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1.
用蛋白质工程方法改变葡萄糖异构酶最适pH和最适温度   总被引:5,自引:2,他引:3  
用寡核苷酸诱导的定点突变方法构建了葡萄糖异构酶基因的突变体(N184D和A198C)。含突变体的重组质粒pTKD-GI1(N184D)和pTKD-GI2(A198c)在E.coliK38菌株中表达,用DEAE-Sepharose FF和Sephacryl S-300HR柱层析分离纯化突变酶。与野生型葡萄糖异构酶比较实验表明:(1)突变酶N184D的最适pH值下降了1个单位;等电点下降了0.6个单位  相似文献   

2.
人表皮生长因子 (hEGF)是由 5 3个氨基酸组成的单链多肽 ,内有 3个二硫键。它具有多种重要的生物学功能 ,能够促进表皮细胞的生长繁殖 ,加速皮肤和角膜创伤的修复 ,加速胃溃疡的治疗和抑制胃酸的分泌 ,具有广阔的医疗应用前景[1 ] 。最近又发现hEGF在化妆品产业中有潜在的重大应用[2 ] 。这些都促进了人们试图采用基因工程技术大规模地生产hEGF。1982年Smith等人首先在E .coli中以融合蛋白形式实现了hEGF的表达[3 ] ,随后在枯草杆菌和酵母菌中亦相继实现了表达[4] 。由于hEGF在酵母菌中表达水平很低 ,大肠杆…  相似文献   

3.
表达LacZ基因重组火鸡疱疹病毒(HVT)的构建   总被引:3,自引:0,他引:3  
赵军  张秀根 《病毒学报》1999,15(3):244-248
将不含任何启动子的E.coli LacZ基因,插入火鸡疱疹平素FC-126株胸苷激酶编码区末尾的NheⅠ位点,构建成转移载体质粒pTKLacZ。用此质粒和HVT感染的细胞基因组总DNA共感染鸡胚成纤维细胞,在X-gal存在下,通过蓝斑筛选,分离到重组体HVT。rHVT与野生型HVT-FC-126株在CEF中的生长特性完全相似,且在连续传代过程中能稳定表达LacZ基因。  相似文献   

4.
用豌豆叶绿体陪伴蛋白(ch-cpn60)的杭体对衣藻和多种蓝藻细胞提取液作West-ernblot分析表明:真核的衣藻和原核的有异形胞丝状蓝藻的一种鱼腥藻中存在一种与豌豆ch-cpn60抗体有交叉反应的蛋白,其一个亚基的分子量与豌豆该蛋白的分子量相似,而另一亚基的分子量高于豌豆的β亚基分子量。绿藻和丝状蓝藻中这一蛋白在热处理后含量增加,而经-20℃冷处理12h后含量明显下降。在单细胞蓝藻中则检测不到这种蛋白。  相似文献   

5.
MCP-1结构与功能的分子基础   总被引:7,自引:0,他引:7  
MCP 1 (monocytechemoattractantprotein 1 )是第一个被克隆鉴定的CC家族趋化因子 ,最早的研究起源于Cochran等[1] 报道的小鼠基因JE ,该基因从经血小板衍生因子 (PDGF)刺激的成纤维细胞中克隆 ,编码的蛋白质具有趋化白细胞功能。 1 989年 ,Yoshimura等[2 ]从神经胶质瘤系U 1 0 5MG筛到一cDNA克隆 ,其核苷酸和氨基酸序列与鼠JE同源 ,命名为MCP 1。同年Furutani[3] 和Robinson[4] 亦报道相同的趋化因子 ,分别命名为MCAF(hu manmonocy…  相似文献   

6.
era基因是 1986年在测定大肠杆菌基因组序列时发现的一个开放读框 .由于其编码的氨基酸序列与人及酵母的RAS蛋白有部分相似之处 ,命名为E .coliRAS like(era)基因[1] .era基因广泛存在于原核生物中 ,与细胞周期和细胞分裂有关 ,是细菌生存繁殖所必需的重要基因[2 ] .在真核生物如蠕虫、小鼠和人的组织中都发现有与细菌era高度同源基因存在[3 ,4 ] .人era基因 (hera)的全长cDNA于 1998年被克隆 ,与原核era基因具有很高的同源性[5] .人Era蛋白与大肠杆菌Era蛋白的氨基酸序列有 30 %相同 ,两者…  相似文献   

