不同温度下酿酒酵母细胞分裂周期蛋白Cdc5在有丝分裂中的分子动力学研究

[目的] 探究不同温度下酿酒酵母细胞分裂周期蛋白Cdc5蛋白在有丝分裂中的分子动力学变化。[方法] 本研究以酿酒酵母（Saccharomyces cerevisiae）为材料,采用活细胞成像的方法,探究Cdc5蛋白在不同温度下在酿酒酵母有丝分裂过程中的精细分子动力学变化;通过测量OD₅₉₅绘制生长曲线图,看其宏观的分裂情况是否与微观下Cdc5蛋白的分子动力学变化一致;利用流式细胞术检测细胞的细胞周期变化的情况。[结果] 在胞质分裂时,Cdc5蛋白从母细胞进入子细胞,并在芽颈处发生聚集。25℃条件下细胞中Cdc5蛋白在芽颈处的聚集时间长,37℃条件下Cdc5蛋白在芽颈处聚集时间短,两者间存在显著差异;但两个温度下,细胞中Cdc5蛋白的表达量没有显著性差异。同时,温度也会影响Cdc5蛋白在降解过程中的动力学行为,包括Cdc5蛋白在母细胞与子细胞中荧光强度峰值出现的次数和时间。生长曲线结果显示,酿酒酵母单一细胞分裂周期的变化影响了其宏观的细胞生长,且酵母分裂速度越快,子细胞长宽比越小;细胞周期结果表明,37℃下Cdc5蛋白的动力学变化与酿酒酵母细胞周期变化一致,酿酒酵母细胞周期从G₀/G₁期进入S期,亦加速了酿酒酵母的分裂。[结论] 本研究首次探究了不同温度下酿酒酵母有丝分裂中Cdc5蛋白的精细分子动力学及对应的酵母的宏观生长情况,结果表明温度会对Cdc5蛋白的动力学产生影响,且其精细分子动力学与酿酒酵母的分裂速度成正相关,该结果为进一步研究其在细胞有丝分裂中的功能提供了前期研究基础。     

A kinesin-related protein, KRP(180), positions prometaphase spindle poles during early sea urchin embryonic cell division

We have investigated the intracellular roles of an Xklp2-related kinesin motor, KRP(180), in positioning spindle poles during early sea urchin embryonic cell division using quantitative, real-time analysis. Immunolocalization reveals that KRP(180) concentrates on microtubules in the central spindle, but is absent from centrosomes. Microinjection of inhibitory antibodies and dominant negative constructs suggest that KRP(180) is not required for the initial separation of spindle poles, but instead functions to transiently position spindle poles specifically during prometaphase.     

The proliferating cell marker monoclonal antibody Ki-67 recognizes specific antigens associated with the nuclear envelope of the early Drosophila embryo

Summary— Immunofluorescence and immunoelectron microscopy indicated that the antibody raised against the nuclear antigen Ki-67 of mammalian cells recognized antigenic determinants of early Drosophila embryos, localized on the outside of the nuclear envelope. Hence, the nuclear envelope of Drosophila appears to share a similar epitope with the chromosome scaffold of mitotic mammalian cells. With the progression of mitosis the antigen persisted around the mitotic spindle region and was also found in the pole regions at metaphase and anaphase. The antibody also stained the equatorial regions of the spindles from anaphase to late telophase. The antibody may therefore be used as a biochemical marker of the nuclear envelope for studying nuclear membrane biogenesis and behavior during the mitotic divisions of the Drosophila embryo.     

