烟草和衣藻中ftsZ 基因的克隆及功能分析

作为一种广泛存在于原核细胞中的原始的细胞骨架蛋白，FtsZ在植物中的发现为我们研究植物细胞中质体的分裂机制提供了可能。已有的研究证明了FtsZ与质体的分裂和形态维持有关，但高等植物中FtsZ在质体分裂和形态维持中的作用机制仍不十分清楚，同时高等植物中多个ftsZ成员的存在也使得对FtsZ功能的研究更加复杂。我们从烟草中克隆了两个ftsZ基因，序列和谱系分析表明二者均属于高等植物中的FtsZl基因家族，这也是首次在高等植物中发现多个FtsZl家族的成员。杂交分析表明ftsZ在烟草基因组中是以多拷贝形式存在，并且这两个基因具有相似的表达谱，这些结果暗示着高等植物中FtsZ在质体分裂中的作用更为复杂。GFP标记的原核定位表明二者具有与原核FtsZ类似的功能。此外，利用反义和正义表达的方法研究了二者在烟草质体分裂和形态维持中的作用。反义转化并未对烟草细胞叶绿体的数目和形态造成明显的影响，相反，二者的正义表达均导致细胞中叶绿体数目和形态上的明显变化，这一结果预示着二者在控制质体分裂和形态方面可能具有不同的功能。同时，这些结果也为高等植物中多样化的FtsZ可能具有除质体分裂之外的功能，如质体骨架．提供了证据。 　利用简并引物PCR和RACE从衣藻中扩增得到了一个ftsZ基因的部分cDNA序列，命名为CrFtsZ。序列分析表明该基因编码的蛋白具有FtsZ的典型特点，但同时还有一个与目前已知FtsZ均不同的突出c-末端：分子谱系分析认为CrFtsZ与线粒体进化祖先a -proteobacteria中的FtsZ有着共同起源，因此CrFtsZ可能是一个控制线粒体分裂的FtsZ。此外，CrFtsZ的c-端突出序列还具有目前已知真核生物线粒体分裂相关蛋白dynamin的某些特征，考虑到FtsZ在原核细胞分裂和真核细胞器分裂中的作用，我们推测CrFtsZ可能是FtsZ向dynamin过度的一种中间进化形式。这一发现为线粒体分裂机制的起源和进化提供了新的分子证据，对于认识真核线粒体分裂机制的起源与演化具有重要意义。

Cell division protein ftsZ: running rings around bacteria, chloroplasts and mitochondria

Of all the proteins involved in prokaryotic cell division FtsZ is one of the earliest acting and most widely distributed, being found in all but a few species. We discuss several recent discoveries of FtsZ in eukaryotic cells and the protein’s role in the division of chloroplasts and mitochondria, organelles that are of bacterial origin.

Two dictyostelium orthologs of the prokaryotic cell division protein ftsZ localize to mitochondria and are required for the maintenance of normal mitochondrial morphology

In bacteria, the protein FtsZ is the principal component of a ring that constricts the cell at division. Though all mitochondria probably arose through a single, ancient bacterial endosymbiosis, the mitochondria of only certain protists appear to have retained FtsZ, and the protein is absent from the mitochondria of fungi, animals, and higher plants. We have investigated the role that FtsZ plays in mitochondrial division in the genetically tractable protist Dictyostelium discoideum, which has two nuclearly encoded FtsZs, FszA and FszB, that are targeted to the inside of mitochondria. In most wild-type amoebae, the mitochondria are spherical or rod-shaped, but in fsz-null mutants they become elongated into tubules, indicating that a decrease in mitochondrial division has occurred. In support of this role in organelle division, antibodies to FszA and FszA-green fluorescent protein (GFP) show belts and puncta at multiple places along the mitochondria, which may define future or recent sites of division. FszB-GFP, in contrast, locates to an electron-dense, submitochondrial body usually located at one end of the organelle, but how it functions during division is unclear. This is the first demonstration of two differentially localized FtsZs within the one organelle, and it points to a divergence in the roles of these two proteins.

