衣藻叶绿体分裂基因CrFtsZ1在E.coli中的表达

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。     

衣藻CrFtsZ2-GFP融合蛋白在E.coli中的表达及其定位

FtsZ蛋白在细菌的分裂中担任着重要作用 ,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。胞内FtsZ蛋白浓度的异常升高或降低均可阻断正常的细胞分裂过程进而形成分裂异常的丝状菌体。为了研究衣藻FtsZ蛋白的生物学活性 ,构建了衣藻CrFtsZ2cDNA全长与绿色荧光蛋白基因egfp的融合表达质粒 ,并对其在大肠杆菌中的表达与定位做了初步分析。在大肠杆菌JM10 9中 ,融合表达质粒的过量表达导致宿主菌形成了丝状菌体 ,通过荧光显微镜观察发现CrFtsZ2 EGFP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 ,暗示衣藻CrFtsZ2蛋白能够识别宿主菌内分裂位点的定位信号并参与其细胞分裂过程 ,初步验证了衣藻CrFtsZ2蛋白的功能。     

叶绿体分裂相关基因NtFtsZ2-1在大肠杆菌中的表达与定位

FtsZ蛋白在细菌的分裂中担任着重要作用,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。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。我们为了研究烟草FtsZ蛋白与大肠杆菌FtsZ蛋白的异同,构建了烟草全长ftsZ2-1与绿色荧光蛋白EGFP的融合表达质粒并转化大肠杆菌JM109。融合表达质粒的过量表达导致宿主菌形成了丝状菌体。通过荧光显微镜观察发现NtFtsZ2-1-EGFP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,说明烟草FtsZ2-1蛋白能够识别宿主菌内分裂位点的定位信号并参与其细胞分裂复合物的组装。     

FtsZ在钝顶螺旋藻形态建成中的作用

为了研究细胞骨架蛋白FtsZ在螺旋藻形态建成中的作用,通过PCR克隆了ftsZ基因并进行原核表达,对表达的融合蛋白进行了纯化。通过免疫小鼠制备了FtsZ的多克隆抗体。分别用Western blot和免疫荧光技术检测螺旋藻不同形态藻丝体中ftsZ的表达和定位。结果表明,在两株不同螺旋藻Spirulina platensisFACHB869和FACHB882中,ftsZ在直线形藻丝体中的表达量都高于螺旋形藻丝体。免疫荧光定位结果显示,FtsZ蛋白在藻细胞中呈环状分布于细胞膜上,且这种环状结构在直线形藻丝体中排列较密而在螺旋形藻丝体中排列疏松。ftsZ在不同形态藻丝体中的表达量和细胞定位差异说明,细胞骨架蛋白FtsZ可能通过改变细胞刚性而参与螺旋藻形态建成。     

烟草质体分裂蛋白NtFtsZs在大肠杆菌中的定位分析

分别构建了两个烟草 (NicotianatabacumL .)质体分裂基因NtFtsZ1和NtFtsZ2与编码绿色荧光蛋白的gfpS65A、V68L、S72A基因相融合的原核表达载体 ,并导入大肠杆菌 (Escherichiacoli)JM10 9菌株中进行表达。全长NtFtsZs∶GFP融合蛋白在菌体中有规律地定位 ,暗示NtFtsZs能识别大肠杆菌潜在的分裂位点 ,并能与大肠杆菌的内源FtsZ发生聚合作用 ;融合蛋白的诱导表达抑制了宿主菌的分裂 ,形成了明显的丝状菌体 ,证明真核生物的ftsZ基因与大肠杆菌的ftsZ基因有相似的作用。同时构建了NtFtsZs不同缺失的原核表达载体 ,对这两个基因所编码蛋白不同结构域的功能做了初步分析。实验结果表明 ,烟草FtsZ蛋白的C端结构域与其在大肠杆菌细胞中的正确定位有关 ;而N端结构域与NtFtsZs∶GFP融合蛋白的聚合有关。     

木薯ftsZ基因分离及在原核生物中功能初步鉴定

ftsZ基因是控制细胞分裂的关键基因,其蛋白能够在分裂位点形成一个环状结构而影响细胞分裂.为了研究木薯质体分裂与木薯淀粉品质形成的关系,根据木薯基因组数据库上的预测序列,设计引物,从木薯基因组中分离了与质体分裂相关的ftsZ家族3个新基因(ftsZ1,ftsZ2,ftsZ3).分别将它们与荧光蛋白基因(GFP)融合,构建了3个原核表达载体pET-fisZ1-GFP、pET-fisZ2-GFP、pET-fisZ3-GFP,并转化大肠杆菌BL21(DE3).通过荧光显微镜观察菌体的表型和分裂,初步鉴定了木薯质体分裂相关基因ftsZ家族对细胞分裂的作用.结果显示:尽管木薯与大肠杆菌的亲缘关系较远,ftsZ基因的同源性较低,但是两者表现出相似的功能,木薯ftsZ基因的表达能严重影响大肠杆菌细胞分裂.这一结果为进一步研究木薯ftsZ家族基因的功能奠定了基础.     

