施氏假单胞菌A1501铁吸收调节蛋白Fur的表达特性与功能鉴定

铁是细胞生理代谢的重要金属元素,是多种氧化还原酶类、过氧化物酶类的重要组成部分。革兰氏阴性菌中,胞内铁转运、储存和利用主要由铁吸收调节蛋白Fur（ferric uptake regulator）调控。水稻根际联合固氮菌——施氏假单胞菌（Pseudomonas stutzeri）A1501基因组中含有一个fur同源基因,但其具体功能尚不清楚。比较了A1501中fur基因及其编码蛋白的序列特征,并通过实时荧光定量PCR（real-time quantitative,qRT-PCR）和Western blotting杂交方法测定了fur基因和Fur蛋白的表达特性,结果发现fur基因表达水平与环境中铁浓度呈负相关,在铁限制条件下高表达,铁过量条件下表达受抑制。构建了fur基因突变株和功能回补株,表型测定发现fur突变影响了A1501的碳源代谢谱,并导致A1501对过氧化氢胁迫敏感。胞内铁含量测定表明,fur突变株内的铁含量为野生型的1.3倍,进一步qRT-PCR测定表明fur突变影响了铁离子转运系统及过氧化物酶编码基因的表达。研究结果为解析根际微生物胞内铁平衡调节及根际环境适应机制奠定了理论基础。     

匍枝根霉纤维二糖合成酶胞内糖基供体的初探及结构功能研究

纤维二糖可有效诱导丝状真菌产纤维素酶,前期研究表明匍枝根霉Rhizopus stolonifer TP-02具有纤维二糖合成酶(CBS),可以尿苷二磷酸葡萄糖(UDPG)为糖基供体合成纤维二糖,从而开启纤维素酶的自诱导合成途径。为研究R. stolonifer中纤维二糖的胞内合成途径,通过重叠PCR在GDP-葡糖焦磷酸化酶基因ggp中引入硫胺吡啶抗性基因ptrA,分别转化原菌TP-02和△ugp突变株,构建△ggp和△ugp/△ggp突变株。利用液质联用(LC-MS)检测突变株的胞内糖组分,发现ggp的缺失对胞内纤维二糖合成的影响较弱,但同时缺失ugp则将直接导致二糖合成受阻。RT-qPCR结果显示△ggp突变株中纤维素酶基因转录水平较原株TP-02下调20%左右,而△ugp/△ggp突变株中被测基因的转录水平则出现了高达80%左右的下调。同时对突变株纤维素酶表达水平进行研究,发现△ugp/△ggp突变株中几乎检测不到纤维素酶活力。结果显示,UDPG为R. stolonifer胞内合成纤维二糖的主要糖基供体,而GDPG可能是UDPG的替代物,在UDPG不足时维持胞内二糖合成。此外,利用生物信息学方法对CBS结构功能深入分析,经丙氨酸扫描确定其合成纤维二糖的关键作用残基为Asp210和Asp300,为后续进一步研究及理性改造提供方向和理论依据。     

上皮细胞极性的建立和维持

细胞极性是生物中广泛存在的一个特征。上皮细胞是构成表皮、腺体、气管和消化道等组织的一类特化细胞。上皮细胞通常沿顶端-基底端轴向发生极化,形成紧密连接、粘附连接等胞间结构,同时细胞膜、细胞骨架和中心体、内膜系统、细胞核等也发生不对称分布,使细胞能行使分泌、吸收和屏障等多种重要的生理功能。有许多分子参与上皮细胞极性的建立和维持,其中最主要的是3个极性复合物,即Par-aPKC复合物,Scribble(Lg1-Dlg-Scrib)复合物和Crb(Crb-Pals-PATJ)复合物,三者共同配合发挥功能。     

