Ovarian follicular activity in Booroola lambs with and without a fecundity gene

The ovaries of 3-month-old Booroola lambs which were heterozygous carriers of a major gene (F) influencing the ovulation rate in mature ewes (i.e. F + lambs) were compared to those ofsimilarly-aged Booroola lambs which were non-carriers of the F-gene (i.e. ++ lambs). The ovaries of the F + Booroola lambs were significantly lighter (P less than 0.01) than those of ++ lambs even though the mean +/- s.e.m. number of follicles (greater than or equal to 1 mm diam.) in the F + lambs was greater than that in the ++ lambs (i.e. F + lambs, 30.2 +/- 2.5 follicles; ++ lambs, 18.4 +/- 1.2 follicles; P less than 0.01). In granulosa cells from non-atretic follicles (greater than or equal to 1 mm diam.) from F + and ++ Booroola lambs, FSH (NIAMDD-FSH-S16) doses of 100 and 1000 ng/ml caused significant stepwise increases (P less than 0.05) in cyclic adenosine 3',5'-monophosphate (cAMP) production compared to that achieved at FSH doses of 0 and 1 ng/ml or at any FSH dose in cells from atretic follicles. However, no significant differences in FSH-induced cAMP production were noted with regard to Booroola genotype or follicular diameter. None of the granulosa cell preparations from non-atretic follicles of 1-2.5 mm diameter from F + lambs (N = 13) or from non-atretic follicles of 1-4.5 mm diameter from ++ lambs (N = 16) responded to LH (NIAMDD-LH-S24; 10 or 1000 ng/ml) to produce significantly more cAMP than did the controls. In contrast, the granulosa cell preparations from non-atretic follicles of 3-4.5 mm diameter from F + lambs (N = 4) and from non-atretic follicles of greater than or equal to 5 mm diameter of ++ lambs (N = 4) produced significantly more cAMP (P less than 0.05) in response to LH (1000 and/or 10 ng/ml) relative to that in the controls. The theca interna from follicles of lambs of both genotypes had functional LH receptors as judged by the androstenedione responses to exogenous LH although no genotypic differences were noted. In F + lambs, the follicular fluid concentrations of testosterone but not oestradiol (i.e. in 1-4.5 mm diam. follicles) and granulosa cell aromatase activity (i.e. in 3-3.5 mm diam. follicles) were significantly higher (both P less than 0.05) than in corresponding follicles or cells from ++ lambs. Collectively the results suggest that the Booroola F-gene influences the composition and function of sheep ovaries before puberty.     

MSI4/FVE is required for accumulation of 24-nt siRNAs and DNA methylation at a subset of target regions of RNA-directed DNA methylation

DNA methylation is an important epigenetic mark. In plants, de novo DNA methylation occurs mainly through the RNA-directed DNA methylation (RdDM) pathway. Researchers have previously inferred that a flowering regulator, MULTICOPY SUPPRESSOR OF IRA1 4 (MSI4)/FVE, is involved in non-CG methylation at several RdDM targets, suggesting a role of FVE in RdDM. However, whether and how FVE affects RdDM genome-wide is not known. Here, we report that FVE is required for DNA methylation at thousands of RdDM target regions. In addition, dysfunction of FVE significantly reduces 24-nucleotide siRNA accumulation that is dependent on factors downstream in the RdDM pathway. By using chromatin immunoprecipitation and sequencing (ChIP-seq), we show that FVE directly binds to FVE-dependent 24-nucleotide siRNA cluster regions. Our results also indicate that FVE may function in RdDM by physically interacting with RDM15, a downstream factor in the RdDM pathway. Our study has therefore revealed that FVE, by associating with RDM15, directly regulates DNA methylation and siRNA accumulation at a subset of RdDM targets.     

诱导神经再生治疗阿尔茨海默病的机制研究进展

阿尔茨海默病(Alzheimer's disease,AD)是一种以进行性认知障碍和记忆减退为主要特征的中枢神经退行性疾病,已经成为老年医学中最棘手的、亟待解决的问题之一. AD的病理机制仍不清楚,尚无特效治疗药物.目前,探索AD神经再生逐渐成为研究的热点领域,通过诱导神经再生可以有效地改善AD的症状.研究表明,运用药物、物理刺激或干细胞移植方法,可以提高大脑成体神经再生,是延缓AD的病理症状和认知障碍的有效治疗策略.本文综述诱导神经再生的方法及其治疗AD的作用机制,为神经再生治疗实施提供理论依据.     

