卵泡发生过程中GDF-9和BMP-15(GDF-9B)的作用

近年来,对卵巢内卵泡发育,即从原始卵泡募集、优势选择、卵泡生长、排卵到黄体形成的调控研究取得了很大的进展.研究表明TGF-β超家族的许多成员在各阶段卵泡发生中起重要的调节作用.TGF-β超家族的两个成员-生长分化因子-9(GDF-9)和骨形态因子-15(BMP-15,或GDF-9B)主要在哺乳动物卵母细胞中表达,对卵泡生长发育起关键作用.本文对GDF-9和BMP-15在卵泡发育过程中的作用进行了综述.     

大熊猫9个BAC的测序、注释和进化分析

本文构建了相当于大熊猫10倍基因组覆盖度的BAC文库, 并随机挑选了其中9个BAC进行测序和组装, 9个BAC的选择满足更多基因更少重复序列的原则. 这9个BAC的组装将为评估基于新一代Illumina GA测序技术的大熊猫全基因组测序及组装的准确性提供有效资源. 运用同源比对和从头预测的方法, 对9个BAC, 共约878 kb的序列进行了基因和重复序列的注释以及进化分析. 一共预测到12个蛋白编码基因, 其中, 7个基因匹配到同源基因的功能注释. 这7个基因平均大小约41 kb, 编码区平均大小约1.2 kb, 每个基因平均约含6个外显子. 同时预测到7个tRNA基因. 大约27%的序列被注释为重复序列. 同时, 基于邻接法, 构建了包含人、小鼠、狗、猫以及大熊猫5个物种的物种进化树, 结果显示狗的基因与其他4个物种相比距大熊猫最近. 本实验结果提供了大熊猫9个BAC的详细序列及注释信息, 为对大熊猫的研究提供了数据资源.     

体外培养小鼠卵母细胞及其生长分化因子-9基因表达

体外培养小鼠的窦前卵泡以得到第二次减数分裂中期(MⅡ)卵母细胞,比较体外发育卵母细胞与体内生长的卵母细胞生长分化因子-9(GDF-9)的基因表达量,探讨GDF-9的表达对卵母细胞体外发育成熟的影响。选择体外培养第2天(D2)、D4、D6、D8、D10、D12卵母细胞作为体外发育组;同窝雌性小鼠出生后D12、D14、D16、D18、D20、D22卵母细胞作为体内发育组;半定量逆转录多聚酶链反应技术分别检测两组MⅠ卵母细胞GDF-9基因表达量。结果体外培养小鼠窦前卵泡可以得到MⅡ期卵母细胞,卵泡成活率、窦腔形成率、卵母细胞成熟率分别达到89·5%、51·8%和56·6%。小鼠卵母细胞GDF-9基因表达量随发育时间的改变而发生变化,而体外发育D8—12卵母细胞GDF-9表达量显著低于同期体内发育卵母细胞(P<0·05)。体外发育D8—12卵母细胞GDF-9基因表达量低于同期体内发育的卵母细胞的原因之一可能是其发育潜能较低。     

鸡生长分化因子GDF-8 cDNA的克隆、表达及蛋白纯化

1997年John Hopkins大学的McPherron AC等[1]从小鼠骨骼肌cDNA文库中克隆得到1个新基因GDF-8(Growth andDifferential Factor-8),从蛋白结构来看,GDF-8因子具有TGF-β超家族的典型结构特征,其中包括分泌用的信号肽、蛋白酶水解加工位点及含9个半胱氨酸残基的高度保守的C端区域.进一步研究发现GDF-8主要在小鼠的骨骼肌中表达,并且GDF-8基因敲除鼠的骨骼肌是正常野生型小鼠的3倍以上2],这是继1982年Palmiter等将大鼠的生长激素基因转入小鼠体内产生"超级小鼠"之后的又一只"超级小鼠".     