七鳃鳗KCTD基因家族成员全基因组鉴定及进化分析

钾离子通道四聚化结构域(KCTD)蛋白基因家族是一个保守的基因家族,该家族成员的共同特征是具有一个含有BTB保守结构域的N-末端和一个可变的C-末端。KCTD基因的突变或不正常调控与人类多种疾病相关。七鳃鳗是现存最原始的脊椎动物,作为联系无脊椎动物和脊椎动物之间的桥梁,在生物进化研究中占有重要地位。本研究通过对海七鳃鳗(Petromyzon marinus)和日本七鳃鳗(Lethenteron japonicum)基因组和转录组数据分析,全面系统地鉴定了海七鳃鳗和日本七鳃鳗KCTD基因家族成员,并对其基因结构特征、蛋白保守基序和基因表达模式进行了分析。在海七鳃鳗和日本七鳃鳗中分别鉴定出13个和14个KCTD基因,基因长度和外显子数目在不同KCTD基因间变化很大,KCTD蛋白中4个基序保守性显著,大多数KCTD基因呈泛表达模式,并且在胚胎发育时期明显高表达。除七鳃鳗外,对12个无脊椎动物和脊椎动物代表物种KCTD基因家族成员进行了鉴定,并对KCTD基因家族成员的进化关系进行了分析。根据进化树聚类情况,将KCTD基因家族成员分为11个亚家族。进化分析结果显示,KCTD基因家族从低等的无脊椎动物线虫和果蝇到高等的人类都存在;线虫中仅有5个成员,果蝇中有8个成员,随着物种进化程度由低到高,KCTD家族成员数目呈现增加的趋势;从爬行类开始,脊椎动物KCTD基因数目稳定在24个左右。硬骨鱼类特有的全基因组复制事件影响鱼类KCTD基因数目。本研究结果不仅丰富了七鳃鳗KCTD基因家族信息,同时也对KCTD家族基因间的进化关系进行了探究,为深入研究该家族基因功能提供了一定的依据。     

普通烟草CESA基因家族成员的鉴定、亚细胞定位及表达分析

纤维素合成酶蛋白(cellulose-synthase proteins, CESA)是一类质膜定位蛋白,以蛋白复合体的形式存在于质膜上合成纤维素,在细胞壁建成和植物生长发育过程中起着非常重要的作用。本研究利用CESA蛋白保守域序列PF03552检索普通烟草(Nicotiana tabacum L.)蛋白序列,并通过拟南芥(Arabidopsis thaliana)10个CESA蛋白序列在普通烟草基因组数据库中利用TBLASTN程序进行比对,共获得21条NtCESA基因候选序列,对这些序列进行蛋白序列理化性质分析、系统进化树构建、基因结构分析、保守结构域及跨膜区分析和组织表达模式分析,并对NtCESA9和NtCESA14两个蛋白进行了亚细胞定位实验。结果表明：获得的21条NtCESA蛋白序列的理化性质相似;系统进化分析将21个NtCESA基因和10个AtCESA基因分成5个分支,每一个分支各成员之间的进化相对保守,基因结构类似,不同分支之间的基因结构差异也较小;NtCESA蛋白结构域相对保守,都含有CESA蛋白典型的N端锌指结构、C端跨膜区和DDD-QXXRW保守功能域;组织表达分析结果表明,大部分NtCESA基因在幼苗和成熟期烟草的根、叶、胚芽和愈伤组织中都有表达,同一个分支中的基因表达模式基本一致,并且NtCESA基因参与初/次生细胞壁纤维素的合成与该基因编码蛋白的跨膜区数目存在关联,表明NtCESA基因家族成员功能上的复杂性;亚细胞定位结果证实NtCESA9和NtCESA14为质膜定位蛋白。本研究为烟草CESA基因家族功能的深入研究奠定了基础。     

