酵母PHO85结合蛋白PAP1基因的克隆和表达

酵母调探因了ＰＨＯ８５是一个依赖于细胞周期蛋白（ｃｙｃｌｉｎ）的蛋白酶（ＣＤＫ）,参与对细胞周期和酸性磷酸酯酶基因表达的调控。冯ＰＨＯ８５为靶分子,利用酵母的染色体基因文库中克隆到了一个新的与ＰＨＯ８５相结合的蛋白因子的基因,利用酵母双杂交（ｔｗｏ－ｈｙｂｒｉｄ）系统从酵母的染色体基因文加中克隆到了一个新的与ＰＨＯ８５相结合的蛋白因子的基因,将此蛋白质命名为ＰＡＰ１（ＰＨＯ８５ａｓｓｏｃｉａｔｅｄ     

p16抑癌基因和肿瘤

ｐ１６基因（又名ＭＴＳ１,多肿瘤抑制基因）所编码的蛋白是细胞周期蛋白Ｄ／ＣＤＫ激酶的抑制蛋白,在细胞周期的调控中起着重要作用,能和ＣＤＫ４结合,抑制ＣＤＫ４／ｃｙｃｌｉｎＤ复合物的催化活性。当ｐ１６基因发生突变和缺失,就会丧失对细胞分裂的调控,导致细胞的异常增殖,最终形成肿瘤。研究表明：ｐ１６基因的突变和缺失,和许多肿瘤的发生有关。ｐ１６基因是一种新的肿瘤抑制基因。     

细胞周期后期促进因子DIG9对植物侧根发生的调控研究

在植物体内,细胞周期对于植物的萌发、生长、开花、结实等各个生长发育阶段具有重要作用。细胞周期正常运转需要依赖一些细胞周期蛋白,但是目前关于细胞周期蛋白调控根发育的分子机制还不清楚。通过筛选模式植物拟南芥的根发育异常突变体,分离鉴定了1个突变体dig9（drought inhibition of lateral root growth）,该突变体表现为主根短、侧根少、发育迟缓、顶端分生组织变小、叶片扭曲、无主茎等表型。通过图位克隆,成功定位并克隆了DIG9基因,该基因编码一个细胞周期蛋白,是有丝分裂后期促进复合体的一个亚基APC8 （anaphase-promoting complex）。通过亚细胞定位发现DIG9定位于细胞核;qRT-PCR检测发现DIG9基因在根中有较高的表达量,进一步通过启动子-GUS报告系统发现DIG9在根尖、侧根和顶端分生组织等细胞分裂旺盛区域表达。外施IAA能恢复dig9突变体的侧根表型但不能恢复根短表型。dig9突变体对干旱及盐胁迫反应不敏感。研究结果表明DIG9基因可能通过影响IAA的产生来调控植物的侧根发育。     

CIP—KIP细胞周期蛋白依赖激酶抑制因子家族

细胞周期蛋白依赖激酶抑制因子（ＣＫＩ）是细胞周期调控网络的主要成员之一,在周期调控中具有重要的地位,ＣＩＰ－ＣＫＩ两个家族之一,与细胞生长分化和肿瘤发生方面有极其重要的关系。本文对ＣＩＰ－ＫＩＰ家族的细胞周期调控,基因敲除以及促ＣＤＫ／ＣｙｃｌｉｎＤ装配功能的最新进展进行综述。     

细胞周期蛋白B1、D1和E真核表达载体的构建及表达

目的：为研究细胞周期蛋白（cyclin）在肿瘤形成过程中的分子机制,构建带FLAG标签的细胞周期蛋白B1、D1、E的真核表达载体,并检测其在293T细胞中的表达。方法：以乳腺cDNA文库为模板,分别扩增细胞周期蛋白B1、D1、E基因全长编码区序列,克隆到pcDNA3-FLAG真核表达载体上;用脂质体介导的基因瞬时转染法,将重组正确的表达载体转染293T细胞,检测细胞中的FLAG融合蛋白的表达。结果：酶切鉴定和DNA序列分析显示构建了正确的FLAG-Cyclin真核表达载体,Western印迹分析表明克隆的载体都能在真核细胞中表达分子大小相符的重组蛋白。结论：构建了FLAG-CyclinBl、FIAG-CyclinDl、FLAG-CyclinE真核表达载体,为细胞周期蛋白及其相关蛋白的研究奠定了基础。     