The cost of copy number in a selfish genetic element: the 2‐μm plasmid of Saccharomyces cerevisiae

Many autonomously replicating genetic elements exist as multiple copies within the cell. The copy number of these elements is often assumed to have important fitness consequences for both element and host, yet the forces shaping its evolution are not well understood. The 2 μm is a multicopy plasmid of Saccharomyces yeasts, encoding just four genes that are solely involved in plasmid replication. One simple model for the fitness relationship between yeasts and 2 μm is that plasmid copy number evolves as a trade‐off between selection for increased vertical transmission, favouring high copy number, and selection for decreased virulence, favouring low copy number. To test this model, we experimentally manipulated the copy number of the plasmid and directly measured the fitness cost, in terms of growth rate reduction, associated with high plasmid copy number. We find that the fitness burden imposed by the 2 μm increases with plasmid copy number, such that each copy imposes a fitness burden of 0.17% (± 0.008%), greatly exceeding the cost expected for it to be stably maintained in yeast populations. Our results demonstrate the crucial importance of copy number in the evolution of yeast per 2 μm associations and pave the way for future studies examining how selection can shape the cost of multicopy elements.     

Activity of single-stranded DNA endonucleases in mung bean is associated with cell division

A single-strand-specific endonuclease from mung bean sprouts is widely usedin molecular biology. However, the biological role of this enzyme is unknown. We studied the spatial and temporal activity of single-stranded DNA endonucleases in mung bean seedling by following enzyme activity that linearizes supercoiled plasmid DNA, a characteristic of this type of enzyme. The formation of a linear molecule from supercoiled DNA was found to occur in two distinguishable steps. The first, which involves introducing a nick into the supercoiled DNA and relaxing it, is very rapid and complete within a few seconds. The second step of cleaving the opposite strand to generate a unit-length linear duplex DNA is a relatively slow process. Analysis of the DNA cleavage sites showed the nuclease preferentially cuts supercoiled DNA at an AT-rich region. Varying levels of nuclease activity could be detected in different tissues of the mung bean seedling. The highest activity was in the root tip and was correlated with histone H1 kinase activity. This implies a link between nuclease activity and cell division. Induction of cell division in mung bean hypocotyls with auxin promoted formation of root primordia and considerably increased the activity of single-stranded DNA endonucleases. The nuclease activity and histone H1 kinase activity were reduced in mung bean cuttings treated with hydroxyurea, but not in cuttings treated with oryzalin. The potential function of single-stranded DNA endonucleases is discussed.     

Accumulation of single strand interruptions within the yeast 2 microns DNA plasmid during replication in a DNA ligase mutant

Summary We have investigated the fate of the yeast 2 m DNA plasmid in strains with a temperature sensitive mutation of DNA ligase. At the restrictive temperature the plasmid DNA collects as an open circular form with single strand interruptions. Both alpha factor pheromone, which arrests cells before the start of S phase, and hydroxyurea, which blocks progression through S phase, prevent the appearance of the open circular form. Thus, interrupted plasmid DNA does not accumulate in the absence of DNA replication. On average the interrupted molecules contain four to five interruptions per newly replicated strand. Most of the interruptions are nicks (breaks in a single phosphate ester bond) rather than gaps (absence of one or more nucleotides in a strand) as judged by the in vitro conversion of the interrupted molecules into a covalently closed form by DNA ligase. Mapping of the position of the interruptions reveals no predominate sites.     

Dolichos biflorus agglutinin-binding site expression in basal keratinocytes is associated with cell differentiation

A basal layer of squamous epithelia such as epidermis contains stem cells, transit amplifying cells as well as postmitotic differentiating cells. A detailed knowledge of the transition among these cell types in the course of epidermal renewal is important. It would help in better understanding of many pathological processes, including cancer, and in employment of epidermal cells for therapeutic purposes. In this study we analyzed the possible role of Dolichos biflorus agglutinin (DBA)-reactive alpha-N-acetylgalactosamine glycosylation in behavior of the human epidermal basal cells under in vivo and in vitro conditions. The data received from porcine epidermis were also included. Part of basal cells was positive for DBA-binding sites and these cells exhibited a lower presence of beta1 integrin in their basal surface connected to the basement membrane. The perinuclear Golgi-like accumulation of beta1 integrin was observed in some cultured keratinocytes. The co-localization of integrin with DBA-binding sites and 58 kDa protein suggests that alpha-N-acetylgalactosamine glycosylation could be related to beta1 integrin retention in the endoplasmatic reticulum Golgi intermediate compartment (ERGIC) at the beginning of the secretory pathway. The lack of anchorage in culture elevated the number of DBA-binding site positive cells without significant influence on cell growth when cells isolated directly from epidermis were employed in study. Some role of DBA-reactive glycoligand expressions in a suprabasal movement of differentiated basal cells can be hypothesized.     