浙江地区青霉素耐药肺炎链球菌PBPs基因及氨基酸序列研究

为阐明温州地区青霉素耐药肺炎链球菌（PRSP）的青霉素结合蛋白（PBPs）的基因和氨基酸序列的变异特点,对温州医学院自2000年11月～2004年1月收集的26份肺炎链球菌进行分离、鉴定及青霉素药敏实验,并对每株链球菌的PBP1A、PBP2B、PBP2X基因进行PCR扩增和直接测序,通过序列比对与生物信息学分析。结果表明,研究中的PBP1A的主要突变位点是保守基序KTG之后的4个连续氨基酸替换Thr574Ala、Ser575Thr、Gln576Gly、Phe577Tyr和保守序列STMK内的氨基酸替换Thr371Ser;PBP2B的主要突变位点是保守序列SSN之后的氨基酸替换Thr451Ala;PBP2X的主要突变位点是保守基序STMK 内的氨基酸替换Thr338Ala。以上突变类型以及菌株的青霉素耐药水平与文献报道相符。研究检测的PRSP的PBPs基因中暂未发现本地区特有的（新的）基因突变,也未检测出文献报道的某些与青霉素抗性相关的氨基酸替换。     

HPV基因的进化分析

目的:HPV有许多类型,其大致可分为高危型和低危型,高危型HPV感染是导致宫颈癌发生的首要原因,在HPV基因组中,E6基因是促进宫颈细胞癌变的关键基因,本文主要研究HPV中的E6基因在各种不同型别的HPV中的进化关系,并对E2基因碱基替换率进行分析,探讨高危型HPV与低危型HPV的区别.方法:本文对不同类型HPV E6氨基酸序列构建系统发生树,探讨识别高危型HPV可能的一致序列,对E6基因其中一处能导致恶性程度增加的突变进行分析.并对HPV16与其位于同一颗树的HPV35和HPV31计算相对碱基替换率.结果:高危型HPV均源自同一株病毒株的进化.各种HPV型别中,高危型HPV E6蛋白对应于HPV16E6蛋白的第83位氨基酸为缬氨酸更为保守,HPV中除E2以外的其他基因的非同义替换率均小于同义替换率.结论:HPV E6蛋白对应于HPV16E6蛋白的第83位氨基酸为缬氨酸能更好地实现HPV E6蛋白的致癌作用.HPV基因中除E2以外的基因在进化过程中都较为保守,是HPV增殖生长的关键基因,而E2部分区域非同义替换率大于同义替换率,说明E2这部分区域的突变能够更好的促进HPV的增殖和生长.     

非典型蛋白激酶C复合物在细胞极性建立中的作用

极性是多数细胞的共同特征,是细胞分化和细胞行使正常功能的基础,细胞极性的建立对于生物体的生长发育至关重要。过去十年的研究显示,进化上保守的非典型蛋白激酶C(aPKC)复合物在许多生物的多种细胞中都参与了细胞极性的建立,并且在其中扮演着相当重要的角色,这为揭示极性建立的机制提供了重要的线索。以线虫合子前-后极(anterior-posterior)的形成、哺乳动物和果蝇上皮细胞顶-底极(apical-basal)的建立以及果蝇神经母细胞不对称分裂中细胞命运决定子的分配这3个典型的极性过程为主线,综述了aPKC复合物在细胞极性建立中的作用,并探讨其中的分子机制。     

大仓鼠β2肾上腺素受体基因的套叠PCR法多点突变研究

对大仓鼠β1肾上腺素受体蛋白(β2 adrenergic receptor,β2AR)进行立体结构与疏水性分析.利用套叠,PCR法对β2AR基因进行多点突变,将保守跨膜域疏水氨基酸突变为正电荷亲水氨基酸,成功构建分别带有7个与3个突变氨基酸的原核突变表达载体.探讨了套叠,PCR的反应条件,为β2AR基因在体外的高量表达提供基础.     

水稻ECT基因家族的全基因组研究

真核生物mRNA转录后修饰可调控许多基因的遗传信息,植物m⁶A甲基化研究正成为关注的新热点。m⁶A结合蛋白 (m⁶A readers) 调节m⁶A修饰的特异性,通常具有YTH (YT521-B homology) 结构域,在拟南芥中被命名为ECT结构域 (evolutionarily conserved C-terminal region ECT domain) 。目前ECT基因已在拟南芥和水稻等植物中检测到,但该基因家族在水稻中的成员及生物学功能还缺乏研究。本研究通过水稻ECT基因家族的全基因组分析,鉴定出12个OsECT基因,具有1个保守的基序,多位于蛋白质氨基酸序列C-端。共线性分析表明,在水稻基因组内OsECT-c与OsECT-e发生了重复事件,在物种间ECT同源基因对可能是在双子叶和单子叶植物分化后形成。同源基因对OsECT的Ka/Ks < 1,表明OsECT基因家族在进化过程中可能经历了较强的纯化选择压力。表达模式分析显示,OsECT-b、OsECT-c、OsECT-e和OsECT-j在水稻生长初期各个组织均保持较高的表达水平,OsECT-g在干旱处理后表达量显著下调。因此,OsECT基因在水稻生长发育和逆境胁迫中可能发挥着重要作用。本研究为今后OsECT基因在水稻的节水抗旱机制研究和相关抗逆育种提供了重要的理论基础。     