烟草质体分裂相关基因NtFtsZ1和NtFtsZ2对叶绿体分裂和形态的影响

质体作为植物细胞中一类重要的细胞器,控制其分裂的分子机制一直都不清楚.最近的研究表明,植物细胞中与原核细胞分裂基因ftsZ类似的同源基因控制着质体的分裂过程.通过正反义转化分析了两个烟草的ftsZ基因(NtFtsZ1和NtFtsZ2)在转基因烟草中的功能.二者的反义表达并未对转化烟草细胞中叶绿体的分裂和形态产生明显影响,但二者过表达转化植株中叶绿体的数目和形态都发生了明显的变化,在某些转化植株的叶肉细胞中甚至只有1～2个巨大的叶绿体存在.对不同转化植株的电镜观察和叶绿素含量分析认为,NtFtsZs基因可能对叶绿体的正常发育和功能没有影响,叶绿体形态的变化是对其数目减少的一种补偿.正反义转化植株中叶绿体的不同表型暗示高等植物中同一家族的ftsZ基因可能在控制质体分裂方面具有相同的功能.同时,过表达植株中叶绿体形态的变化被认为是高等植物FtsZ质体骨架功能的体现.     

烟草质体分裂蛋白NtFtsZs在大肠杆菌中的定位分析

分别构建了两个烟草(Nicotiana tabacum L.)质体分裂基因NtFtsZ1和NtFtsZ2与编码绿色荧光蛋白的gfpS65A、V68L、S72A基因相融合的原核表达载体,并导入大肠杆菌( Escherichia coli ) JM109菌株中进行表达.全长NtFtsZs∶GFP融合蛋白在菌体中有规律地定位,暗示NtFtsZs能识别大肠杆菌潜在的分裂位点,并能与大肠杆菌的内源FtsZ发生聚合作用;融合蛋白的诱导表达抑制了宿主菌的分裂,形成了明显的丝状菌体,证明真核生物的 ftsZ 基因与大肠杆菌的 ftsZ 基因有相似的作用.同时构建了NtFtsZs不同缺失的原核表达载体,对这两个基因所编码蛋白不同结构域的功能做了初步分析.实验结果表明,烟草FtsZ蛋白的C端结构域与其在大肠杆菌细胞中的正确定位有关;而N端结构域与NtFtsZs∶GFP融合蛋白的聚合有关.     

烟草质体分裂相关基因NtFtsZ1和NtFtsZ2对叶绿体分裂和形态的影响

质体作为植物细胞中一类重要的细胞器,控制其分裂的分子机制一直都不清楚。最近的研究表明,植物细胞中与原核细胞分裂基因fisZ类似的同源基因控制着质体的分裂过程。通过正反义转化分析了两个烟草的ftsZ基因（NtFtsZ1和NtFtsZ2)在转基因烟草中的功能。二的反义表达并未对转化烟草细胞中叶绿体的分裂和形态产生明显影响,但二过表达转化植株中叶绿体的数目和形态都发生了明显的变化,在某些转化植株的叶肉细胞中甚至只有1－2个巨大的叶绿体存在。对不同转化植株的电镜观察和叶绿素含量分析认为,NtFtsZs基因可能对叶绿体的正常发育和功能没有影响,叶绿体形态的变化是对其数目减少的一种补偿。正反义转化植株中叶绿体的不同表型暗示高等植物中同一家族的ftsZ基因可能在控制质体分裂方面具有相同的功能。同时,过表达植株中叶绿体形态的变化被认为是高等植物的FtsZ质体骨架功能的体现。     

钝顶螺旋藻FtsZ在大肠杆菌中的表达和定位北大核心CSCD

FtsZ是与真核微管蛋白类似的原核骨架蛋白,能在细胞分裂位点聚合组装成环状结构而调控细胞分裂过程。为了研究钝顶螺旋藻(Spirulina platensis)FtsZ蛋白的功能,构建了钝顶螺旋藻FtsZ与绿色荧光蛋白GFP融合表达的质粒,并在大肠杆菌中进行了表达和定位研究,结果发现,表达融合蛋白GFP-FtsZ的大肠杆菌细胞由短杆状变为长丝状,且菌丝体长度与融合蛋白的表达量呈正比。在荧光显微镜下观察到融合蛋白GFP-FtsZ在长丝状体细菌中呈有规律的点状分布,这说明FtsZ蛋白功能高度保守,钝顶螺旋藻FtsZ蛋白能识别大肠杆菌分裂位点并装配成环状结构调控大肠杆菌细胞分裂,FtsZ蛋白的过量表达能抑制大肠杆菌正常的细胞分裂而导致长丝状体细胞的形成。     