7.
拟南芥AtJ3基因的克隆和分析   总被引:2,自引:0,他引:2  
克隆并分析了拟南芥(Arabidopsisthaliana(L.)Heynh.)AtJ3的cDNA核苷酸序列,并证明其翻译产物与E.coli的DnaJ蛋白高度同源。AtJ3蛋白分子中具有全部这类蛋白的典型特征包括J结构域(domain)、G或GF结构域、富含半胱氨酸的锌指结构域,C末端的CAQQ是一个蛋白法尼基化信号。用AtJ3的cDNA序列末端非翻译区作探针,从拟南芥基因组文库中分离获得AtJ3基因。基因序列分析表明该基因是由被5个内含子分隔的6个外显子组成。根据Southern杂交分析,AtJ3基因为单拷贝基因。Northern分析结果表明,AtJ3在子叶、叶、根、花及长角果中都表达。35℃的热激能增加叶中AtJ3的mRNA表达  相似文献   

8.
用耐高温的DNA聚合酶进行酶法DNA序列测定在分子生物学中具有十分重要的实际应用价值,从耐高温的嗜热脂肪芽孢杆菌(Bacillusstearothermophilus)特异菌株中克隆分离了编码去除了5′-3′外切酶活性的BstDNA聚合酶大片段的结构基因。经重组于表达质粒载体pYZ23,在大肠杆菌JF1125(EscherichiacoliJF1125)中得到了稳定的高效表达,经分离纯化,此基因工  相似文献   

9.
藻类植物时间 3月中旬第一课时课题绿藻——衣藻实验内容从绿色池水中寻找衣藻和其他绿色单细胞藻类。目的要求认识衣藻的形态结构和生活习性。第二课时课题绿藻——水绵实验内容 1.制作水绵装片进行观察;2.制作水绵接合生殖的装片进行观察。目的要求在观察过程中要求学生比较水绵与衣藻的异同;从相同点提出绿藻的特征,从不同点说明水绵是多细胞丝状体。第三课时课题其它藻类植物  相似文献   

10.
利用突变的绿化荧光蛋白基因为标记构建新型克隆载体   总被引:1,自引:0,他引:1  
将三位点替换突变的绿色荧光蛋白(GFP-S65T、V68L、S72A)基因插入到pBluescript SK(+)的XbaⅠ和SacⅠ之间,构建为新型的克隆载体pGreenLD。此质粒载体在E.coli中表达后使菌落在日光下呈现黄绿色,而在长波紫外光照射下呈现亮绿荧光,当外源DNA片段插入该载体的多克隆位点使gfp基因表达受阻时,转化的E.coli菌落为白色。因此可以用E.coli菌落黄绿色/绿色  相似文献   

11.
FtsZ蛋白在原核细胞以及植物细胞叶绿体的分裂过程中发挥着重要作用。为了研究叶绿体分裂装置的进化 ,运用RT PCR方法从莱茵衣藻中克隆了叶绿体分裂相关基因CrFtsZ3。由于已经从衣藻细胞中克隆了一个ftsZ基因 ,所以CrFtsZ3的克隆表明衣藻中已经存在两类不同的 ftsZ基因 ,这说明 ftsZ基因的复制与分歧发生于绿藻的分化之前。序列分析结果显示 ,CrFtsZ3所编码的蛋白质具有FtsZ蛋白的典型模体。进一步的原核表达与定位分析表明CrFtsZ3 GFP融合蛋白沿着宿主菌体的纵轴方向有规律地聚集成荧光点或荧光带 ,并且CrFtsZ3蛋白过量表达明显干挠了宿主菌正常的细胞分裂过程 ,说明衣藻CrFtsZ3蛋白能够识别宿主细胞内的分裂位点并影响细胞分裂过程 ,从而初步验证了它的生物学功能  相似文献   

12.
衣藻叶绿体分裂基因CrFtsZ1在E.coli中的表达   总被引:1,自引:0,他引:1  
FtsZ蛋白在细菌的分裂中起着重要作用,能够在分裂位点形成一个环状结构而控制细菌的分裂过程。细胞内FtsZ蛋白浓度的明显降低或异常升高均可阻断正常的细胞分裂过程进而导致丝状菌体的产生。为了研究衣藻叶绿体分裂基因ftsZ的功能,构建了衣藻CrFtsZ1的原核表达重组质粒。试验结果表明,衣藻ftsZ的表达严重影响了大肠杆菌的分裂,初步证明衣藻FtsZ蛋白不仅与E.coli FtsZ蛋白在序列上相似,而且也有着相似的功能,同时这一结果也为真核细胞中质体的内共生起源提供了直接的证据。  相似文献   