Division of Mitochondria Requires a Novel DNM1-interacting Protein, Net2p

Mitochondria are dynamic organelles that undergo frequent division and fusion, but the molecular mechanisms of these two events are not well understood. Dnm1p, a mitochondria-associated, dynamin-related GTPase was previously shown to mediate mitochondrial fission. Recently, a genome-wide yeast two-hybrid screen identified an uncharacterized protein that interacts with Dnm1p. Cells disrupted in this new gene, which we call NET2, contain a single mitochondrion that consists of a network formed by interconnected tubules, similar to the phenotype of dnm1Δ cells. NET2 encodes a mitochondria-associated protein with a predicted coiled-coil region and six WD-40 repeats. Immunofluorescence microscopy indicates that Net2p is located in distinct, dot-like structures along the mitochondrial surface, many of which colocalize with the Dnm1 protein. Fluorescence and immunoelectron microscopy shows that Dnm1p and Net2p preferentially colocalize at constriction sites along mitochondrial tubules. Our results suggest that Net2p is a new component of the mitochondrial division machinery.

Regulation of mitochondrial division by the Drp1 receptors

Mitochondria can remodel their membranes by fusing or dividing. These processes are required for the proper development and viability of multicellular organisms. At the cellular level, fusion is important for mitochondrial Ca2+ homeostasis, mitochondrial DNA maintenance, mitochondrial membrane potential, and respiration. Mitochondrial division, which is better known as fission, is important for apoptosis, mitophagy, and for the proper allocation of mitochondria to daughter cells during cellular division.

The functions of proteins involved in fission have been best characterized in the yeast model organism Sarccharomyces cerevisiae. Mitochondrial fission in mammals has some similarities. In both systems, a cytosolic dynamin-like protein, called Dnm1 in yeast and Drp1 in mammals, must be recruited to the mitochondrial surface and polymerized to promote membrane division. Recruitment of yeast Dnm1 requires only one mitochondrial outer membrane protein, named Fis1. Fis1 is conserved in mammals, but its importance for Drp1 recruitment is minor. In mammals, three other receptor proteins—Mff, MiD49, and MiD51—play a major role in recruiting Drp1 to mitochondria. Why mammals require three additional receptors, and whether they function together or separately, are fundamental questions for understanding the mechanism of mitochondrial fission in mammals.

We have determined that Mff, MiD49, or MiD51 can function independently of one another to recruit Drp1 to mitochondria. Fis1 plays a minor role in Drp1 recruitment, suggesting that the emergence of these additional receptors has replaced the system used by yeast. Additionally, we found that Fis1/Mff and the MiDs regulate Drp1 activity differentially. Fis1 and Mff promote constitutive mitochondrial fission, whereas the MiDs activate recruited Drp1 only during loss of respiration.

To better understand the function of the MiDs, we have determined the atomic structure of the cytoplasmic domain of MiD51, and performed a structure-function analysis of MiD49 based on its homology to MiD51. MiD51 adopts a nucleotidyl transferase fold, and binds ADP as a co-factor that is essential for its function. Both MiDs contain a loop segment that is not present in other nucleotidyl transferase proteins, and this loop is used to interact with Drp1 and to recruit it to mitochondria.

Pollen ultrastructure in anther cultures of Datura innoxia. I. Division of the presumptive vegetative cell.

Ultrastructural features of embryogenic pollen in Datura innoxia are described, just prior to, during, and after completion of the first division of the presumptive vegetative cell. In anther cultures initiated towards the end of the microspore phase and incubated at 28 degrees C in darkness, the spores divide within 24 h and show features consistent with those of dividing spores in vivo. Cytokinesis is also normal in most of the spores and the gametophytic cell-plate curves round the presumptive generative nucleus in the usual highly ordered way. Further differentiation of the 2 gametophytic cells does not take place and the pollen either switches to embryogenesis or degenerates. After 48-72 h, the remaining viable pollen shows the vegetative cell in division. The cell, which has a large vacuole and thin layer of parietal cytoplasm carried over from the microspore, divides consistently in a plane parallel to the microspore division. The dividing wall follows a less-ordered course than the gametophytic wall and usually traverses the vacuole, small portions of which are incorporated into the daughter cell adjacent to the generative cell. The only structural changes in the vegetative cell associated with the change in programme appear to be an increase in electron density of both plastids and mitochondria and deposition of an electron-dense material (possibly lipid) on the tonoplast. The generative cell is attached to the intine when the vegetative cell divides. Ribosomal density increases in the generative cell and exceeds that in the vegetative cell. A thin electron-dense layer also appears in the generative-cell wall. It is concluded that embryogenesis commences as soon as the 2 gametophytic cells are laid down. Gene activity associated with postmitotic synthesis of RNA and protein in the vegetative cell is switched off. The data are discussed in relation to the first division of the embryogenic vegetative cells in Nicotiana tabacum.

