猪TAF7基因的克隆、染色体定位和组织表达谱分析

 【目的】通过对猪TAF7基因初步的研究,为猪分子遗传育种提供基础分子生物学信息,为猪的遗传育种提供分子标记。【方法】以五指山猪为研究对象,克隆TAF7基因,分析该基因结构特点,然后利用IMpRH（the INRA-University of Minnesota porcine radiation hybrid,法国农业科学院-明尼苏达大学的辐射杂种克隆板）分析该基因在猪染色体上定位信息,利用半定量RT-PCR方法,分析该基因在成年五指山猪16个不同组织（心脏、背肌、淋巴、脾脏、肝脏、肾脏、肺脏、子宫、睾丸、胃、小肠、大肠、卵巢、胸腺、脑、脂肪）的表达谱信息。【结果】克隆得到长1 701 bp的五指山猪TAF7基因序列,其中包括1 050 bp完整CDS（Coding Sequence,编码序列）区域,分析表明其编码含349个氨基酸的蛋白质。利用IMpRH分析结果表明,猪TAF7基因与分子标记SW1879和IL4（interleukin-4,白细胞介素-4）紧密连锁,LOD（Limit of Detection,检测极限）值分别为6.69和6.15。组织表达谱分析结果显示该基因在大多数组织中均有表达,其中在睾丸表达量较高,而在心脏、背肌中表达很低。【结论】猪TAF7基因5’UTR中有一个短的内含子,而在该基因CDS区域没有内含子。猪TAF7蛋白序列与人TAF7蛋白序列的相似性较高,二者在生物系统发育树中的距离最接近。猪TAF7基因在大多数组织中均表达,在猪睾丸中表达量较高,证实它调节目的基因转录具有广泛性。     

番茄Trihelix家族SIP1亚家族基因(SlGT-33)克隆表达分析及功能研究

【目的】对番茄三螺旋（Trihelix）家族SIP1亚家族基因（SlGT-33）进行克隆表达及功能研究,为深入解析SIP1亚家族成员调控植物生长发育机制及选育矮化番茄品种提供理论依据。【方法】以番茄品种AC++为材料,PCR扩增SlGT-33基因,对其进行序列分析,采用实时荧光定量PCR（qRT-PCR）检测SlGT-33基因在不同组织及外源激素和非生物胁迫下的表达模式,并利用RNAi技术鉴定SlGT-33基因的生物学功能。【结果】扩增获得的SlGT-33基因（GenBank登录号Solyc12g043090）开放阅读框（ORF）为1125 bp,编码374个氨基酸残基,与已知同源基因序列（GenBank登录号XP004252336.1）仅缺少3个碱基。SlGT-33与SlGT-17（GenBank登录号Solyc05g018350）位于同一分支,虽然二者的氨基酸序列相似性最高,但仅为45.8%。SlGT-33基因在茎和成熟叶中的相对表达量较高,显著高于其他组织（P<0.05,下同）;经IAA、GA₃和MeJA处理后,SlGT-33基因的相对表达量均与对照无显著差异（P>0.05,下同）;SlGT-33基因经ABA处理2～24 h时相对表达量均显著高于对照。SlGT-33基因在高盐胁迫和机械损伤下的相对表达量与对照无显著差异;高温胁迫和低温胁迫下SlGT-33基因表达整体上均呈逐渐降低的变化趋势;在脱水胁迫下SlGT-33基因表达整体上均呈逐渐升高的变化趋势。通过农杆菌介导转化法获得6个SlGT-33-RNAi沉默株系,沉默效率为64%～83%。生长70 d的SlGT-33-RNAi沉默株系RNAi-4和RNAi-5幼苗株高和节间长度均为野生型的60%左右,且复叶结构尺寸明显变小,花序提前形成。茎尖组织中顶端分生组织关键转录因子基因KNOX2和WUS基因在SlGT-33-RNAi沉默株系的相对表达量均极显著（P<0.01）或显著高于野生型,腺苷酸异戊烯转移酶（CTK合成的关键酶）编码基因IPT2和茎尖生长点调控基因PHAN基因在2个SlGT-33-RNAi沉默株系的相对表达量均显著低于野生型。顶端分生组织关键转录因子基因KNOX1基因在2个SlGT-33-RNAi沉默株系的相对表达量与野生型无显著差异。SlGT-33-RNAi沉默株系茎尖组织的细胞分裂素（CTK）含量显著低于野生型。【结论】SlGT-33基因属于环境敏感型基因,其表达受ABA和脱水胁迫诱导,但受极端温度的抑制。抑制SlGT-33基因表达会导致了番茄植株矮化和生殖生长加速,其作用机制与顶端分生组织的CTK合成受抑制及茎尖组织调控基因KNOX2、PHAN和WUS的异常表达密切相关。     

