中华鳖vasa基因克隆及在卵母细胞中的表达分析

研究利用中华鳖为研究模型进行爬行类生殖细胞发育分化成熟等生物学研究,克隆了中华鳖vasa基因的cDNA序列,全长3865 bp,包括5'端非编码区90 bp,3'端非编码区1699 bp,开放阅读框长2076 bp,共编码691个氨基酸。中华鳖Vasa氨基酸序列包含DEAD-box家族蛋白8个保守保守功能域,在N末端有4个RGG重复序列和2个GG富集区,与小鼠Vasa蛋白的同源性较高（72%）。荧光定量PCR的结果表明,中华鳖vasa mRNA主要精巢和卵巢中表达,其他体组织中均难检测到表达。卵巢冰冻切片原位杂交结果显示:中华鳖vasa mRNA在生殖细胞中特异表达;在卵子发生过程中的不同发育期卵母细胞中呈现动态的变化。即vasa mRNA在初级卵母细胞及生长期卵母细胞中表达最强,且均匀分布在细胞质中,随着卵母细胞的逐渐增大,信号逐渐减弱,直至在成熟的卵母细胞中几乎检测不到表达信号,说明vasa可能在中华鳖早期卵母细胞发育中起重要作用。同时,vasa基因可作为中华鳖生殖细胞分子标记物,根据其mRNA的表达水平来鉴别不同发育时期的卵母细胞。研究结果为进一步开展中华鳖胚胎生殖细胞发育及配子生成,特别是研究中华鳖,乃至爬行类原始生殖细胞（Primordial Germ Cells,PGCs）的起源、迁移、分化等研究奠定了基础。     

Differential interaction of PRMT1 with RGG‐boxes of the FET family proteins EWS and TAF15

The FET sub‐family (F US/T LS, E WS, T AF15) of RNA‐binding proteins have remarkably similar overall structure but diverse biological and pathological roles. The molecular basis for FET protein specialization is largely unknown. Gly‐Arg‐Rich regions (RGG‐boxes) within FET proteins are targets for methylation by Protein‐Arginine‐Methyl‐Transferase‐1 (PRMT1) and substrate capture is thought to involve electrostatic attraction between positively charged polyRGG substrates and negatively charged surface channels of PRMT1. Unlike FUS and EWS, a high proportion of TAF15 RGG‐boxes are embedded within neutrally charged YGGDR(S/G)G repeats, suggesting that they might not bind well to PRMT1. This notion runs contrary however to a report that YGGDR(S/G)G repeats are methylated by PRMT1. Using peptide‐based polyRGG substrates and a novel 2‐hybrid binding assay, we find that the Asp residue in YGGDR(S/G)G repeats confers poor binding to PRMT1. Our results therefore indicate that YGGDR(S/G)G repeats may contribute to TAF15 specialization by enabling differential interactions with PRMT1 and reduced overall levels of TAF15 methylation compared with other FET proteins. By analogy with molecular recognition of other disordered polyvalent ligands by globular protein partners, we also propose a dynamic polyelectrostatic model for substrate capture by PRMT1.     

vasa基因研究进展

DEAD-box家族基因编码一类ATP依赖的RNA解旋酶。经系统进化分析可将该家族蛋白分为VASA、PL10和p68三个亚家族。其中,vasa基因最先在果蝇(Drosophila melanogaster)中被发现,在许多动物中都已经克隆得到其同源基因,研究显示,vasa基因在生殖细胞系中特异性表达,在许多生物中为生殖细胞形成和配子发生所需。有趣的是在果蝇中VASA蛋白是生殖质的组成部分,而在斑马鱼(Danio rerio)中vasa mRNA才是生殖质的组成部分。本文主要综述了vasa基因及其蛋白的结构、功能、表达和作为原生殖细胞分子标记物的应用等方面的内容,并展望了其研究前景。     