取食转Bt基因棉花上的棉蚜对丽草蛉发育和繁殖的影响

为明确转Bt基因抗虫棉对捕食性天敌的生态效应 ,比较了 2种转Bt基因抗虫棉 (GK-1 2和新棉99B)和 1种常规棉上的棉蚜Chrysopaformosa对丽草蛉Aphisgossypii发育和繁殖的影响。结果表明 ,(1 )与取食常规棉泗棉 3号上棉蚜的对照相比 ,取食转Bt基因抗虫棉GK- 1 2上棉蚜的第 1代和第 2代丽草蛉 ,其幼虫期和茧期的死亡率、幼虫和茧的发育历期、茧重及成虫性比等均与对照无显著差异 ;但第 1代成虫的产卵量减少了 2 88 0粒 ,与对照差异显著 (P <0 . 0 1 ) ;第 2代成虫所产卵的孵化率为 64. 0 % ,显著低于对照的 77. 7% (P <0 0 1 )。 (2 ) 2个品种的Bt棉对丽草蛉发育和繁殖的影响也略有差异。与取食GK 1 2上棉蚜的个体相比 ,取食新棉 99B上蚜虫的第 1代丽草蛉 ,其幼虫发育历期缩短了 0 . 6d(P <0 0 1 ) ,茧期缩短了 0 . 7d(P <0 . 0 1 ) ,茧重降低了 1 . 2mg(P <0 .0 1 ) ,幼虫期和茧期的死亡率、茧重以及成虫性比等则无显著差异 ;雌成虫产卵前期、产卵期、产卵量和寿命等繁殖学特性也无显著差异 ,但其成虫所产卵的孵化率为 65. 0 % ,显著低于取食GK- 1 2上棉蚜的处理 (72 . 7% ,P <0. 0 1 )。分别取食 2种Bt棉上的棉蚜 ,对第 2代丽草蛉的发育和繁殖的影响差异则较小。     

植酸酶phyAm基因结构延伸突变改善酶的热稳定性

将来源于黑曲霉N25的植酸酶基因phyA^m重组于大肠杆菌表达载体pET-30b（＋）,以重组表达载体pET30b-FphyA^e为模板经PCR扩增获得结构延伸突变植酸酶基因phyA^m（在植酸酶基因C端增加了来源于pET-30b-FphyA^m载体上13氨基酸残基）。含突变基因的重组表达载体pPIC9k-phyA^e在GS115酵母中表达。纯化的突变酶pp-NP^e与野生型酶PP-NP^m-8相比：PP-NPA^e的最适反应温度上升了3气,75℃处理10min,热稳定性提高21％,比活力略有提高。最适反应pH为5．6,有效pH范围pH4,6到pH6．6。比未突变酶扩大了0.4单位。     

人类新基因WDR70的克隆及初步研究

运用基于基因组数据库的特定基因同源新基因的克隆策略得到一个人类新基因WDR70 ,该基因编码一个包含 12个WD4 0结构域的蛋白。WDR70的cDNA序列长 2 2 6 6bp ,预测编码蛋白含 6 30个氨基酸 ,理论分子量为 70× 10 3 u ,染色体定位为 17p13.1。以小鼠胚胎为模型进行整体原位杂交 ,结果显示WDR70基因在 8.5d小鼠胚胎中没有表达 ,而在 9.5d和 10 .5d的小鼠胚胎的脑部有特异表达。由此推断该基因对胚胎期脑部的发育有重要的影响。     

无饲养层培养人胚胎干细胞方法的建立

人胚胎干细胞(human embryonic stem cell,hES细胞)是当前医学研究的热点之一．然而hES细胞培养条件苛刻,通常需要采用鼠胚胎成纤维细胞(mouse embryonic fibroblast,MEFs)饲养层来维持其未分化状态,成为目前hES细胞研究的瓶颈之一、本实验成功地将hES细胞接种在细胞外基质包被的六孔板上培养,传代20次后细胞仍然保持良好的未分化状态,各种hES细胞生物学特性(如表面标志物SSEA-3、SSEA-4、TRA-1-60和TRA-1-8l,OCT-4,碱性磷酸酶及体内外分化潜能等)均无改变;其冻存、复苏效果与生长在饲养层上的hES细胞无明显差异．因此,该无饲养层培养体系可以用于培养hES细胞,并为hES细胞转基因研究及大规模培养打下良好的基础．     