The expression pattern of a mouse doublesex-related gene is consistent with a role in gonadal differentiation

The signal for somatic sex determination in mammals, Caenorhabditis elegans and Drosophila melanogaster is chromosomal, but the overall mechanisms do not appear to be conserved between the phyla. However it has been found quite recently that the C. elegans sex-determining gene Mab-3 contains a domain highly homologous to the Drosophila sex-determining gene doublesex (dsx) and shares a similar role. These data suggest that at least some aspects of the regulation of sex determination might be conserved. In humans, a doublesex-related gene (DMRT1) was identified at less than 30 kb from the critical region for sex reversal on chromosome 9p24 (TD9). In order to get insights into the role of DMRT1 in sex determination/differentiation, we have isolated DMRT1 mouse homologue (Dmrt1) and analysed its expression pattern. The gene is expressed in the genital ridges of both sexes during the sex-determining switch and it shows male/female dimorphism at late stages of sex differentiation.     

Identification and characterization of a serine hydroxymethyltransferase isoform in Caenorhabditis briggsae

In the nematode Caenorhabditis elegans, the maternal effect lethal gene mel-32 encodes a serine hydroxymethyltransferase isoform. Since interspecies DNA comparison is a valuable tool for identifying sequences that have been conserved because of their functional importance or role in regulating gene activity, mel-32(SHMT) genomic DNA from C. elegans was used to screen a genomic library from the closely related nematode Caenorhabditis briggsae. The C. briggsae genomic clone identified fully rescues the Mel-32 phenotype in C. elegans, indicating functional and regulatory conservation. Computer analysis reveals that CbMEL-32(SHMT) is 92% identical (97% similar) to CeMEL-32(SHMT) at the amino acid level over the entire length of the protein (484 amino acids), whereas the coding DNA is 82.5% identical (over 1455 nucleotides). Several highly conserved non-coding regions upstream and downstream of the mel-32(SHMT) gene reveal potential regulatory sites that may bind trans-acting protein factors.     

Localization and Function of Pals1-associated Tight Junction Protein in Drosophila Is Regulated by Two Distinct Apical Complexes

The transmembrane protein Crumbs (Crb) and its intracellular adaptor protein Pals1 (Stardust, Sdt in Drosophila) play a crucial role in the establishment and maintenance of apical-basal polarity in epithelial cells in various organisms. In contrast, the multiple PDZ domain-containing protein Pals1-associated tight junction protein (PATJ), which has been described to form a complex with Crb/Sdt, is not essential for apical basal polarity or for the stability of the Crb/Sdt complex in the Drosophila epidermis. Here we show that, in the embryonic epidermis, Sdt is essential for the correct subcellular localization of PATJ in differentiated epithelial cells but not during cellularization. Consistently, the L27 domain of PATJ is crucial for the correct localization and function of the protein. Our data further indicate that the four PDZ domains of PATJ function, to a large extent, in redundancy, regulating the function of the protein. Interestingly, the PATJ-Sdt heterodimer is not only recruited to the apical cell-cell contacts by binding to Crb but depends on functional Bazooka (Baz). However, biochemical experiments show that PATJ associates with both complexes, the Baz-Sdt and the Crb-Sdt complex, in the mature epithelium of the embryonic epidermis, suggesting a role of these two complexes for the function of PATJ during the development of Drosophila.     