Relocation of a cytoplasmic yeast linear plasmid to the nucleus is associated with circularization via nonhomologous recombination involving inverted terminal repeats

The linear plasmid pCLU1 from the yeast Kluyveromyces lactis normally replicates in the cytoplasm, with the aid of the helper linear plasmid pGKL2, using terminal protein (TP) as a primer. However, it relocates to the nucleus when selection is applied for the expression of a plasmid-borne nuclear marker. Migration to the nucleus occurred in K. lactis at a frequency of about 10⁻³/cell ten or more times higher than the rate observed in Saccharomyces cerevisiae. The nuclear plasmids existed only in a circularized form in K. lactis, while in S. cerevisiae a telomere-associated linear form is also found. Sequence analysis showed that circularization in K. lactis was caused by non-homologous recombination between the inverted terminal repeat (ITR) at the ends of the linear form and non-specific internal target sites in pCLU1. No sequence similarity existed among the junction sites, indicating that the free ITR end plays a crucial role in circularization. In S. cerevisiae, circular plasmids were generated not only by non-homologous recombination, but also by homologous recombination between short direct repeats within pCLU1. Circularization via the ITR end was observed independently of RAD52 activity. Sequences highly homologous to ARS core elements, 5′-ATTTATTGTTTT-3′ for K. lactis and 5′-(A/T)TTTAT(T/G)TTT(A/T)-3′ for S. cerevisiae, were detected at multiple sites in the nuclear forms of the plasmids. Received: 25 October 1999 / Accepted: 13 March 2000     

DNA damage-induced RPA focalization is independent of gamma-H2AX and RPA hyper-phosphorylation

Replication protein A (RPA) is the major eukaryotic single stranded DNA binding protein that plays a central role in DNA replication, repair and recombination. Like many DNA repair proteins RPA is heavily phosphorylated (specifically on its 32 kDa subunit) in response to DNA damage. Phosphorylation of many repair proteins has been shown to be important for their recruitment to DNA damage-induced intra-nuclear foci. Further, phosphorylation of H2AX (gamma-H2AX) has been shown to be important for either the recruitment or stable retention of DNA repair proteins to these intra-nuclear foci. We address here the relationship between DNA damage-induced hyper-phosphorylation of RPA and its intra-nuclear focalization, and whether gamma-H2AX is required for RPA's presence at these foci. Using GFP-conjugated RPA, we demonstrate the formation of extraction-resistant RPA foci induced by DNA damage or stalled replication forks. The strong DNA damage-induced RPA foci appear after phosphorylated histone H2AX and Chk1, but earlier than the appearance of hyper-phosphorylated RPA. We demonstrate that while the functions of phosphoinositol-3-kinase-related protein kinases are essential for DNA damage-induced H2AX phosphorylation and RPA hyper-phosphorylation, they are dispensable for the induction of extraction-resistant RPA and RPA foci. Furthermore, in mouse cells genetically devoid of H2AX, DNA damage-induced extraction-resistant RPA appears with the same kinetics as in normal mouse cells. These results demonstrate that neither RPA hyper-phosphorylation nor H2AX are required for the formation in RPA intra-nuclear foci in response to DNA damage/replicational stress and are consistent with a role for RPA as a DNA damage sensor involved in the initial recognition of damaged DNA or blocked replication forks.     