13.
Wang D  Kong D  Wang Y  Hu Y  He Y  Sun J 《Journal of experimental botany》2003,54(384):1115-1116
In order to elucidate the origin of the plastid division gene ftsZ in green plant lineage, and to understand the significance of this divergence for the function of FtsZ proteins in plants, two full-length cDNAs (accession numbers AF449446 and AB084236) were isolated from Chlamydomonas reinhardtii, a base species of green plant lineage. A phylogenetic analysis based on amino acid sequences of eukaryotic FtsZs reveals that an ancient duplication of the ftsZ gene occurred after the endosymbiotic event. The ancient duplication implies that two ftsZ families might play an indispensable role at the early endosymbiotic stage.  相似文献   

14.
杜氏盐藻psaB基因cDNA的克隆与序列分析   总被引:3,自引:0,他引:3  
根据真核生物莱茵衣藻(Chlamydornonas reinhardtii)、Chlamydomonas moewusii、Chlorella vulgaris以及Mesostigma viride的psaB基因的氨基酸高度保守序列,设计一对简并引物,利用TRIzol试剂提取杜氏盐藻(Dunaliella salina)细胞的总RNA,通过RTPCR,得到的一段长为1.8kb左右的cDNA片段。PCR产物经T-A克隆并测序分析以及测序结果推导成氨基酸序列进行同源性比较.表明所克隆的1815bp序列为杜氏盐藻psaB cDNA片段,GenBank收录号为AY820754。根据已经得到的psaB序列推导成氨基酸序列与一些已知物种的psaB基因相比较,同源性分别为Chlamydomonas reinhardtii 92%,Chlamydornonas moewusii 91%,Chlorella vulgaris 86%,Mesostigma viride 85%,Physcomitrella patens subsp.Patens 85%,Nephroselmis olivacea 84%。据此可推断本实验中所克隆的序列为杜氏盐藻psaB cDNA序列.  相似文献   

15.
The FtsZ protein is a GTPase that is essential for cell division. We have cloned, sequenced, and expressed the FtsZ (PgFtsZ) gene from the Porphyromonas gingivalis, an oral, anaerobic, rod-shaped bacterium implicated in progressive periodontal disease. The PgFtsZ gene consisted of 1374 bp and coded for an acidic protein with a calculated molecular mass of 50,253 Da. The deduced amino acid sequence exhibited a significant homology with E. coli FtsZ (54% identical residues). Like other prokaryotic FtsZs, PgFtsZ possessed the clear motifs for GTP binding (GGGTGTG) and hydrolysis (NLDFADV). When PgFtsZ was overexpressed in E. coli, cell division was inhibited. Recombinant PgFtsZ was purified to homogeneity and characterized. The purified PgFtsZ exhibited GTPase activity even in the absence of Mg2+, and completely retained its activity with EDTA. Furthermore, Na+ and K+ ions inhibited its GTPase activity in a dose-dependent manner. These results suggest that PgFtsZ contains an atypical GTPase activity that has not been previously described. Received: 25 May 2001 / Accepted: 8 August 2001  相似文献   

16.
FtsZ ring: the eubacterial division apparatus conserved in archaebacteria   总被引:12,自引:2,他引:10  
FtsZ is a tubulin-like protein that is essential for cell division in eubacteria. It functions by forming a ring at the division site that directs septation. The archaebacteria constitute a kingdom of life separate from eubacteria and eukaryotes. Like eubacteria, archaebacteria are prokaryotes, although they are phylogenetically closer to eukaryotes. Here it is shown that archaebacteria also possess FtsZ and that it is biochemically similar to eubacterial FtsZs. Significantly, FtsZ from the archaebacterium Haloferax volcanii is a GTPase that is localized to a ring that coincides with the division constriction. These results indicate that the FtsZ ring was part of the division apparatus of a common prokaryotic ancestor that was retained by both eubacteria and archaebacteria.  相似文献   