Bacterial lessons on organelle division

Mitochondria and chloroplasts arose from bacterial endosymbionts about a billion years ago. This ancestry is now showing us how these organelles divide in modem cells.

Diverse eukaryotes have retained mitochondrial homologues of the bacterial division protein FtsZ

Mitochondrial fission requires the division of both the inner and outer mitochondrial membranes. Dynamin-related proteins operate in division of the outer membrane of probably all mitochondria, and also that of chloroplasts – organelles that have a bacterial origin like mitochondria. How the inner mitochondrial membrane divides is less well established. Homologues of the major bacterial division protein, FtsZ, are known to reside inside mitochondria of the chromophyte alga Mallomonas, a red alga, and the slime mould Dictyostelium discoideum, where these proteins are likely to act in division of the organelle. Mitochondrial FtsZ is, however, absent from the genomes of higher eukaryotes (animals, fungi, and plants), even though FtsZs are known to be essential for the division of probably all chloroplasts. To begin to understand why higher eukaryotes have lost mitochondrial FtsZ, we have sampled various diverse protists to determine which groups have retained the gene. Database searches and degenerate PCR uncovered genes for likely mitochondrial FtsZs from the glaucocystophyte Cyanophora paradoxa, the oomycete Phytophthora infestans, two haptophyte algae, and two diatoms – one being Thalassiosira pseudonana, the draft genome of which is now available. From Thalassiosira we also identified two chloroplast FtsZs, one of which appears to be undergoing a C-terminal shortening that may be common to many organellar FtsZs. Our data indicate that many protists still employ the FtsZ-based ancestral mitochondrial division mechanism, and that mitochondrial FtsZ has been lost numerous times in the evolution of eukaryotes.

The evolution of organelle division proteins in diverse eukaryotes

The evolutionary distribution of chloroplast and mitochondrial division proteins has been investigated, gleaning new insights to the evolution of organelle division: specifically the use and features of FtsZ and dynamin-like proteins. Additional novel proteins that are potentially involved in mitochondrial division have been identified in Dictyostelium discoideum.

Mitochondrial division in dictyostellium discoideum

Mitochondria, the cellular powerplants, are essential to eukaryotic life and have evolved from free-living bacteria. Using molecular biology, this thesis has deepened our understanding of the evolution of mitochondrial division through the study of two, key bacterially-derived proteins in the slime mold, dictyostelium.

Evolution of RNA editing in trypanosome mitochondria

Two different RNA editing systems have been described in the kinetoplast-mitochondrion of trypanosomatid protists. The first involves the precise insertion and deletion of U residues mostly within the coding regions of maxicircle-encoded mRNAs to produce open reading frames. This editing is mediated by short overlapping complementary guide RNAs encoded in both the maxicircle and the minicircle molecules and involves a series of enzymatic cleavage-ligation steps. The second editing system is a C34 to U34 modification in the anticodon of the imported tRNATrp, thereby permitting the decoding of the UGA stop codon as tryptophan. U-insertion editing probably originated in an ancestor of the kinetoplastid lineage and appears to have evolved in some cases by the replacement of the original pan-edited cryptogene with a partially edited cDNA. The driving force for the evolutionary fixation of these retroposition events was postulated to be the stochastic loss of entire minicircle sequence classes and their encoded guide RNAs upon segregation of the single kinetoplast DNA network into daughter cells at cell division. A large plasticity in the relative abundance of minicircle sequence classes has been observed during cell culture in the laboratory. Computer simulations provide theoretical evidence for this plasticity if a random distribution and segregation model of minicircles is assumed. The possible evolutionary relationship of the C to U and U-insertion editing systems is discussed.

Knowledge management strategy for the Building Division, Queensland Department of Public Works

This paper describes the background and methodology developed and employed in undertaking research developing a Knowledge Management Strategy for a key construction focused government agency. This paper reviews this methodology and examines a likely Knowledge Management Strategy. Two central objectives structure this Case Study: 1. Identify categories of important information generated by the Building Division, Queensland Department of Public Works in its service delivery to internal and external stake-holders, and 2. Formulate an appropriate and targeted Knowledge Management Strategy to meet the needs of the Queensland Building Capital Works program. The structure of this paper includes: *Description of the Queensland construction industry setting *Review the relevant literature *Design an appropriate research methodology *Analyse results *Formulate conclusions, contributions and implications of the targeted strategy.