哺乳动物核移植中供核与受体卵胞质细胞周期的相互关系

就供核与受体卵胞质细胞周期的相互关系问题进行了综述.核移植技术不管是在基础理论,还是在应用研究中都具有广泛的应用价值,但核移植的效率却很低,其根本原因是与核移植相关的许多基础理论问题尚不清楚,对这些问题的研究发现,维持重构卵核的正确倍性,并使其重新程序化是核移植成功的关键,不同的胞质受体及不同的供体细胞及其状态均对重构胚的发育有影响.     

Cold-Shock and the Mammalian Cell Cycle

no abstract available     

Cell Cycle Regulates Nuclear Stability of AID and Determines the Cellular Response to AID

AID (Activation Induced Deaminase) deaminates cytosines in DNA to initiate immunoglobulin gene diversification and to reprogram CpG methylation in early development. AID is potentially highly mutagenic, and it causes genomic instability evident as translocations in B cell malignancies. Here we show that AID is cell cycle regulated. By high content screening microscopy, we demonstrate that AID undergoes nuclear degradation more slowly in G1 phase than in S or G2-M phase, and that mutations that affect regulatory phosphorylation or catalytic activity can alter AID stability and abundance. We directly test the role of cell cycle regulation by fusing AID to tags that destabilize nuclear protein outside of G1 or S-G2/M phases. We show that enforced nuclear localization of AID in G1 phase accelerates somatic hypermutation and class switch recombination, and is well-tolerated; while nuclear AID compromises viability in S-G2/M phase cells. We identify AID derivatives that accelerate somatic hypermutation with minimal impact on viability, which will be useful tools for engineering genes and proteins by iterative mutagenesis and selection. Our results further suggest that use of cell cycle tags to regulate nuclear stability may be generally applicable to studying DNA repair and to engineering the genome.     

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell′s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry¹ offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-β1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture⁵. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.     

Mammalian Formin Fhod3 Regulates Actin Assembly and Sarcomere Organization in Striated Muscles

Actin filament assembly in nonmuscle cells is regulated by the actin polymerization machinery, including the Arp2/3 complex and formins. However, little is known about the regulation of actin assembly in muscle cells, where straight actin filaments are organized into the contractile unit sarcomere. Here, we show that Fhod3, a myocardial formin that localizes to thin actin filaments in a striated pattern, regulates sarcomere organization in cardiomyocytes. RNA interference-mediated depletion of Fhod3 results in a marked reduction in filamentous actin and disruption of the sarcomeric structure. These defects are rescued by expression of wild-type Fhod3 but not by that of mutant proteins carrying amino acid substitution for conserved residues for actin assembly. These findings suggest that actin dynamics regulated by Fhod3 are critical for sarcomere organization in striated muscle cells.In striated muscle, thin actin filaments and thick filaments of myosin are highly organized to form myofibrils (1) (Fig. 1A). During myofibrillogenesis, actin cytoskeleton undergoes dynamic remodeling to produce uniform lengths of straight filaments packaged in the sarcomere, a contractile unit of myofibrils (2–4). In nascent sarcomeres, a filamentous actin-containing structure, referred to as the Z-body or I-Z-I structure, emerges as a precursor of the Z-line that anchors actin filaments. Subsequent alignment of the precursors leads to formation of a striated pattern of the Z-line, and myosin filaments are incorporated between Z-lines. Finally, the M-line that serves as an anchoring site for myosin filaments becomes visible; the appearance is accompanied by alignment of the unanchored end of actin filaments (5). Thus, the mature distribution pattern of actin filaments is constructed at the final step in myofibril assembly, indicating that actin filaments continue to develop throughout myofibrillogenesis. However, the regulation of actin dynamics in this process has remained poorly understood. In nonmuscle cells, organization of actin cytoskeleton is achieved by two major actin nucleating-polymerizing systems, formins and the Arp2/3 complex, with the former producing long straight actin filaments and the latter producing branched actin network (6, 7). Because an unbranched straight actin filament is the major form in striated muscle cells, it is possible that a formin family protein serves as the key regulator of actin dynamics in myofibrils.Open in a separate windowFIGURE 1.Localization of Fhod3 in cultured rat cardiomyocytes. A, shown is a representation of the sarcomere structure (upper panel) and relative localization of Fhod3 and other sarcomeric proteins from B–D (lower panel). B–D, neonatal rat cardiomyocytes were subjected to immunofluorescent double staining for endogenous Fhod3 (red) and α-actinin (green) (B), myomesin (green) (C), or phalloidin (green) (D). For Fhod3 staining, the anti-Fhod3-(650–802) polyclonal antibodies were used. Scale bar, 10 μm.Formins are characterized by the presence of two conserved regions, the formin homology 1 and 2 domains (FH1 and FH2 domains, respectively)² (8, 9). The FH2 domain associates with the barbed end of an actin filament and promotes actin nucleation and polymerization. The FH2 domain continues to associate with the barbed end during polymerization; this processive association protects the growing barbed end from capping proteins that inhibit actin elongation. The FH1 domain, located N-terminally to the FH2 domain, accelerates the FH2-mediated actin elongation via recruiting profilin complexed with an actin monomer. Through cooperation of the FH1 and FH2 domains, formins produce long straight actin filaments even in the presence of capping proteins. Here, we focused on the role of the mammalian formin Fhod3 (previously designated as Fhos2L), which is expressed predominantly in the heart (10), in actin assembly in myofibrils.     