衣藻质体分裂相关基因CrFtsZ2的克隆及其进化分析

ＦｔｓＺ(ｆｉｌａｍｅｎｔｉｎｇｔｅｍｐｅｒａｔｕｒｅｓｅｎｓｉｔｉｖｅ)是一类从大肠杆菌温度敏感型突变体中分离到的基因 .该基因与Ｅ .ｃｏｌｉ细胞分裂密切相关 .突变体由于细胞分裂受阻而呈现“长丝状”[1] .此类基因于 1980年首次被克隆[2 ] .随后的研究表明 ,ＦｔｓＺ蛋白在Ｅ .ｃｏｌｉ分裂细胞的凹陷处形成环状多聚体 ,Ｚ环 ,是Ｅ .ｃｏｌｉ细胞分裂的限制因子[3 ] .衣藻属于绿藻 ,在现存的所有单细胞真核藻类中 ,绿藻是与陆生植物亲缘关系最近的一支[4] .由于衣藻为单细胞真核生物 ,并且仅含有一个巨大的叶绿体 ,因而是研究…     

A new essential gene of the 'minimal genome' affecting cell division

The complete sequencing of bacterial genomes has offered new opportunities for the identification of essential genes involved in the control and progression of the cell cycle. For this purpose, we have disrupted ten E. coli genes belonging to the so-called 'minimal genome'. One of these genes, yihA, was necessary for normal cell division. The yihA gene possesses characteristic GTPase motifs and its homologues are present in eukaryotes, archaea and most prokaryotes. Depletion of YihA protein led to a severe reduction in growth rate and to extensive filamentation, with a block beyond the stage of nucleoid segregation. Filamentation was correlated with reduced FtsZ levels and could be specifically suppressed by overexpression of ftsQI, ftsA and ftsZ, and to some extent by ftsZ alone. We hypothesize that YihA, like the Era GTPase, may participate in a checkpoint mechanism that ensures a correct coordination of cell cycle events.     

Cloning and characterization of Bacillus subtilis homologs of Escherichia coli cell division genes ftsZ and ftsA.

The Bacillus subtilis homolog of the Escherichia coli ftsZ gene was isolated by screening a B. subtilis genomic library with anti-E. coli FtsZ antiserum. DNA sequence analysis of a 4-kilobase region revealed three open reading frames. One of these coded for a protein that was about 50% homologous to the E. coli FtsZ protein. The open reading frame just upstream of ftsZ coded for a protein that was 34% homologous to the E. coli FtsA protein. The open reading frames flanking these two B. subtilis genes showed no relationship to those found in E. coli. Expression of the B. subtilis ftsZ and ftsA genes in E. coli was lethal, since neither of these genes could be cloned on plasmid vectors unless promoter sequences were first removed. Cloning the B. subtilis ftsZ gene under the control of the lac promoter resulted in an IPTGs phenotype that could be suppressed by overproduction of E. coli FtsZ. These genes mapped at 135 degrees on the B. subtilis genetic map near previously identified cell division mutations.     

Analysis of cell division gene ftsZ (sulB) from gram-negative and gram-positive bacteria.

The ftsZ (sulB) gene of Escherichia coli codes for a 40,000-dalton protein that carries out a key step in the cell division pathway. The presence of an ftsZ gene protein in other bacterial species was examined by a combination of Southern blot and Western blot analyses. Southern blot analysis of genomic restriction digests revealed that many bacteria, including species from six members of the family Enterobacteriaceae and from Pseudomonas aeruginosa and Agrobacterium tumefaciens, contained sequences which hybridized with an E. coli ftsZ probe. Genomic DNA from more distantly related bacteria, including Bacillus subtilis, Branhamella catarrhalis, Micrococcus luteus, and Staphylococcus aureus, did not hybridize under minimally stringent conditions. Western blot analysis, with anti-E. coli FtsZ antiserum, revealed that all bacterial species examined contained a major immunoreactive band. Several of the Enterobacteriaceae were transformed with a multicopy plasmid encoding the E. coli ftsZ gene. These transformed strains, Shigella sonnei, Salmonella typhimurium, Klebsiella pneumoniae, and Enterobacter aerogenes, were shown to overproduce the FtsZ protein and to produce minicells. Analysis of [35S]methionine-labeled minicells revealed that the plasmid-encoded gene products were the major labeled species. This demonstrated that the E. coli ftsZ gene could function in other bacterial species to induce minicells and that these minicells could be used to analyze plasmid-endoced gene products.     

