列,通过序列多重比对和系统发育分析研究序列的保守性和基因起源,通过阶段收样,组织解剖和qPCR技术获得这两个基因的时空表达。【结果】从马铃薯甲虫Leptinotarsa decemlineata的4龄幼虫中克隆得到LdeSid-1a和LdeSid-1c,其m RNA全长为2 887和3 733 bp,编码759和782个氨基酸,分子量为86.19和90.27 ku,两条蛋白质序列的相似性为59%。与其它昆虫的系统性RNA干扰缺失基因的比对结果显示,LdeSid-1a和LdeSid-1c分属于鞘翅目系统性RNA干扰缺失基因的两个分支,均具有典型的11个跨膜域结构,在蛋白质序列的N端具有4段高度保守的基序。qPCR的时序分析表明LdeSid-1a和LdeSid-1c的表达从初孵幼虫开始逐渐上升,而LdeSid-1c在卵中的表达也较高,组织中的分布结果表明,两个基因在所有组织中均表达,在前肠、中肠和后肠以及生殖系统中表达较高,在神经系统中为优势表达。LdeSid-1a和LdeSid-1c的Gen Bank登录号为KR153284和KR153285。【结论】LdeSid-1a和LdeSid-1c具有典型的系统性RNA干扰缺失基因家族的结构,时空表达和系统发育的结果均表明这两条基因可能在幼虫的高龄阶段起到重要作用。     

东亚飞蝗fem-1基因的克隆与表达分析

秀丽隐杆线虫Caenorhabditis elegans fem-1基因是性别决定的关键基因。本研究基于生物信息学方法从东亚飞蝗Locusta migratoria manilensis的转录组数据库中克隆出了线虫fem-1的3个同源基因, 将其分别命名为Lmfem-1a, Lmfem-1b和Lmfem-1c (GenBank登录号分别为AB698670, AB698671和AB698672)。其cDNA序列长度分别为2 233, 2 625和2 142 bp, 分别编码662, 642和638个氨基酸。生物信息学分析显示, Lmfem-1a, Lmfem-1b和Lmfem-1c分别含有6, 8和8个典型的锚蛋白重复序列模体。组织表达谱分析发现, Lmfem-1a, Lmfem-1b和Lmfem-1c基因在检测的所有组织中都有表达, 但均在精巢中的表达水平最高, 说明Lmfem-1a, Lmfem-1b和Lmfem-1c基因可能参与东亚飞蝗的多种生理过程, 并受到严格的表达调控。而且, 随着精巢的发育, Lmfem-1a, Lmfem-1b和Lmfem-1c的表达均逐渐增强, 可能与东亚飞蝗的精子形成有关, 但这3个基因是否参与东亚飞蝗的性别决定还有待进一步研究。     

从江香猪λ1干扰素基因的克隆及序列分析

[目的]对从江香猪λ1干扰素(interferon-λ1,IFN-λ1)基因进行扩增、克隆和生物信息学分析。[方法]根据Gen Bank登录的猪IFN-λ1基因序列(FJ455508)设计合成特异性引物,通过RT-PCR从淋巴细胞中扩增IFN-λ1基因CDS区并进行克隆和生物信息分析。[结果]从江香猪IFN-λ1基因CDS全长576 bp,共编码191个氨基酸,其分子式为C949H1543N277O270S7,相对分子质量21.38 k Da;该蛋白等电点为9.42,为亲水性蛋白。从江香猪IFN-λ1含有丰富的二级结构,以α-螺旋(64.40%)和无规卷曲(25.65%)为主,蛋白质三级结构中主要以α-螺旋为主,同源性及系统进化树分析结果表明从江香猪与野猪IFN-λ1核苷酸同源性最高(为100%),亲缘关系最近。[结论]从江香猪IFN-λ1基因的克隆和生物信息学分析为进一步研究其抗病毒功能奠定了基础。     

