外源性双链RNA对小鼠卵母细胞basonuclin基因表达的影响

dsRNA能阻抑卵母细胞中同源基因的表达,其作用相当于基因敲除。质粒表达的发夹环型dsRNA也可以有效降解basonuclin转录产物,这为研究basonuclin在卵母细胞发育早期的功能提供了新的手段。

Effects of exogenous double-stranded RNA on the basonuclin gene expression in mouse oocytes

In plants and less-advanced animal species, such as C.elegans, introduction of exogenous double-stranded RNA (dsRNA) into cells would trigger degradation of the mRNA with homologous sequence and interfere with the endogenous gene expression. It might represent an ancient anti-virus response which could prevent the mutation in the genome that was caused by virus infection or mobile DNA elements insertion. This phenomenon was named RNA interference, or RNAi. In this study, RNAi was used to investigate the function of basonuclin gene during oogenesis. Microinjection of dsRNA directed towards basonuclin into mouse germinal-vesicle-intact (GV) oocytes brought down the abundance of the cognate mRNA effectively in a time- and concentration-dependent manner. This reduction effect was sequence-specific and showed no negative effect on other non-homologous gene expression in oocytes, which indicated that dsRNA can recognize and cause the degradation of the transcriptional products of endogenous basonuclin gene in a sequence-specific manner. Immunofluorescence results showed that RNAi could reduce the concentration of basonuclin protein to some extent, but the effect was less efficient than the dsRNA targeting towards tPA and cMos which was also expressed in oocytes. This result might be due to the long half life of basonuclin protein in oocytes and the short reaction time which was posed by the limited life span of GV oocytes cultured in vitro. In summary, dsRNA could inhibit the expression of the cognate gene in oocytes at both mRNA and protein levels. The effect was similar to Knock-out technique which was based on homologous recombination. Furthermore, hairpin-style dsRNA targeting basonuclin gene could be produced by transcription from a recombinant plasmid and worked efficiently to deplete the cognate mRNA in oocytes. This finding offered a new way to study the function of basonuclin in the early stage of oogenesis by infection of primordial oocytes with the plasmid expressing hairpin-style basonuclin dsRNA.

Non-monophyly of Rhacophorus rhodopus,Theloderma and Philautus albopunctatus Inferred from Mitochondrial 16S rRNA Gene Sequences

测定了4个种(红蹼树蛙、黑蹼树蛙、白斑小树蛙和红吸盘小树蛙)共11个种群的16S rRNA基因片段.双斑树蛙、马来棱皮树蛙、越南棱皮树蛙以及日本溪树蛙的同源序列通过GenBank检索获得.去除所有插入、缺失及模糊位点后,比对序列长度为500 bp,其中变异位点115个,简约信息位点92个.以日本溪树蛙为外群,运用Bayesian法、MP法和ML法构建了系统发育树.结果表明红蹼树蛙和白斑小树蛙在种级水平上均不是单系.红蹼树蛙海南种群与双斑树蛙亲缘关系更近,并且来自云南不同地理种群的红蹼树蛙可以分为两大支系;越南棱皮树蛙与红吸盘小树蛙聚为一支,马来棱皮树蛙嵌套在白斑小树蛙不同地理种群中.进而认为白斑小树蛙是马来棱皮树蛙的同物异名,建议将红吸盘小树蛙并入棱皮树蛙属.

Glucocorticoid receptor and protein/RNA synthesis-dependent mechanisms underlie the control of synaptic plasticity by stress

Learning and memory are exquisitely sensitive to behavioral stress, but the underlying mechanisms are still poorly understood. Because activity-dependent persistent changes in synaptic strength are believed to mediate memory processes in brain areas such as the hippocampus we have examined the means by which stress affects synaptic plasticity in the CA1 region of the hippocampus of anesthetized rats, Inescapable behavioral stress (placement on an elevated platform for 30 min) switched the direction of plasticity, favoring low frequency stimulation-induced decreases in synaptic transmission (long-term depression, LTD), and opposing the induction of long-term potentiation by high frequency stimulation, We have discovered that glucocorticoid receptor activation mediates these effects of stress on LTD and longterm potentiation in a protein synthesis-dependent manner because they were prevented by the glucocorticoid receptor antagonist RU 38486 and the protein synthesis inhibitor emetine. Consistent with this, the ability of exogenously applied corticosterone in non-stressed rats to mimic the effects of stress on synaptic plasticity was also blocked by these agents, The enablement of low frequency stimulation-induced LTD by both stress and exogenous corticosterone was also blocked by the transcription inhibitor actinomycin D, Thus, naturally occurring synaptic plasticity is liable to be reversed in stressful situations via glucocorticoid receptor activation and mechanisms dependent on the synthesis of new protein and RNA, This indicates that the modulation of hippocampus-mediated learning by acute inescapable stress requires glucocorticoid receptor-dependent initiation of transcription and translation.