体外培养小鼠卵母细胞及其生长分化因子-9基因表达

体外培养小鼠的窦前卵泡以得到第二次减数分裂中期(MⅡ)卵母细胞，比较体外发育卵母细胞与体内生长的卵母细胞生长分化因子-9(GDF-9)的基因表达量，探讨GDF-9的表达对卵母细胞体外发育成熟的影响。选择体外培养第2天(D2)、D4、D6、D8、D10、D12卵母细胞作为体外发育组；同窝雌性小鼠出生后D12、D14、D16、D18、D20、D22卵母细胞作为体内发育组；半定量逆转录多聚酶链反应技术分别检测两组MⅠ卵母细胞GDF-9基因表达量。结果体外培养小鼠窦前卵泡可以得到MⅡ期卵母细胞，卵泡成活率、窦腔形成率、卵母细胞成熟率分别达到89.5%、51.8%和56.6%。小鼠卵母细胞GDF-9基因表达量随发育时间的改变而发生变化，而体外发育D8—12卵母细胞GDF-9表达量显著低于同期体内发育卵母细胞(P<0.05)。体外发育D8—12卵母细胞GDF-9基因表达量低于同期体内发育的卵母细胞的原因之一可能是其发育潜能较低。     

生长分化因子-9对牛卵丘细胞增殖的影响

研究生长分化因子-9（growth differentiation factor-9,GDF-9）对牛卵丘细胞增殖的影响。采用MTT法检测不同浓度GDF-9对卵丘细胞增殖的影响,结果表明,GDF-9能促进卵丘细胞的增殖,且GDF-9与卵丘细胞增殖效应存在浓度梯度关系;在卵丘细胞增殖过程中,FSH在一定程度上与GDF-9发挥协同作用。在GDF-9和FSH的作用下,去除卵母细胞的卵丘细胞复合体（oocytectomized cumulus cell complexes,OOX）也可以保持较好的发育形态。实时定量PCR结果表明,随着GDF-9浓度的增加,卵丘细胞扩展相关基因PTX3、HAS2及PTGS2的表达量也增加。总之,以上的研究结果表明,GDF-9可以促进卵丘细胞的增殖,对卵丘细胞功能的发挥起着重要的作用。     

大熊猫RPS15 cDNA的克隆及序列分析

本研究运用RT-PCR技术,首次从大熊猫 Ailuropoda melanoleuca的肌肉组织总RNA中成功克隆了核糖体蛋白S15 (RPS15)基因的表达序列,并对其进行了初步分析.结果 表明:大熊猫RPS15基因的表达序列全长为442 bp,开放阅读框(ORF)为438 bp,编码145个氨基酸,该蛋白的分子量为17.0401 KDa, 等电点为10.3,含有2个依赖于cAMP和cGMP的蛋白激酶磷酸化位点, 5个蛋白激酶C磷酸化位点,4个N-酰基化位点及1个RPS19蛋白signature位点.进一步分析发现,大熊猫RPS15基因的表达序列及其编码的氨基酸序列与已报道的部分哺乳动物具有很高的相似性.     

大熊猫核糖体蛋白S11亚基基因(RPS11)cDNA的克隆及序列分析

RPS11是核糖体小亚基40S的组成部分,由RPS11基因所编码,属于核糖体蛋白S17p家族,主要存在于真核生物中.为了解大熊猫核糖体蛋白亚基RPS11基因的结构特点及其与已报道的人和其他哺乳动物核糖体蛋白亚基RPS11 基因的异同,本研究根据已报道的部分哺乳动物核糖体蛋白S11亚基基因(RPS11)的相关信息设计引物,运用RT-PCR 技术从大熊猫的肌肉组织总RNA中成功克隆了核糖体蛋白亚基RPS11基因,并进行了测序和序列分析.结果表明:大熊猫RPS11亚基基因的开放阅读框(ORF)长为477 bp,编码158 个氨基酸的蛋白质,该蛋白的相对分子量为18.4275 kDa,pI为10.96.拓扑预测显示该蛋白含有14个功能位点:即2 个N-糖基化位点,6个蛋白激酶C磷酸化位点,4个酪蛋白激酶Ⅱ磷酸化位点,1个酪氨酸激酶磷酸化位点和1个核糖体蛋白S17 signature位点.进一步分析发现,大熊猫RPS11基因与已报道的部分哺乳动物的表达序列及其编码的氨基酸序列都具有很高的相似性.本研究结果为丰富和完善哺乳动物RPS11基因资源库提供了基础资料.     