单酶切cDNA-AFLP改良法分离草菇冷诱导基因

采用单酶切cDNA-AFLP改良法克隆分离了十条草菇冷诱导基因片段VC7、VC8、VC9、VC10、VC11、VC12、VC13、VC14、VC15、VC16。DNA序列测定结果表明,克隆分离的十条DNA片段分别为504bp、549bp、555bp、301bp、189bp、602bp、605bp、834bp、703bp、1085bp。同源性BLAST程序搜索结果显示,除VC14与粗糙脉孢菌Neurosporacrassa的钙磷型ATP酶基因有较高的同源性外,其余九条DNA片段目前都没有搜索到同源的核酸序列;而其翻译的蛋白质序列,除了VC11和VC15目前未能搜索到同源的蛋白质序列外,其余八条DNA片段翻译的蛋白质序列与粗糙脉孢菌Neurosporacrassa、裂殖酵母Schizosaccharomycespombe、链霉菌Streptomycesavermitilis等的有关蛋白质序列具有较高的同源性。     

Role of intracellular pH in muscle fatigue

Intracellular pH of in vitro diaphragm preparations was determined following low- (5 Hz, 1.5 min) and high- (75 Hz, 1 min) frequency stimulation, using glass microelectrodes of the liquid membrane type (pHm). Results were compared with values obtained by the standard homogenate technique (pHh). High- and low-frequency stimulation reduced peak tetanic tension to 21 +/- 1 (SE) and 71 +/- 2% of initial values, respectively. Peak tetanic tension returned to resting values after 10- to 15-min recovery from high- or low-frequency stimulation. Resting pHm was 7.063 +/- 0.011 (n = 72), and after fatiguing stimulation declined to values as low as 6.33. During recovery pHm significantly increased and by 10 min had returned to prefatigue values. No difference was observed in the recovery of pHm between the low- and high-frequency stimulation groups (analysis of variance test, ANOVA), and in both groups pHm recovery was highly correlated to the recovery of peak tetanic tension (r = 0.94, P less than 0.001). Resting pHh was 7.219 +/- 0.023 (n = 13), which was significantly higher than the pHm value. In contrast to pHm, intracellular pHh was significantly higher during recovery from 75- vs. 5-Hz stimulation (P less than 0.05). For both groups pHh increased significantly with time and by 10 min returned to prestimulation values. The ANOVA test demonstrated that pHh values were significantly higher than pHm values during recovery from fatigue. The results from this study support our hypothesis that fatigue from both high- and low-frequency stimulation is at least partially due to the deleterious effects of intracellular acidosis on excitation-contraction coupling.     

Cell cycle dependent genes inducible by different mitogens in cells from different species

Summary A number of genes and cDNA sequences (including at least four oncogenes) are known to be expressed in a cell cycle-dependent manner, i.e. the levels of specific mRNAs vary with the phases of the cell cycle. In order to explore the significance of some of these sequences in the mitogenic response, we have investigated the expression of 8 cell cycle-dependent sequences (plus two control sequences, not expressed in a cell cycle-dependent manner) under a variety of conditions. These conditions included cells of different types, from different species, stimulated to proliferate by different mitogens. The genes (or sequences) studied included five cDNA clones whose sequences are preferentially expressed in early G₁, i.e. two cDNA clones inducible by platelet-derived growth factor (JE-3 and KC-1), and three cDNA clones inducible by serum (2A9, 2F1, 4F1); and three oncogenes (c-myc, c-ras^Ha and p53) whose expression is known to be cell cycle-dependent. All of the tested genes, except 2A9, c-ras^Ha and the control genes, are expressed in a cell cycle-dependent manner in human peripheral blood mononuclear cells stimulated by phytohemagglutinin and in serum-stimulated mouse and Syrian hamster fibroblasts. The inducibility of these genes by different mitogens in cells of different types and from different species strongly suggests that these genes play a role in cell cycle progression. This conclusion is further supported by the known structural and functional similarities between cell-cycle dependent genes, oncogenes and genes coding for cell-cycle related molecules.     

Primary structure, regioselectivity, and evolution of the membrane-bound fatty acid desaturases of Claviceps purpurea