Splice-isoform specific immunolocalization of neuronal nitric oxide synthase in mouse and rat brain reveals that the PDZ-complex-building nNOSalpha beta-finger is largely exposed to antibodies

Knock out mice deficient for the splice-isoform alphaalpha of neuronal nitric oxide synthase (nNOSalphaalpha) display residual nitric oxide synthase activity and immunosignal. To attribute this signal to the two minor neuronal nitric oxide synthase splice variants, betabeta and gammagamma, we generated isoform-specific anti-peptide antibodies against the nNOSalphaalpha specific betabeta-finger motif involved in PDZ domain scaffolding and the nNOSbetabeta specific N-terminus. The nNOSalphaalpha betabeta-finger-specific antibody clearly recognized the 160-kDa band of recombinant nNOSalphaalpha on Western blots. Using immunocytochemistry, this antibody displayed, in rats and wild-type mice, a labeling pattern similar to but not identical with that obtained using a commercial pan-nNOS antibody. This similarity indicates that the majority of immunocytochemically detectable nNOS is not likely to be complexed with PDZ-domain proteins via the betabeta-finger motif. This conclusion was confirmed by the inhibition of PSD-95/nNOS interaction by the nNOSalphaalpha betabeta-finger antibody in pull-down assays. By contrast, nNOSalphaalpha betabeta-finger labeling was clearly reduced in hippocampal and cortical neuropil areas enriched in NMDA receptor complex containing spine synapses. In nNOSalphaalpha knock out mice, nNOSalphaalpha was not detectable, whereas the pan-nNOS antibody showed a distinct labeling of cell bodies throughout the brain, most likely reflecting betabeta/gammagamma-isoforms in these cells. The nNOSbetabeta antibody clearly detected bacterial expressed nNOSbetabeta fusion protein and nNOSbetabeta in overexpressing HEK cells by Western blotting. Immunocytochemically, individual cell bodies in striatum, cerebral cortex, and in some brain stem nuclei were labeled in knock out but not in wild-type mice, indicating an upregulation of nNOSbetabeta in nNOSalphaalpha deficient animals.     

A global but stable change in HeLa cell morphology induces reorganization of DNA structural loop domains within the cell nucleus

DNA of higher eukaryotes is organized in supercoiled loops anchored to a nuclear matrix (NM). The DNA loops are attached to the NM by means of non-coding sequences known as matrix attachment regions (MARs). Attachments to the NM can be subdivided in transient and permanent, the second type is considered to represent the attachments that subdivide the genome into structural domains. As yet very little is known about the factors involved in modulating the MAR-NM interactions. It has been suggested that the cell is a vector field in which the linked cytoskeleton-nucleoskeleton may act as transducers of mechanical information. We have induced a stable change in the typical morphology of cultured HeLa cells, by chronic exposure of the cells to the polar compound dimethylsulfoxide (DMSO). Using a PCR-based method for mapping the position of any DNA sequence relative to the NM, we have monitored the position relative to the NM of sequences corresponding to four independent genetic loci located in separate chromosomes representing different territories within the cell nucleus. Here, we show that stable modification of the NM morphology correlates with the redefinition of DNA loop structural domains as evidenced by the shift of position relative to the NM of the c-myc locus and the multigene locus PRM1 --> PRM2 --> TNP2, suggesting that both cell and nuclear shape may act as cues in the choice of the potential MARs that should be attached to the NM.     