Protein tyrosine kinase Wee1B is essential for metaphase II exit in mouse oocytes

Waves of cyclin synthesis and degradation regulate the activity of Cdc2 protein kinase during the cell cycle. Cdc2 inactivation by Wee1B-mediated phosphorylation is necessary for arrest of the oocyte at G2-prophase, but it is unclear whether this regulation functions later during the metaphase-to-anaphase transition. We show that reactivation of a Wee1B pathway triggers the decrease in Cdc2 activity during egg activation. When Wee1B is down-regulated, oocytes fail to form a pronucleus in response to Ca(2+) signals. Calcium-calmodulin-dependent kinase II (CaMKII) activates Wee1B, and CaMKII-driven exit from metaphase II is inhibited by Wee1B down-regulation, demonstrating that exit from metaphase requires not only a proteolytic degradation of cyclin B but also the inhibitory phosphorylation of Cdc2 by Wee1B.     

Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex

Deregulation of Akt/protein kinase B (PKB) is implicated in the pathogenesis of cancer and diabetes. Akt/PKB activation requires the phosphorylation of Thr308 in the activation loop by the phosphoinositide-dependent kinase 1 (PDK1) and Ser473 within the carboxyl-terminal hydrophobic motif by an unknown kinase. We show that in Drosophila and human cells the target of rapamycin (TOR) kinase and its associated protein rictor are necessary for Ser473 phosphorylation and that a reduction in rictor or mammalian TOR (mTOR) expression inhibited an Akt/PKB effector. The rictor-mTOR complex directly phosphorylated Akt/PKB on Ser473 in vitro and facilitated Thr308 phosphorylation by PDK1. Rictor-mTOR may serve as a drug target in tumors that have lost the expression of PTEN, a tumor suppressor that opposes Akt/PKB activation.     

Acetylation by Tip60 is required for selective histone variant exchange at DNA lesions

Phosphorylation of the human histone variant H2A.X and H2Av, its homolog in Drosophila melanogaster, occurs rapidly at sites of DNA double-strand breaks. Little is known about the function of this phosphorylation or its removal during DNA repair. Here, we demonstrate that the Drosophila Tip60 (dTip60) chromatin-remodeling complex acetylates nucleosomal phospho-H2Av and exchanges it with an unmodified H2Av. Both the histone acetyltransferase dTip60 as well as the adenosine triphosphatase Domino/p400 catalyze the exchange of phospho-H2Av. Thus, these data reveal a previously unknown mechanism for selective histone exchange that uses the concerted action of two distinct chromatin-remodeling enzymes within the same multiprotein complex.     

The yeast cell cycle gene CDC34 encodes a ubiquitin-conjugating enzyme

Mutants in the gene CDC34 of the yeast Saccharomyces cerevisiae are defective in the transition from G1 to the S phase of the cell cycle. This gene was cloned and shown to encode a 295-residue protein that has substantial sequence similarity to the product of the yeast RAD6 gene. The RAD6 gene is required for a variety of cellular functions including DNA repair and was recently shown to encode a ubiquitin-conjugating enzyme. When produced in Escherichia coli, the CDC34 gene product catalyzed the covalent attachment of ubiquitin to histones H2A and H2B in vitro, demonstrating that the CDC34 protein is another distinct member of the family of ubiquitin-conjugating enzymes. The cell cycle function of CDC34 is thus likely to be mediated by the ubiquitin-conjugating activity of its product.     

Bora and the kinase Aurora a cooperatively activate the kinase Plk1 and control mitotic entry

A central question in the study of cell proliferation is, what controls cell-cycle transitions? Although the accumulation of mitotic cyclins drives the transition from the G2 phase to the M phase in embryonic cells, the trigger for mitotic entry in somatic cells remains unknown. We report that the synergistic action of Bora and the kinase Aurora A (Aur-A) controls the G2-M transition. Bora accumulates in the G2 phase and promotes Aur-A-mediated activation of Polo-like kinase 1 (Plk1), leading to the activation of cyclin-dependent kinase 1 and mitotic entry. Mechanistically, Bora interacts with Plk1 and controls the accessibility of its activation loop for phosphorylation and activation by Aur-A. Thus, Bora and Aur-A control mitotic entry, which provides a mechanism for one of the most important yet ill-defined events in the cell cycle.