人类CD34^＋造血干／祖细胞的分子生物学特性

造血干／祖细胞（ＨＳＣ／ＨＰＣ）以有序的不同年龄等级结构状态存在于体内,它们的增殖、分化、成熟及程序死亡是一个连续的动态过程。ＣＤ３４抗原是目前所能认识表达在ＨＳＣ／ＨＰＣ表面上的最早抗原分子,对其分子结构的研究使人们更加认识到ＣＤ３４＾＋ＨＳＣ／ＨＰＣ的不同功能亚群、表达范围及其它生物学特征,诸如细胞周期状态、光散射性、造血生长因子受体（ＨＧＦｓＲ）Ｐ糖蛋白（Ｐｇｐ）或多药耐药基因（ＭＤＲ）的表     

基于DNA序列信息的贞琵甲属物种界定分析（鞘翅目：拟步甲科：琵甲亚科）

贞琵甲属Agnaptoria是小琵甲亚族Gnaptorinina中第三大属和青藏高原特有类群,已知36种/亚种。本文选取3个线粒体基因（COI; Cytb; 16S rDNA）和1个核基因（28S rDNA-D2）片段,采用最大似然法（Maximum likelihood,ML）构建了该属的系统发育树;运用ASAP（Assemble Species by Automatic Partitioning）、GMYC（Generalized Mixed Yule Coalescent）和PTP（Poisson Tree Processes）3种方法对该属进行了分子物种界定分析。结果表明：综合运用3种分子物种界定方法的界定结果与形态鉴定结果基本吻合。依据形态特征与分子物种界定技术相结合的综合鉴定方法,大大提高了该类群的物种鉴定效率,为该类群未来在系统发育、地理分布格局演化等方面的研究提供了可靠的分子数据。     

ACC合酶基因（ACSG）可能是黄瓜雌性系的分子标记

利用本实验室根据已知序列分离得到的ＡＣＣ合酶基因（ＡＣＳＧ）为探针对不同黄瓜（Ｃｕｃｕｍｉｓ ｓａｔｉｖｕｓ Ｌ．）品种（系）的基因组ＤＮＡ进行Ｓｏｕｔｈｅｒｎ杂交,初步分析了该基因与黄瓜性别表型之间的相关性。发现在所检测的１０个不同品系中,ＡＣＳＧ与雌性系表型之间存在明显的相关关系,而且这种相关关系在不同的实验中具有良好的重复怀。ＡＣＳＧ基因可能是鉴定黄瓜雌性系的一个分子标记。     

硫化氢与细胞的增殖和凋亡

硫化氢是内源性气体分子家族中的一员,是一种气体递质（gasotransmitter）。近年来,内源性硫化氢的产生及生理意义已经被认识,其代谢异常与许多疾病有关。本文综述了最近发现的硫化氢对细胞增殖和凋亡的调节作用,并重点概述硫化氢细胞效应的分子机制,包括丝裂原活化蛋白激酶、细胞周期相关激酶、细胞死亡相关基因以及离子通道等的改变。对硫化氢调节细胞生长或死亡的深入了解将为新药设计及许多疾病的治疗提供新的思路。     

ＣＹＰ２１基因启动子结构功能及突变研究进展

２１羟化酶基因（ＣＹＰ２１基因）缺陷可引起２１羟化酶缺乏。近年对ＣＹＰ２１基因的研究取得了一定进展,本文介绍了该基因启动子区的位置、结构、调节因子,重要功能域及突变等方面的进展,从分子水平进一步加深了对ＣＡＨ的发病机理的了解。     

Differential contribution of inhibitory phosphorylation of CDC2 and CDK2 for unperturbed cell cycle control and DNA integrity checkpoints

Inhibition of cyclin-dependent kinases (CDKs) by Thr14/Tyr15 phosphorylation is critical for normal cell cycle progression and is a converging event for several cell cycle checkpoints. In this study, we compared the relative contribution of inhibitory phosphorylation for cyclin A/B1-CDC2 and cyclin A/E-CDK2 complexes. We found that inhibitory phosphorylation plays a major role in the regulation of CDC2 but only a minor role for CDK2 during the unperturbed cell cycle of HeLa cells. The relative importance of inhibitory phosphorylation of CDC2 and CDK2 may reflect their distinct cellular functions. Despite this, expression of nonphosphorylation mutants of both CDC2 and CDK2 triggered unscheduled histone H3 phosphorylation early in the cell cycle and was cytotoxic. DNA damage by a radiomimetic drug or replication block by hydroxyurea stimulated a buildup of cyclin B1 but was accompanied by an increase of inhibitory phosphorylation of CDC2. After DNA damage and replication block, all cyclin-CDK pairs that control S phase and mitosis were to different degrees inhibited by phosphorylation. Ectopic expression of nonphosphorylated CDC2 stimulated DNA replication, histone H3 phosphorylation, and cell division even after DNA damage. Similarly, a nonphosphorylation mutant of CDK2, but not CDK4, disrupted the G2 DNA damage checkpoint. Finally, CDC25A, CDC25B, a dominant-negative CHK1, but not CDC25C or a dominant-negative WEE1, stimulated histone H3 phosphorylation after DNA damage. These data suggest differential contributions for the various regulators of Thr14/Tyr15 phosphorylation in normal cell cycle and during the DNA damage checkpoint.     