Generic allocation of Indian and Sri Lankan Philautus (Anura: Rhacophoridae) inferred from 12S and 16S rRNA genes

The generic allocation of Indian and Sri Lankan Philautus needs further examination. In this study, a comprehensive understanding of the phylogeny of Indian and Sri Lankan Philautus is obtained based on 125 and 16S rRNA genes. All phylogenetic analyses indicate that Indian-Sri Lankan Philautus, Philautus menglaensis, Philautus longchuanensis, and Philautus gryllus form a well supported clade, separate from Philautus of Sunda Islands that form another well supported clade representing true Philautus. This result supports the designation of the genus Pseudophilautus to accommodate the Indian and Sri Lankan species. Pseudophilautus consists of two major lineages, one comprises the majority of Indian species, Chinese species, and Southeast Asian species, and one comprises all Sri Lankan species and a few Indian species. Pseudophilautus may have originated in South Asia and dispersed into Southeast Asia and China. Based on the results, we further suggest that Philautus cf. gryllus (MNHN1997.5460) belongs to the genus Kurixalus. (C) 2010 Published by Elsevier Ltd.

Identification of a Giardia krr1 homolog gene and the secondarily anucleolate condition of Giaridia lamblia

Giaridia lamblia was long considered to be one of the most primitive eukaryotes and to lie close to the transition between prokaryotes and eukaryotes, but several supporting features, such as lack of mitochondrion and Golgi, have been challenged recently. It was also reported previously that G. lamblia lacked nucleolus, which is the site of pre-rRNA processing and ribosomal assembling in the other eukaryotic cells. Here, we report the identification of the yeast homolog gene, krr1, in the anucleolate eukaryote, G. lamblia. The krr1 gene, encoding one of the pre-rRNA processing proteins in yeast, is actively transcribed in G. lamblia. The deduced protein sequence of G. lamblia krr1 is highly similar to yeast KRR1p that contains a single-KH domain. Our database searches indicated that krr1 genes actually present in diverse eukaryotes and also seem to present in Archaea. However, only the eukaryotic homologs, including that of G. lamblia, have the single-KH domain, which contains the conserved motif KR(K)R. Fibrillarin, another important pre-rRNA processing protein has also been identified previously in G. lamblia. Moreover, our database search shows that nearly half of the other nucleolus-localized protein genes of eukaryotic cells also have their homologs in Giardia. Therefore, we suggest that a common mechanism of pre-RNA processing may operate in the anucleolate eukaryote G. lamblia and in the other eukaryotes and that like the case of "lack of mitochondrion," "lack of nucleolus" may not be a primitive feature, but a secondarily evolutionary condition of the parasite.

Tobramycin与16S rRNA A位点复合物的分子动力学模拟

采用分子动力学方法(Molecular dynamics,MD)对托普霉素(Tobramycin)与16S rRNA的A位点复合物的特异性识别机制进行了理论模拟研究,模拟时间为3.6 ns. 结果表明,A位点中波动最大的部位是两个环外碱基A1492和A1493;tobramycin的环Ⅰ和环Ⅱ是其最保守的结构单元,可能参与了Tobramycin与16S rRNA的A位点之间的特异性识别. 另外,发现一个残存时间为3.6 ns的"结构化"水分子,它桥接了Tobramycin环Ⅱ的N3与环Ⅰ的N6′之间的氢键,稳定了Tobramycin的结构;Tobramycin周围水合密度较高的位点出现在环Ⅰ和环Ⅱ附近,这也正是晶体结构中形成较多水媒介氢键及动力学模拟中结构化水分子出现的位置. 动力学模拟证实Tobramycin与16S rRNA间的结合是大量氢键及水分子相互作用的结果,这有助于设计和开发以Tobramycin为基础,具有高亲和力及特异性的16S rRNA抑制剂.

RNA 的历史与未来

核酶的发现使得人们有理由相信生命起源于RNA ,通过试管演化实验获得的各种各样的催化性RNA 更使人们 对地球历史早期的RNA 世界有了越来越多的了解。同时,随着RNA 结构和功能上非凡的多样性的日益被揭示,RNA 在未来的临床应用研究中所具有的巨大潜力也正逐渐显现出来。

稳定的RNA 发夹结构在人类88 个mRNA 编码区中的分布

　以UNCG, GNRA , CUU G (N = A , U , C 或G; R = G或A) 为端环能够形成稳定的、保 守的发夹结构. 它们具有特殊的结构特征, 并在体内发挥着重要的生物学功能. 这些稳定的发夹 广泛分布于体内rRNA , 催化RNA 和非编码mRNA 中. 但对人类88 个编码区mRNA 二级结构的 研究当中, 却没有发现C(UUCG) G发夹. 而且, 与rRNA 不同, 这些编码区mRNA 四环序列的 分布没有明显的偏好性.

RNA A_6膨胀环的动力学特征

通过对RNA A6膨胀环在水溶液中的动力学模拟发现,在A6膨胀环中,环区构象主要以非堆积构象为主,环区具有较大的构象柔性,膨胀环区链的构象波动对已形成的RNA分子弯折的影响不大,弯折角只是在小范围内变动,提示作为全局结构,带大尺寸膨胀环的RNA分子仍然具有一定的刚性,柔性主要表现在膨胀环区域.