Disparate cell types use a shared complex of PDZ proteins for polarized protein localization

Based on their morphology and function, epithelial cells and neurons appear to have very little in common; however, growing evidence indicates that these two disparate cell types share an underlying polarization pathway responsible for sorting proteins to specific subcellular sites. An evolutionarily conserved complex of PDZ domain-containing proteins thought to be responsible for polarized protein localization has been identified from both brain and epithelial tissue, both from mammals and from the nematode C. elegans. Some of the most recent data on PDZ proteins and the proteins with which they interact are summarized. In particular, some of the more recently proposed models for their function in cells, and the in vivo and in vitro data that support these models are focussed upon.     

DE-Cadherin, a Core Component of the Adherens Junction Complex Modifies Subcellular Localization of the Drosophila Gap Junction Protein Innexin2

The Drosophila innexin multigene family of gap junction encoding proteins consists of eight family members whose function in epithelial morphogenesis is mostly unknown. We have recently shown that innexin2 plays a crucial role in the organization of embryonic epithelia. Innexin2 protein accumulates in the epidermis in the apico-lateral membrane domain and colocalizes with core proteins of adherens junctions, such as DE-cadherin and Armadillo, the β -catenin homolog. Innexin2 localization is altered in both armadillo and DE-cadherin mutants Biochemical interaction studies point to a direct interaction of DE-cadherin and Armadillo with innexin2 suggesting a close link between gap junction and adherens junction biogenesis. We have used the Drosophila Schneider cell tissue culture system to further study the interaction of innexin2 with DE-cadherin. Our results provide evidence that DE-cadherin may be a key component to control trafficking, and localization of Innexin2 to the plasma membrane.     

A highly conserved nematode protein folding operon in Caenorhabditis elegans and Caenorhabditis briggsae

In the free-living model nematode, Caenorhabditis elegans, a protein-folding co-transcribed gene pair has previously been described. The degree and form of trans-splicing, orientation and spacing of the genes, and the co-ordinate co-expression of protein folding catalysts in the nematode's hypodermis indicated this to be a functionally important operon. This gene pair has now been cloned and compared in the related organism Caenorhabditis briggsae to identify evolutionarily conserved, functionally important features. The corresponding C. briggsae gene pair was found to share the operon-specific features, including sequence homology blocks in the upstream 5′ flanking regions. The intergenic regions were not conserved. The homology block closest to the translational initiation codon of the upstream gene was found to contain a known Ceanorhabbitis promoter element site, and may therefore be an important cis-regulatory region directing the hypodermis-specific expression of this operon gene of C. elegans. This study also provides further confirmation of the high degree of chromosomal synteny between these nematode species.     

Identification, sequence and developmental expression of invertebrate flotillins from Drosophila melanogaster

Caveolae are vesicular organelles that represent a sub-compartment of the plasma membrane. Caveolins (Cav-1, -2 and -3) and flotillins {FLO-1 and FLO-2 [also known as epidermal surface antigens (ESAs)]} are two families of mammalian caveolae-associated integral membrane proteins. Although a caveolin gene family has recently been described in the invertebrate Caenorhabditis elegans, it remains unknown as to whether flotillin homologues exist in invertebrates.

Here, we report the identification, cDNA sequence and embryonic expression pattern of the first invertebrate flotillin, i.e. flotillin from Drosophila melanogaster (FLO^Dm). FLO^Dm is most closely related to mammalian flotillin-1. Remarkably, the invertebrate FLO^Dm protein behaves like mammalian flotillins and is targeted to the caveolae-enriched membrane fraction after transient expression in mammalian cells. Localization of the FLO^Dm message in D. melanogaster embryos reveals that expression of FLO^Dm is confined primarily to the developing nervous system. This is consistent with our previous observation that mammalian flotillin-1 mRNA and protein is expressed abundantly in brain tissue. Interestingly, the FLO^Dm gene is localized to chromosomal region 52 B1–B2. In addition, we find that at least two flotillin-related genes are expressed in D. melanogaster. Our current results provide a starting point and systematic basis for dissecting the role of flotillin in caveolae and neuronal development using Drosophila as a genetic system.