Phosphorylation of mammalian CDC6 by cyclin A/CDK2 regulates its subcellular localization.

Cyclin-dependent kinases (CDKs) are essential for regulating key transitions in the cell cycle, including initiation of DNA replication, mitosis and prevention of re-replication. Here we demonstrate that mammalian CDC6, an essential regulator of initiation of DNA replication, is phosphorylated by CDKs. CDC6 interacts specifically with the active Cyclin A/CDK2 complex in vitro and in vivo, but not with Cyclin E or Cyclin B kinase complexes. The cyclin binding domain of CDC6 was mapped to an N-terminal Cy-motif that is similar to the cyclin binding regions in p21(WAF1/SDI1) and E2F-1. The in vivo phosphorylation of CDC6 was dependent on three N-terminal CDK consensus sites, and the phosphorylation of these sites was shown to regulate the subcellular localization of CDC6. Consistent with this notion, we found that the subcellular localization of CDC6 is cell cycle regulated. In G1, CDC6 is nuclear and it relocalizes to the cytoplasm when Cyclin A/CDK2 is activated. In agreement with CDC6 phosphorylation being specifically mediated by Cyclin A/CDK2, we show that ectopic expression of Cyclin A, but not of Cyclin E, leads to rapid relocalization of CDC6 from the nucleus to the cytoplasm. Based on our data we suggest that the phosphorylation of CDC6 by Cyclin A/CDK2 is a negative regulatory event that could be implicated in preventing re-replication during S phase and G2.     

Late-G1 cyclin-CDK activity is essential for control of cell morphogenesis in budding yeast

The accurate spatial and temporal coordination of cell polarization with DNA replication and segregation guarantees the fidelity of genetic transmission. Here we report that in Saccharomyces cerevisiae, a build-up or burst of G1 cyclin-dependent kinase (CDK) activity through activation of the cyclin genes CLN1,2 and PCL1,2 is essential for cell morphogenesis, but not for other events associated with the G1-S-phase transition, including DNA replication. Strains lacking a burst of late-G1 cyclin-CDK activity (LG1C(-)) undergo a catastrophic morphogenesis and halt the nuclear cycle at the morphogenesis checkpoint in G2 phase. Consistent with a role in morphogenesis, the Pho85 G1 cyclins Pcl1 and Pcl2 show a unique pattern of localization to sites of polarized cell growth, and strains lacking PCL1 and PCL2 show genetic interactions with the cell polarity GTPase Cdc42, its regulators and downstream effectors. Our data suggest that inability to assemble a septin ring and localize the GTP exchange factor Cdc24 at the incipient bud site may be the primary morphogenetic defects in LG1C-depleted cells. We conclude that a burst of late G1 cyclin-CDK activity is essential for establishing cell polarity and development of the cleavage apparatus.     

CDC6 requirement for spindle formation during maturation of mouse oocytes

A master regulator of DNA replication, CDC6 also functions in the DNA-replication checkpoint by preventing DNA rereplication. Cyclin-dependent kinases (CDKs) regulate the amount and localization of CDC6 throughout the cell cycle; CDC6 phosphorylation after DNA replication initiation leads to its proteolysis in yeast or translocation to the cytoplasm in mammals. Overexpression of CDC6 during the late S phase prevents entry into the M phase by activating CHEK1 kinase that then inactivates CDK1/cyclin B, which is essential for the G2/M-phase transition. We analyzed the role of CDC6 during resumption of meiosis in mouse oocytes, which are arrested in the first meiotic prophase with low CDK1/cyclin B activity; this is similar to somatic cells at the G2/M-phase border. Overexpression of CDC6 in mouse oocytes does not prevent resumption of meiosis. The RNA interference-mediated knockdown of CDC6, however, reveals a new and unexpected function for CDC6; namely, it is essential for spindle formation in mouse oocytes.     