Beta-endorphin regulation of MAPKs in cultured human articular chondrocytes: MAPK inhibitors prevent the increase of IL-1 beta protein levels during beta-endorphin stimulation

We investigated the effect of β-endorphin on the activities of mitogen-activated protein kinases in cultured human articular chondrocytes in order to elucidate its effect on cartilage. Monolayer cultures of chondrocytes obtained from patients undergoing total knee arthroplasty were treated with 60, 600, or 6000 ng/ml β-endorphin, or 100 ng/ml naltrexone combined with 600 ng/ml β-endorphin. The regulation of three major mitogen-activated protein kinases phosphorylation, ERKp44/p42, p38, and JNK, was determined by Western blotting. We also examined the influence of specific mitogen-activated protein kinase inhibitors on IL-1β protein levels during β-endorphin stimulation. The results demonstrate that β-endorphin, dependent on concentration and duration of stimulation, significantly affected the activation of the three mitogen-activated protein kinases in cultured human articular chondrocytes. Naltrexone in some cases significantly regulated the mitogen-activated protein kinases in different ways when added to β-endorphin 600 ng/ml. Furthermore, specific mitogen-activated protein kinase inhibitors hindered the increase of IL-1β during β-endorphin incubation. The effect of β-endorphin seen in this study is considered critical for the production of several mediators of cartilage damage in an arthritic joint.     

DNA damage during mitosis invokes a JNK‐mediated stress response that leads to cell death

Mitotic catastrophe is a phenomenon displayed by cells undergoing aberrant mitosis to eliminate cells that fail to repair the errors. Why and how mitotic catastrophe would lead to cell death remains to be resolved and the answer will prove valuable in design of better therapeutic agents that specifically target such cells in mitosis. The antibiotic actinomycin D has been shown to induce chromosomal lesions in lower order organisms as well as in human interphase cells. Relatively few studies have been conducted to elucidate molecular events in the context of mitotic DNA damage. We have previously established a model of mitotic catastrophe in human HeLa cells induced by actinomycin D. Here, we show that actinomycin D induce cellular stress via DNA damage during mitosis. The higher order packing of chromosomes during mitosis might impede efficient DNA repair. γH2AX serves as a marker for DNA repair and active JNK interacts with γH2AX in actinomycin D‐treated mitotic extracts. We believe JNK might be in part, responsible for the phosphorylation of H2AX and thereby, facilitate the propagation of a positive signal for cell death, when repair is not achieved. The mitotic cell activates JNK‐mediated cell death response that progresses through a caspase cascade downstream of the mitochondria. In the mean time, remaining checkpoint signals may be sufficient to put a restraining hand on entry into anaphase and the cell eventually dies in mitosis. J. Cell. Biochem. 110: 725–731, 2010. © 2010 Wiley‐Liss, Inc.     

Properties of a tobacco DNA methyltransferase, NtMET1 and its involvement in chromatin movement during cell division

BACKGROUND AND AIMS: Plants possess three types of DNA methyltransferase, among which methyltransferase type 1 (MET1) is considered to play a major role by maintaining the CpG methylation patterns. However, little information is available as to its enzymatic activity, interacting proteins and spatial and temporal behaviours during DNA replication. In the present study, one example, NtMET1 from tobacco plants, was selected and an analysis was made of its biochemical properties and cellular localization. METHODS: NtMET1 was expressed in Sf9 insect cells, and a purified sample was subjected to a standard in vitro methylation assay. Intramolecular interaction was examined by the yeast two-hybrid and pull-down assays. Transgenic tobacco plants (Nicotiana tabacum) over-expressing NtMET1 were constructed via Agrobacterium-mediated transformation. Cellular localization was examined by fluorescence protein fusion, which was expressed in tobacco bright yellow 2 cells. KEY RESULTS: In vitro assays showed no detectable methylation activity when both hemimethylated and unmethylated DNA samples were used as the substrate. In planta assays with over-expressing transgenic lines showed no hypermethylation but rather hypomethylation of genomc DNA. The inability of methylation was conceivably due to a tight intramolecular interaction between the N- and C-terminal regions with the catalytic domain residing on the C-terminus being completely masked. Cellular localization analyses indicated that NtMET1 localized to the nucleus in the resting stage and migrates to the cytoplasm during mitosis, particularly at metaphase. The pattern observed resembled that of Ran GTPase, and in vitro pull-down assays showed a clear interaction between NtMET1 and AtRAN3, an Arabidopsis orthologue of tobacco Ran GTPase, NtRan-A1. CONCLUSIONS: The results suggest that enzymatic activity of NtMET1 is well adjusted by its own intra/intermolecular interaction and perhaps by interactions with other proteins, one of which was found to be Ran GTPase. Results also revealed that NtMET1 becomes localized to the vicinity of chromatin with the aid of Ran GTPase during cell division, and may play an important role in progress through mitosis independently of methylation activity.     