F43Y及I354M,L358F定点突变对植酸酶热稳定性及酶活性的改善

对重组酵母PPNPm8的植酸酶phyAm基因进行PCR介导的定点突变,即将植酸酶43位的苯丙氨酸替换为酪氨酸(F43Y),将其354、358位的异亮氨酸、亮氨酸分别替换为甲硫氨酸和苯丙氨酸(I354M,L358F),得到了2个突变体PPNPm-1(F43Y)及PPNPm-2(I354M,L358F).含突变基因的重组表达载体pPIC9kphyAm-1,pPIC9kphyAm-2在毕赤酵母GS115中表达,对表达产物进行酶活性测定及热稳定性检测.结果表明:突变体PPNPm-1最适反应温度比未突变体PPNPm8上升了3℃,75℃处理10min,热稳定性提高15%,比活力提高11%;PPNPm-2最适反应温度未改变,热稳定性比PPNPm8仅提高3%,比活力降低6.5%.对突变前后的植酸酶空间结构进行比较预测,发现突变氨基酸Tyr43与空间位置相邻的Asn416之间形成氢键,增强了酶的热稳定性.     

Involvement of Long-Distance Na<Superscript>+</Superscript> Transport in Maintaining Water Potential Gradient in the Medium-Root-Leaf System of a Halophyte <Emphasis Type="Italic">Suaeda altissima</Emphasis>

The aim of this study was to determine the range of NaCl concentrations in the nutrient solution that allow Suaeda altissima (L.) Pall., a salt-accumulating halophyte, to maintain the upward gradient of water potential in the “medium-root-leaf” system. We evaluated the contribution of Na⁺ ions in the formation of water potential gradient and demonstrated that Na⁺ loading into the xylem is involved in this process. Plants were grown in water culture at NaCl concentrations ranging from zero to 1 M. The water potential of leaf and root cells was measured with the method of isopiestic thermocouple psychrometry. When NaCl concentration in the growth medium was raised in the range of 0–500 mM (the medium water potential was lowered accordingly), the root and leaf cells of S. altissima decreased their water potential, thus promoting the maintenance of the upward water potential gradient in the medium-root-leaf system. Growing S. altissima at NaCl concentrations f 750 mM and 1 M disordered water homeostasis and abolished the upward gradient of water potential between roots and leaves. At NaCl concentrations of 0–250 mM, the detached roots of S. altissima were capable of producing the xylem exudate. The concentration of Na⁺ in the exudate was 1.3 to 1.6 times higher than in the nutrient medium; the exudate pH was acidic and was lowered from 5.5 to 4.5 with the rise in the salt concentration. The results indicate that the long-distance Na⁺ transport and, especially, the mechanism of Na⁺ loading into the xylem play a substantial role in the formation of water potential gradient in S. altissima. The accumulation of Na⁺ in the xylem and acidic pH values of the xylem sap suggest that Na⁺ loading into the xylem is carried out by the Na⁺/H⁺ antiporter of the plasma membrane in parenchymal cells of the root stele.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 4, 2005, pp. 549–557.Original Russian Text Copyright © 2005 by Balnokin, Kotov, Myasoedov, Khailova, Kurkova, Lun’kov, Kotova.     

铜绿假单胞菌色素代谢相关基因的研究

首次应用Mu转座重组技术研究铜绿假单胞菌色素合成与调控的机制。通过一系列的表型筛选,得到8株色素合成能力改变的突变子。经基因克隆、核苷酸测序研究,证明转座子分别插入到hmgA、ptsP、sucC、phzS、phzF1五个基因中。hmgA基因转座失活导致酪氨酸分解代谢中间产物尿黑酸的积累,后四种情况转座突变显著地影响了铜绿假单胞菌最重要的色素绿脓素（pyocyanin）的合成,其中PhzS和phzF1是绿脓素合成过程中的结构基因,ptsP基因是1个磷酸转移酶系统的重要组分,sucC基因的产物是三羧酸循环中的琥珀酰辅酶A合成酶,对后两个基因在色素合成的调控方面可能起到重要作用的报道尚属首例。