On the concentrations of cyclins and cyclin-dependent kinases in extracts of cultured human cells

Cyclins and cyclin-dependent kinases (CDKs) are key regulators of the human cell cycle. Here we have directly measured the concentrations of the G(1) and G(2) cyclins and their CDK partners in highly synchronized human cervical carcinoma cells (HeLa). To determine the exact concentrations of cyclins and CDKs in the cell extracts, we developed a relatively simple method that combined the use of (35)S-labeled standards produced in rabbit reticulocyte lysates and immunoblotting with specific antibodies. Using this approach, we formally demonstrated that CDC2 and CDK2 are in excess of their cyclin partners. We found that the concentrations of cyclin A2 and cyclin B1 (at their peak levels in the G(2) phase) were about 30-fold less than that of their partner CDC2. The peak levels of cyclin A2 and cyclin E1, at the G(2) phase and G(1) phase, respectively, were only about 8-fold less than that of their partner CDK2. These ratios are in good agreement with size fractionation analysis of the relative amount of monomeric and complexed forms of CDC2 and CDK2 in the cell. All the cyclin A2 and cyclin E1 are in complexes with CDC2 and CDK2, but there are some indications that a significant portion of cyclin B1 may not be in complex with CDC2. Furthermore, we also demonstrated that the concentration of the CDK inhibitor p21(CIP1/WAF1) induced after DNA damage is sufficient to overcome the cyclin-CDK2 complexes in MCF-7 cells. These direct quantitations formally confirmed the long-held presumption that CDKs are in excess of the cyclins in the cell. Moreover, similar approaches can be used to measure the concentration of any protein in cell-free extracts.     

Intracellular localization of the cyclin-dependent kinase inhibitor p21<Superscript>CDKN1A</Superscript>-GFP fusion protein during cell cycle arrest

The cyclin-dependent kinase (CDK) inhibitor p21^CDKN1A is known to induce cell cycle arrest by inhibiting CDK activity and by interfering with DNA replication through binding to proliferating cell nuclear antigen. Although the molecular mechanisms have been elucidated, the temporal dynamics, as well as the intracellular sites of the activity of p21 bound to cyclin/CDK complexes during cell cycle arrest, have not been fully investigated. In this study we have induced the expression of p21^CDKN1A fused to green fluorescent protein (GFP) in HeLa cells, in order to visualize the intracellular localization of the inhibitor during the cell cycle arrest. We show that p21-GFP is preferentially expressed in association with cyclin E in cells arrested in G1 phase, and with cyclin A more than with cyclin B1 in cells arrested in the G2/M compartment. In addition, we show for the first time that p21-GFP colocalizes with cyclin E in the nucleolus of HeLa cells during the G1 phase arrest.O. Cazzalini and P. Perucca contributed equally to this work     

Complexity of the mechanisms of initiation and maintenance of DNA damage-induced G2-phase arrest and subsequent G1-phase arrest: TP53-dependent and TP53-independent roles

Through a detailed study of cell cycle progression, protein expression, and kinase activity in gamma-irradiated synchronized cultures of human skin fibroblasts, distinct mechanisms of initiation and maintenance of G2-phase and subsequent G1-phase arrests have been elucidated. Normal and E6-expressing fibroblasts were used to examine the role of TP53 in these processes. While G2 arrest is correlated with decreased cyclin B1/CDC2 kinase activity, the mechanisms associated with initiation and maintenance of the arrest are quite different. Initiation of the transient arrest is TP53-independent and is due to inhibitory phosphorylation of CDC2 at Tyr15. Maintenance of the G2 arrest is dependent on TP53 and is due to decreased levels of cyclin B1 mRNA and a corresponding decline in cyclin B1 protein level. After transiently arresting in G2 phase, normal cells chronically arrest in the subsequent G1 phase while E6-expressing cells continue to cycle. The initiation of this TP53-dependent G1-phase arrest occurs despite the presence of substantial levels of cyclin D1/CDK4 and cyclin E/CDK2 kinase activities, hyperphosphoryated RB, and active E2F1. CDKN1A (also known as p21(WAF1/CIP1)) levels remain elevated during this period. Furthermore, CDKN1A-dependent inhibition of PCNA activity does not appear to be the mechanism for this early G1 arrest. Thus the inhibition of entry of irradiated cells into S phase does not appear to be related to DNA-bound PCNA complexed to CDKN1A. The mechanism of chronic G1 arrest involves the down-regulation of specific proteins with a resultant loss of cyclin E/CDK2 kinase activity.     

Human TopBP1 participates in cyclin E/CDK2 activation and preinitiation complex assembly during G1/S transition

Human TopBP1 with eight BRCA1 C terminus domains has been mainly reported to be involved in DNA damage response pathways. Here we show that TopBP1 is also required for G(1) to S progression in a normal cell cycle. TopBP1 deficiency inhibited cells from entering S phase by up-regulating p21 and p27, resulting in down-regulation of cyclin E/CDK2. Although co-depletion of p21 and p27 with TopBP1 restored the cyclin E/CDK2 kinase activity, however, cells remained arrested at the G(1)/S boundary, showing defective chromatin-loading of replication components. Based on these results, we suggest a dual role of TopBP1 necessary for the G(1)/S transition: one for activating cyclin E/CDK2 kinase and the other for loading replication components onto chromatin to initiate DNA synthesis.     