17.
The assembly of the lipid-linked oligosaccharide, Glc(3)Man(9)GlcNAc(2)-P-P-Dol, occurs on the rough ER membrane in an ordered stepwise manner. The process is highly conserved among eukaryotes. In order to isolate the human mannosyltransferase I (MT-I) gene involved in the process, we used the Saccharomyces cerevisiae MT-I gene ( ALG1 ), which has already been cloned. On searching the EST database with the amino acid sequence of the ALG1 gene product, we detected seven related human EST clones. A human fetal brain cDNA library was screened by PCR using gene-specific primers based on the EST nucleotide sequences and a 430 bp cDNA fragment was amplified. The cDNA library was rescreened with this 430 bp cDNA, and two cDNA clones (HR1-3 and HR1-4) were isolated and sequenced. On a homology search of the EST database with the nucleotide sequence of HR1-3, we detected a novel human EST clone, AA675921 (GenBank accession number). Based on the nucleotide sequences of AA675921 and HR1-4, we designed gene-specific PCR primers, which allowed to amplify a 1.8 kb cDNA from human fetal brain cDNA. This cDNA was cloned and shown to contain an ORF encoding a protein of 464 amino acids. We designated this ORF as Hmat-1. The amino acid sequence deduced from the Hmat-1 gene showed several highly conserved regions shared with the yeast and nematode MT-I sequences. Furthermore, this 1.8 kb cDNA successfully complemented the S. cerevisiae alg1-1 mutation, indicating that the Hmat-1 gene encodes the human MT-I and that the function of this enzyme was conserved between yeast and human.  相似文献   

18.
已从西伯利亚蓼叶中cDNA文库中获得的钙调蛋白EST序列,采用cDNA末端快速扩增(RACE)技术克隆了具有完整编码区的钙调蛋白基因的cDNA序列(GenBank登录号GQ988382),命名为PsCaM。该基因全长615bp,编码区为450bp,编码149个氨基酸,5'非翻译区为63bp,3'非翻译区为102bp。同源性分析表明,该蛋白与其他植物钙调蛋白高度保守,氨基酸同源性高达98%。用实时荧光定量PCR研究3%NaHCO3胁迫下西伯利亚蓼基因表达的结果显示,自然条件下,该基因在叶中表达量最高,地下茎次之,茎中最低;盐胁迫下CaM在西伯利亚蓼的地下茎、茎和叶中均有表达,表达模式不同。  相似文献   

19.
作物数量遗传学基础 二、遗传力及其估算   总被引:9,自引:0,他引:9       下载免费PDF全文
两栖类染色体的研究,过去多使用以生殖 细胞和端蚌尾部上皮细胞为材料的水低渗压片 法[6]。然而,生殖细胞和峪蚌组织受季节的限 制颇大,所得的中期分裂相亦不甚多。随着 低等脊稚动物组织培养技术[1,2]与血液培养技 术[3]的发展,近年来已更多的使用离体培养的 细胞来进行研究。  相似文献   

20.
FtsZs from Mycoplasma pulmonis (MpuFtsZ) and Bacillus subtilis (BsFtsZ) are only 46% and 53% identical in amino acid sequence to FtsZ from Escherichia coli (EcFtsZ). In the present study we show that MpuFtsZ and BsFtsZ can function for cell division in E. coli provided we make two modifications. First, we replaced their C-terminal tails with that from E. coli, giving the foreign FtsZ the binding site for E. coli FtsA and ZipA. Second, we selected for mutations in the E. coli genome that facilitated division by the foreign FtsZs. These suppressor strains arose at a relatively high frequency of 10(-3) to 10(-5), suggesting that they involve loss-of-function mutations in multigene pathways. These pathways may be negative regulators of FtsZ or structural pathways that facilitate division by slightly defective FtsZ. Related suppressor strains were obtained for EcFtsZ containing certain point mutations or insertions of yellow fluorescent protein. The ability of highly divergent FtsZs to function for division in E. coli is consistent with a two-part mechanism. FtsZ assembles the Z ring, and perhaps generates the constriction force, through self interactions; the downstream division proteins remodel the peptidoglycan wall by interacting with each other and the wall. The C-terminal peptide of FtsZ, which binds FtsA, provides the link between FtsZ assembly and peptidoglycan remodeling.  相似文献   

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