Baker's yeast mediated reduction of β-keto esters and β-keto amides in an organic solvent system

The baker's yeast mediated reduction of four β-keto esters in petroleum ether indicated that the size of the group attached to the keto carbon affected their reactivity. Ethyl 3-phenyl-3-oxopropanoate (1), which has a phenyl group directly attached to the keto carbon, is incompletely reduced using 20 g yeast/mmol substrate, ethyl 4-phenyl-3-oxobutanoate (2), which has one methylene group between the phenyl and keto carbon, was also incompletely reduced using 20 g yeast/mmol, although the extent of reduction was about double that of (1), ethyl 5-phenyl-3-oxopentanoate (3), which has two methylene groups between the phenyl and keto carbon, is completely reduced using 10 g yeast/mmol and ethyl 3-oxobutanoate (4), which has a methyl group attached to the keto carbon shows complete reduction using only 1 g yeast/mmol. The corresponding β-keto amides are considerably less reactive than the corresponding β-keto esters with only the amides derived from ethyl 3-oxobutanoate indicating any significant reduction using 20 g yeast/mmol.     

Cytotoxic effect of multinucleation in HeLa cell cultures associated with the presence of Vir plasmid in Escherichia coli strains

The occurrence activity and localization of calmodulin in three heterocystous cyanobacteria of the genus Anabaena were studied. Boiled crude extracts caused a Ca2+-dependent stimulation of NAD kinase. Such a stimulation was blocked by EGTA and chlorpromazine, SDS-PAGE and Western blot analysis using antiserum against eukaryotic spinach calmodulin, revealed a polypeptide of about 17 kDa. Immunogold localization of calmodulin gave a dense gold label in both vegetative cells and heterocysts. The label was mainly confined to the centroplasm in vegetative cells, while it was evenly distributed in the cytoplasm of mature heterocysts.     

Sex drives intracellular conflict in yeast

Theory predicts that sex can drive the evolution of conflict within the cell. During asexual reproduction, genetic material within the cell is inherited as a single unit, selecting for cooperation both within the genome as well as between the extra‐genomic elements within the cell (e.g. plasmids and endosymbionts). Under sexual reproduction, this unity is broken down as parental genomes are distributed between meiotic progeny. Genetic elements able to transmit to more than 50% of meiotic progeny have a transmission advantage over the rest of the genome and are able to spread, even where they reduce the fitness of the individual as a whole. Sexual reproduction is therefore expected to drive the evolution of selfish genetic elements (SGEs). Here, we directly test this hypothesis by studying the evolution of two independent SGEs, the 2‐μm plasmid and selfish mitochondria, in populations of Saccharomyces cerevisiae. Following 22 rounds of sexual reproduction, 2‐μm copy number increased by approximately 13.2 (± 5.6) copies per cell, whereas in asexual populations copy number decreased by approximately 5.1 (± 1.5) copies per cell. Given that the burden imposed by this parasite increases with copy number, these results support the idea that sex drives the evolution of increased SGE virulence. Moreover, we found that mitochondria that are respiratory‐deficient rapidly invaded sexual but not asexual populations, demonstrating that frequent outcrossed sex can drive the de novo evolution of genetic parasites. Our study highlights the genomic perils of sex and suggests that SGEs may play a key role in driving major evolutionary transitions, such as uniparental inheritance.