Coordinated Activation of the Origin Licensing Factor CDC6 and CDK2 in Resting Human Fibroblasts Expressing SV40 Small T Antigen and Cyclin E

We have previously shown that SV40 small t antigen (st) cooperates with deregulated cyclin E to activate CDK2 and bypass quiescence in normal human fibroblasts (NHF). Here we show that st expression in serum-starved and density-arrested NHF specifically induces up-regulation and loading of CDC6 onto chromatin. Coexpression of cyclin E results in further accumulation of CDC6 onto chromatin concomitantly with phosphorylation of CDK2 on Thr-160 and CDC6 on Ser-54. Investigation of the mechanism leading to CDC6 accumulation and chromatin loading indicates that st is a potent inducer of cdc6 mRNA expression and increases CDC6 protein stability. We also show that CDC6 expression in quiescent NHF efficiently promotes cyclin E loading onto chromatin, but it is not sufficient to activate CDK2. Moreover, we show that CDC6 expression is linked to phosphorylation of the activating T loop of CDK2 in serum-starved NHF stimulated with mitogens or ectopically expressing cyclin E and st. Our data suggest a model where the combination of st and deregulated cyclin E result in cooperative and coordinated activation of both an essential origin licensing factor, CDC6, and an activity required for origin firing, CDK2, resulting in progression from quiescence to S phase.Upon mitogenic stimulation mammalian G1 CDKs⁴ trigger passage through the restriction point and the transition into DNA replication. In particular, cyclin E/CDK2 is activated in mid to late G1 and phosphorylates a variety of substrates that play critical roles in these processes. CDK2 cooperates with D-type cyclin/CDKs to inactivate E2F/pocket protein repressor complexes inducing the expression of DNA synthesis factors and other cell cycle regulators (reviewed in Refs. 1 and 2). CDK2 also phosphorylates DNA replication factors facilitating prereplication complex assembly and origin firing and plays additional roles in centrosome duplication and histone synthesis (reviewed in Ref. 1). In particular, it has been proposed that CDK2 phosphorylates the essential origin licensing factor CDC6 promoting its stabilization prior to inactivation of the APC^Cdh1 ubiquitin ligase (3). This is thought to ensure that CDC6 accumulation precedes accumulation of other APC substrates that inhibit origin licensing. Moreover, CDK2-independent cyclin E functions have also been reported to be important for prereplication complex assembly in cells in transit from G0 into G1 (4, 5). In keeping with its role as positive regulator of major G1 transitions, deregulation of the cyclin E via gene amplification or defective protein turnover is commonly seen in primary tumors and is associated with poor prognosis (6–8). In normal fibroblasts, ectopic expression of cyclin E has been associated with shortening of the G1 phase of the cell cycle (9, 10), and with induction of DNA damage (reviewed in Ref. 8). Cyclin E deregulation in certain human tumor cell lines and immortalized rat fibroblasts is associated with mitogen-independent cell cycle entry and progression through the cell cycle (11). However, when cyclin E is ectopically expressed in quiescent normal human fibroblasts (NHF), cells remain in G0 (12).We have recently reported that coexpression of SV40 small t antigen (st) in quiescent NHF with deregulated cyclin E expression is sufficient to trigger mitogen-independent cell cycle progression, proliferation beyond cell confluence, and foci formation. The bypass of quiescence induced by the expression of st and cyclin E is dependent on CDK2 activation (12). Thus, contrary to what is seen in normal murine cells (13), CDK2 activity appears essential for cell cycle progression when it is oncogenically driven by cyclin E and st expression (12). Because st is known to target pathways uniquely required for the transformation of human cells (14, 15), tumor cells with altered pathways that mimic st/cyclin E expression could predictably be sensitive to selective inhibition of CDK2 activity.Given the critical role of CDK2 activity in cyclin E and st cooperation in inducing cell proliferation and transformation of NHF, we sought to determine the factors and mechanisms by which st modulates CDK2 activation. In this report we have identified the CDC6 replication licensing factor as a cellular target of st. We also uncover CDC6 as a participant in the events leading to chromatin association of cyclin E and CDK2 and in phosphorylation of CDK2 on its activating T loop both in response to mitogenic stimulation, as well as expression of cyclin E and st in NHF.