圈养大熊猫群体间的基因流状况分析

圈养群体的遗传管理一个非常重要的手段就是实现不同群体间的基因交流.为了全面评估大熊猫圈养群体间的基因流状况,本研究以卧龙中国大熊猫保护中心的31只圈养大熊猫(简称卧龙群体)和成都大熊猫繁育研究基地与楼观台陕西省珍稀野生动物抢救饲养研究中心的37只圈养大熊猫(简称成都群体,其中楼观台1只)为研究对象,以7个大熊猫微卫星位点为分子标记,发现卧龙群体和成都群体间的遗传分化水平很低(Fst=0.041,P=0.001);尽管整个圈养群体的近交程度较低(Fis=0.026),但是成都群体的近交系数(Fis=0.045)远高于卧龙群体的近交系数(Fis=0.002);谱系分析、贝叶斯分析和系统进化法分析均显示,这两个群体间存在着基因流,但是这种基因流是单向的.此结果提示各个大熊猫饲养单位之间必须实现更多的合作,将大熊猫群体作为一个管理单元进行管理,从而实现更多的基因流.     

大熊猫Sox和Zfx基因

采用PCR克隆技术 ,获得了大熊猫 6个Sox基因HMG盒区及 2个Zfx基因锌指区DNA序列 ,并进行了DNA序列测定与分析 .大熊猫Sox基因分别属于两个Sox基因亚族 .大熊猫 pSox和 pZfx基因与哺乳类相应基因具有较高同源性 ,并与灵长类具有相近的DNA突变率 .     

Trisomy 9p with i(9p) and t(9q18p)

Summary The trisomy 9p syndrome in a 2-year-old girl with moderate mental retardation is presented. She has a unique karyotype with a de novo isochromosome 9p and a translocation between 9q and 18p.     

Content Volume 9 (2000)

Volume Contents

Content Volume 9 (2000)     

Preparation and characterization of (9-methyladenine)triammineplatinum(II) and trans-dihydroxo(9-methyladenine)triammineplatinum(IV) complexes

The preparation is reported of [(NH₃)₃Pt(9- MeA)] X₂ (9-MeA = 9-methyladenine) with XCl (1a) and XClO₄ (1b) and of trans-[(OH)₂Pt(NH₃)₃- (9-MeA)]X₂ with XCl (2a) and XClO₄ (2b), and the crystal structure of 1b. [(NH₃)₃Pt(C₆H₇N₅)](ClO₄)₂ crystallizes in space group P2₁/n with a = 20.810(7) Å, b = 7.697(3) Å, c = 10.567(4) Å, β = 91.57(6)°, Z = 4. The structure was refined to R = 0.054, R_w = 0.063. In all four compounds Pt coordination is through N7 of 9-MeA, as is evident from ³J coupling between H8 of the adenine ring and ¹⁹⁵Pt. Pt(II) and Pt(IV) complexes can be differentiated on the basis of different ³J values, larger for Pt(II) than for Pt(IV) by a factor of 1.57 (av). In Me₂SO-d₆, hydrogen bonding occurs between Cl^? and C(8)H of 9-MeA as weil as between Cl^? and the NH₃ groups in the case of the Pt(II) complex 1a. Protonation of the 9-MeA ligands was followed using ¹H NMR spectroscopy and pK_a values for the N1 protonated 9-MeA ligands were determined in D₂O. They are 1.9 for 1a and 1.8 for 2a, which compares with 4.5 for the non-platinated 9-MeA. Possible consequences for hydrogen bonding with the complementary bases thymine or uracil are discussed briefly. Protonation of the OH groups in the Pt(IV) complexes has been shown not to occur above pH 1.     