The Cell Cycle of Early Mammalian Embryos: Lessons from Genetic Mouse Models

Genes coding for cell cycle components predicted to be essential for its regulation have been shown to be dispensable in mice, at the whole organism level. Such studies have highlighted the extraordinary plasticity of the embryonic cell cycle and suggest that many aspects of in vivo cell cycle regulation remain to be discovered. Here, we discuss the particularities of the mouse early embryonic cell cycle and review the mutations that result in cell cycle defects during mouse early embryogenesis, including deficiencies for genes of the cyclin family (cyclin A2 and B1), genes involved in cell cycle checkpoints (Mad2, Bub3, Chk1, Atr), genes involved in ubiquitin and ubiquitin-like pathways (Uba3, Ubc9, Cul1, Cul3, Apc2, Apc10, Csn2) as well as genes the function of which had not been previously ascribed to cell cycle regulation (Cdc2l, E4F and Omcg1).     

RNA沉默抑制子p19调控细胞周期相关蛋白表达

番茄丛矮病毒的P19蛋白不仅是一个重要的病毒致病因子,而且还可作为RNA干扰(RNAi)的抑制子．这种作用是通过限制细胞内的小RNA,比如小干扰RNA(siRNAs)和微RNA(miRNAs)来实现．但是目前对P19蛋白在哺乳动物细胞上的作用还未见报道．构建了一株p19稳定表达的293细胞系,即293-p19．流式细胞仪分析发现在293细胞中过量表达P19蛋白可显著引发细胞周期的G2/M阻滞．细胞增殖实验显示,293-p19细胞的DNA复制及细胞生长均受到显著的抑制． 此外,研究还发现p19可使人胚肾293细胞内的细胞周期调控子的表达谱发生改变． 其中包括上调cyclin A1,CDK2,CDK4,CDK6,p18,cyclin D2,p19INK4d和E2F1,及下调p15,cyclin A,cyclin B1和cyclin E1的表达．上述研究结果提示,p19有可能靶向多个G2/M调控蛋白从而引发细胞的G2/M阻滞．     

细胞周期的调控及在核移植研究中的应用

细胞周期是生命活动中一个最重要的过程.以cyclin、CDK、CKI等细胞周期调控蛋白的相互作用推动着细胞周期时相的进展和时相之间的转变.这一过程受到严密的调控机制所监控.在核移植的研究中,对细胞周期进行调控,使细胞阻滞于某一特定时期有非常重要的意义.     

体细胞克隆哺乳动物胚胎发育的表观遗传学

如何提高克隆效率和体细胞核移植后表观遗传重编程的潜在机制的研究是当前生命科学的热点之一。将处于分化状态而进行核移植的体细胞转变成具有全能型的早期胚胎的关键是表观遗传的重编程。文章从基因印迹,x染色体失活,端粒长度等方面来探讨哺乳动物克隆胚胎在发育过程中的表观遗传重编程的机制。     

The Merkel Cell Polyomavirus Minor Capsid Protein

The surface of polyomavirus virions is composed of pentameric knobs of the major capsid protein, VP1. In previously studied polyomavirus species, such as SV40, two interior capsid proteins, VP2 and VP3, emerge from the virion to play important roles during the infectious entry process. Translation of the VP3 protein initiates at a highly conserved Met-Ala-Leu motif within the VP2 open reading frame. Phylogenetic analyses indicate that Merkel cell polyomavirus (MCV or MCPyV) is a member of a divergent clade of polyomaviruses that lack the conserved VP3 N-terminal motif. Consistent with this observation, we show that VP3 is not detectable in MCV-infected cells, VP3 is not found in native MCV virions, and mutation of possible alternative VP3-initiating methionine codons did not significantly affect MCV infectivity in culture. In contrast, VP2 knockout resulted in a >100-fold decrease in native MCV infectivity, despite normal virion assembly, viral DNA packaging, and cell attachment. Although pseudovirus-based experiments confirmed that VP2 plays an essential role for infection of some cell lines, other cell lines were readily transduced by pseudovirions lacking VP2. In cell lines where VP2 was needed for efficient infectious entry, the presence of a conserved myristoyl modification on the N-terminus of VP2 was important for its function. The results show that a single minor capsid protein, VP2, facilitates a post-attachment stage of MCV infectious entry into some, but not all, cell types.