Borrelia burgdorferi ftsZ plays a role in cell division

ftsZ is essential for cell division in many microorganisms. In Escherichia coli and Bacillus subtilis, FtsZ plays a role in ring formation at the leading edge of the cell division septum. An ftsZ homologue is present in the Borrelia burgdorferi genome (ftsZ(Bbu)). Its gene product (FtsZ(Bbu)) is strongly homologous to other bacterial FtsZ proteins, but its function has not been established. Because loss-of-function mutants of ftsZ(Bbu) might be lethal, the tetR/tetO system was adapted for regulated control of this gene in B. burgdorferi. Sixty-two nucleotides of an ftsZ(Bbu) antisense DNA sequence under the control of a tetracycline-responsive modified hybrid borrelial promoter were cloned into pKFSS1. This construct was electroporated into a B. burgdorferi host strain carrying a chromosomally located tetR under the control of the B. burgdorferi flaB promoter. After induction by anhydrotetracycline, expression of antisense ftsZ RNA resulted in generation of filamentous B. burgdorferi that were unable to divide and grew more slowly than uninduced cells. To determine whether FtsZ(Bbu) could interfere with the function of E. coli FtsZ, ftsZ(Bbu) was amplified from chromosomal DNA and placed under the control of the tetracycline-regulated hybrid promoter. After introduction of the construct into E. coli and induction with anhydrotetracycline, overexpression of ftsZ(Bbu) generated a filamentous phenotype. This suggested interference of ftsZ(Bbu) with E. coli FtsZ function and confirmed the role of ftsZ(Bbu) in cell division. This is the first report of the generation of a B. burgdorferi conditional lethal mutant equivalent by tetracycline-controlled expression of antisense RNA.     

Characterization of the ftsZ gene from Mycoplasma pulmonis, an organism lacking a cell wall.

The ftsZ gene is required for cell division in Escherichia coli and Bacillus subtilis. In these organisms, FtsZ is located in a ring at the leading edge of the septum. This ring is thought to be responsible for invagination of the septum, either causing invagination of the cytoplasmic membrane or activating septum-specific peptidoglycan biosynthesis. In this paper, we report that the cell division gene ftsZ is present in two mycoplasma species, Mycoplasma pulmonis and Acholeplasma laidlawii, which are eubacterial organisms lacking a cell wall. Sequencing of the ftsZ homolog from M. pulmonis revealed that it was highly homologous to other known FtsZ proteins. The M. pulmonis ftsZ gene was overexpressed, and the purified M. pulmonis FtsZ bound GTP. Using antisera raised against this purified protein, we could demonstrate that it was expressed in M. pulmonis. Expression of the M. pulmonis ftsZ gene in E. coli inhibited cell division, leading to filamentation, which could be suppressed by increasing expression of the E. coli ftsZ gene. The implications of these results for the role of ftsZ in cell division are discussed.     

叶绿体分裂相关蛋白CrMinD的保守功能

细胞或质体中部正确分裂位点的选择是MinD蛋白与其他Min蛋白（MinC／E）相互作用的结果,MinD蛋白在原核细胞以及植物叶绿体的分裂过程中发挥着重要的作用。细胞中MinD蛋白浓度的明显升高可影响正常细胞的分裂过程而产生丝状体细胞。为了研究叶绿体分裂蛋白CrMinD的保守功能,构建了衣藻CrMinD-gfp的原核表达重组质粒进行了原核功能验证。试验结果表明,衣藻CrMinD蛋白的过量表达严重影响了大肠杆菌的分裂,其在原核细胞中运动和定位与用GFP标记的原核细胞MinD蛋白具有相似性。更进一步证明了叶绿体分裂同源物CrMinD蛋白与原核细胞MinD蛋白有着相似的功能,是一个进化上功能保守的蛋白。同时,这一结果也为研究植物细胞中质体的分裂机制奠定了一定的基础。     

The proper ratio of FtsZ to FtsA is required for cell division to occur in Escherichia coli.

Interactions among cell division genes in Escherichia coli were investigated by examining the effect on cell division of increasing the expression of the ftsZ, ftsA, or ftsQ genes. We determined that cell division was quite sensitive to the levels of FtsZ and FtsA but much less so to FtsQ. Inhibition of cell division due to an increase in FtsZ could be suppressed by an increase in FtsA. Inhibition of cell division due to increased FtsA could be suppressed by an increase in FtsZ. In addition, although wild-type strains were relatively insensitive to overexpression of ftsQ, we observed that cell division was sensitized to ftsQ overexpression in ftsI, ftsA, and ftsZ mutants. Among these, the ftsI mutant was the most sensitive. These results suggest that these gene products may interact and that the proper ratio of FtsZ to FtsA is critical for cell division to occur.