Complete structure of the chicken alpha 2(VI) collagen gene

Type VI collagen is a hybrid molecule consisting of a short triple helix flanked by two large globular domains. These globular domains are composed of several homologous repeats which show a striking similarity to the collagen-binding motifs found in von Willebrand factor. The alpha 2(VI) subunit contains three of these homologous repeats termed D1, D2 and D3. We have isolated and characterized the entire gene for chicken alpha 2(VI) collagen. This gene, which is present as a single copy in the chicken genome, is 26 kbp long and comprises 28 exons. All exons can be classified in three groups. (a) The triple-helical domain is encoded by 19 short exons (27-90 bp) separated by introns of phase class 0. These exons are multiples of 9 bp and encode an integral number of collagenous Gly-Xaa-Yaa triplets. (b) The homologous repeats D1-D3 are encoded by one or two very long exons each (153-1578 bp). These exons are separated by introns of phase class 1. (c) The homologous repeats and the collagen sequence are linked to each other by three short adapter segments which are each encoded by a single exon (21-46 bp). The modular nature of the polypeptide is thus clearly reflected by the mosaic structure of its gene. The size of the exons and the phase class of the introns suggest that the alpha 2(VI) gene evolved by duplication and shuffling of two different primordial exons, one of 9 bp encoding a collagen Gly-Xaa-Yaa triplet and one of 600 bp encoding the precursor of the homologous repeats.     

Structure of the human laminin B2 chain gene reveals extensive divergence from the laminin B1 chain gene

The exon-intron structure of the human laminin B2 chain gene was elucidated from genomic lambda phage clones spanning 2 kilobase pairs (kb) of the 5'-flanking region, 58 kb of the structural gene and 10 kb of the 3'-flanking region. The entire gene was shown to contain 28 exons. The promoter region has no TATA or CAAT boxes whereas it contains five GC boxes and three AP-2-like binding sites. Comparison with the promoter region of the mouse gene revealed six highly conserved sequences of 14 to 42 base pairs in length. Sequencing of the last exon of the gene showed that the 3'-untranslated region of the mRNA can be up to 2797 nucleotides with five AATAAA potential polyadenylation signals. The similarity of the human 3'-untranslated sequence with that of mouse was shown to be 68.8%. The exon-intron structure of the laminin B2 chain gene demonstrated extensive divergence from the human laminin B1 chain gene, which has 34 exons. Only three intron locations are conserved in these two genes. The overall exon profile of the laminin B2 chain gene correlates only marginally with the pattern of structural domains and internal cysteine-rich repeats in the laminin B2 polypeptide chain.     

Identification of a light-responsive region of the nuclear gene encoding the B subunit of chloroplast glyceraldehyde 3-phosphate dehydrogenase from Arabidopsis thaliana.

We report here the identification of a cis-acting region involved in light regulation of the nuclear gene (GapB) encoding the B subunit of chloroplast glyceraldehyde 3-phosphate dehydrogenase from Arabidopsis thaliana. Our results show that a 664-bp GapB promoter fragment is sufficient to confer light induction and organ-specific expression of the Escherichia coli beta-glucuronidase reporter gene (Gus) in transgenic tobacco (Nicotiana tabacum) plants. Deletion analysis indicates that the -261 to -173 upstream region of the GapB gene is essential for light induction. This region contains four direct repeats with the consensus sequence 5'-ATGAA(A/G)A-3' (Gap boxes). Deletion of all four repeats abolishes light induction completely. In addition, we have linked a 109-bp (-263 to -152) GapB upstream fragment containing the four direct repeats in two orientations to the -92 to +6 upstream sequence of the cauliflower mosaic virus 35S basal promoter. The resulting chimeric promoters are able to confer light induction and to enhance leaf-specific expression of the Gus reporter gene in transgenic tobacco plants. Based on these results we conclude that Gap boxes are essential for light regulation and organ-specific expression of the GapB gene in A. thaliana. Using gel mobility shift assays we have also identified a nuclear factor from tobacco that interacts with GapA and GapB DNA fragments containing these Gap boxes. Competition assays indicate that Gap boxes are the binding sites for this factor. Although this binding activity is present in nuclear extracts from leaves and roots of light-grown or dark-treated tobacco plants, the activity is less abundant in nuclear extracts prepared from leaves of dark-treated plants or from roots of greenhouse-grown plants. In addition, our data show that this binding factor is distinct from the GT-1 factor, which binds to Box II and Box III within the light-responsive element of the RbcS-3A gene of pea.