Structural Characterization of Bardet-Biedl Syndrome 9 Protein (BBS9)

The Bardet-Biedl syndrome protein complex (BBSome) is an octameric complex that transports membrane proteins into the primary cilium signaling organelle in eukaryotes and is implicated in human disease. Here we have analyzed the 99-kDa human BBS9 protein, one of the eight BBSome components. The protein is composed of four structured domains, including a β-stranded N-terminal domain. The 1.8 Å crystal structure of the 46-kDa N-terminal domain reveals a seven-bladed β-propeller. A structure-based homology search suggests that it functions in protein-protein interactions. We show that the Bardet-Biedl syndrome-causing G141R mutation in BBS9 likely results in misfolding of the β-propeller. Although the C-terminal half of BBS9 dimerizes in solution, the N-terminal domain only does so in the crystal lattice. This C-terminal dimerization interface might be important for the assembly of the BBSome.     

Author Index Volume 9 (2000)

Authors Index

Author Index Volume 9 (2000)     

Histone deacetylase 9 (HDAC9) regulates the functions of the ATDC (TRIM29) protein

Histone deacetylase 9 (HDAC9), like most Class II HDACs, catalyzes the removal of acetyl moieties from the ε-amino groups of conserved lysine residues in the N-terminal tail of histones. Biologically, HDAC9 regulates a wide variety of normal and abnormal physiological functions, including cardiac growth, T-regulatory cell function, neuronal disorders, muscle differentiation, development, and cancer. In a biochemical approach to identify non-histone substrates of HDAC9, we found that HDAC9 co-purifies specifically with the ataxia telangiectasia group D-complementing (ATDC; also called TRIM29) protein. HDAC9 deacetylates ATDC, alters the ability of ATDC to associate with p53, and consequently inhibits the cell proliferation-promoting activity of ATDC. These results implicate the importance of non-histone deacetylation by HDAC9 and confirm and further extend the multifunctions of this Class II deacetylase.     

Contents of Volume 9 (2001)

Volume Contents

Contents of Volume 9 (2001)     

Changes in retinal aquaporin-9 (AQP9) expression in glaucoma

The eye contains numerous water channel proteins and the roles of AQPs (aquaporins) in the retina are blurred, especially under disease conditions. The purpose of this study was to investigate the expression of AQP9 gene and proteins affected by elevated IOP (intraocular pressure) in a rat model of glaucoma induced by intravitreous injection of hypertonic saline into the episcleral veins. The gene and protein expressions of AQP9 were investigated by real-time PCR and Western blotting. The immunoreactive expression of AQP9, AQP4 and GFAP (glial fibrillary acidic protein) in the optic nerve of rats exposed to experimentally elevated IOP was detected by immunofluorescence microscopy. The mRNA and protein expression levels of AQP9 were up-regulated in the retina of an animal model of glaucoma. The immunoreactivities of the AQP9, AQP4 and GFAP were also detected and increased in the optic nerve region. The expression of AQP9 was up-regulated in this glaucoma model and the immunoreactivities of the AQP4 and GFAP were also detected as co-localizing with AQP9 in the optic nerve region, indicating retina ganglion cells were surrounded by activated astrocytes. This may indicate that the injured neurons may rely on the astrocytes. The alterations of AQP expression may compensate the glaucomatous damage.     

Molecular characterization of a tandem-repeat galectin-9 (RuGlec9) from Korean rose bitterling (Rhodeus uyekii)

Galectin-9 is a b-galactoside-binding lectin that regulates many cellular functions, ranging from cell adhesion to pathogen recognition. We isolated and characterized the cDNA of tandem-repeat galectin-9 (RuGlec9) from the Korean rose bitterling (Rhodeus uyekii), an endemic Korean fish belonging to the Acheilognathinae subfamily of the Cyprinidae family. RuGlec9 cDNA is 1486 bp long and encodes a polypeptide of 323 amino acids containing two carbohydrate-recognition domains connected by a linker peptide. The deduced amino acid sequence of RuGlec9 shows 45-84% amino acid sequence identity to other galectin-9 sequences, including those from mammals and fish. RuGlec9 appeared in a large cluster with other galectin-9 sequences from fish and is more closely related to galectin-9 from Danio rerio than to those of other fish and mammals. RuGlec9 mRNA was expressed highly in the testis, spleen, intestine, stomach, and liver, and moderately in the brain, kidney, ovary, and gills of normal Korean rose bitterling. RuGlec9 mRNA expression in the spleen was increased by lipopolysaccharide. These results suggest that RuGlec9 plays a role in innate immunity in Korean rose bitterling.