Two cDNAs with sequence similarity to fatty acid desaturase genes were isolated from the phytopathogenic fungus, Claviceps purpurea. The predicted amino acid sequences of the corresponding genes, named CpDes12 and CpDesX, share 87% identity. Phylogenetic analysis indicates that CpDes12 and CpDesX arose by gene duplication of an ancestral Delta(12)-desaturase gene after the divergence of Nectriaceae and Clavicipitaceae. Functional expression of CpDes12 and CpDesX in yeast (Saccharomyces cerevisiae) indicated that CpDes12 is primarily a "Delta(12)"-desaturase, whereas CpDesX is a novel desaturase catalyzing "Delta(12)," "Delta(15)," and "omega(3)" types of desaturation with omega(3) activity predominating. CpDesX sequentially desaturates both 16:1-9c and 18:1-9c to give 16:3-9c,12c,15c and 18:3-9c,12c,15c, respectively. In addition, it could also act as an omega(3)-desaturase converting omega(6)-polyunsaturates 18:3-6c,9c,12c, 20:3-8c,11c,14c, and 20:4-5c,8c,11c,14c to their omega(3) counterparts 18:4-6c,9c,12c,15c, 20:4-8c,11c,14c,17c, and 20:5-5c,8c,11c,14c,17c, respectively. By using reciprocal site-directed mutagenesis, we demonstrated that two residues (isoleucine at 152 and alanine at 206) are critical in defining the catalytic specificity of these enzymes and the C-terminal amino acid sequence (residues 302-477) was also found to be important. These data provide insights into the nature of regioselectivity in membrane-bound fatty acid desaturases and the relevant structural determinants. The authors suggest that the regios-electivity of such enzymes may be best understood by considering the relative importance of more than one regioselective preference. In this view, CpDesX is designated as anu + 3(omega(3)) desaturase, which primarily references an existing double bond (nu + 3 regioselectivity) and secondarily shows preference for omega(3) desaturation.     

梅花鹿源BVDV基因E0克隆与序列分析

对梅花鹿源BVDV基因E0进行了克隆和序列分析。结果表明,梅花鹿源BVDV基因E0的大小为681bp,与报道9株BVDV（VEDEVAC、Bega、C24V、ILLC、NADL、OSLOSS、R1935、SD-1、Y546）和7株猪瘟病毒（ALD、Bres-cia、c、GPE、JL、LN9912、SM）及3株羊边界病毒（BD31、C413、BDVX818）相比,核苷酸序列的同源性依次为98.6%～84.8%、76.1%～74.7%、77.0%～76.7%。梅花鹿源BVDV为Ιb基因亚型。     

Characterization of recognition sites on bacteriophage HP1c1 DNA which interact with the DNA uptake system of Haemophilus influenzae Rd

The 32.4-kb genome of the Haemophilus influenzae bacteriophage HP1c1 contains at least twelve sites, each conferring high affinity for the DNA uptake system of transformable H. influenzae Rd. Five of these high-affinity sites have been located and their nucleotide sequences determined. Three sites contained a contiguous 9-bp sequence identical to the first nine residues of the 11-bp site previously identified as conferring high affinity for the H. influenzae transformation receptor to DNA fragments. The remaining two sites contained complete 11-bp sequences. In contrast, an HP1c1 restriction fragment containing a sequence identical to the final nine residues of the 11-bp uptake site exhibits only a low affinity for the DNA uptake system. An 8-bp sequence consisting of the first eight residues of the 11-bp site was 1% as active as the longer, high-affinity sites. Thus the first 9-bp of the 11-bp site are sufficient to direct high-affinity uptake, while the first 8-bp or the distal 9-bp are not. These results provide an initial assessment of the relative contributions of the individual residues constituting the 11-bp site to the apparent affinity of DNA fragments for the receptor of Haemophilus transformation.     

GP37蛋白结构分析与昆虫病毒分子进化的关系

The gp37 gene from LsMNPV has been sequenced and the deduced amino acid sequence was compared with other GP37 amino acid sequences from 8 insect viruses. The maximum homology of amino acid sequences and the conserved structural regions were analyzed with PROSIS software. The relationship of evolution of 9 insect viruses was discussed and the evolutionary tree was drawned.     

斯氏鼢鼠物种地位有效性的探讨

为了探讨斯氏鼢鼠的物种地位,分别测得了4个斯氏鼢鼠、3个高原鼢鼠、2个秦岭鼢鼠个体的线粒体细胞色素b基因和12SrRNA基因的全序列,结合下载自Gen Bank中的鼢鼠序列,利用MEG A4.0,PH YLIP 3.57c软件包和Mrbayes 3.1.2软件以中华竹鼠做外群分别重建鼢鼠亚科的系统发育关系,并比较了采自3个地区的斯氏鼢鼠、高原鼢鼠和秦岭鼢鼠的头骨形态学特征。分子生物学研究结果支持斯氏鼢鼠的物种地位,且与秦岭鼢鼠和高原鼢鼠的亲缘关系较近。