利用环形染色体构象捕获技术对Bci11b基因座位在细胞核内空间组织的研究

环形染色体构象捕获（4c）技术实现了在全基因组范围内捕获与4c靶位点发生相互作用的基因座位,因而通过4C相关技术可以进一步研究靶基因座位在细胞核内的空间组织形式。该文以ABclllb基因座位作为4C分析的靶位点,通过优化4C分析的反向巢式PCR扩增条件,实现4C分析PCR的高效扩增：并通过有限克隆筛选与普通测序分析相结合的方法,在全基因组范围内捕获到一些与BcHlb基因座位发生潜在相互作用的基因座位。这些基因座位与靶位点间的相互作用既有发生在相同染色体内的,也有发生在不同染色体之间的。这些基因座位间的相互作用表明了Bclllb基因座位在细胞核内复杂的空间组织形式。     

马铃薯Y病毒蚜传辅助成分介导PVX/PVY协生作用

构建了马铃薯Y病毒中国株系（PVY－C）蚜传辅助成分（HC－Pro）基因的正义、反义和缺失三种植物表达载体,通过农杆菌介导法转化烟草品种NC89。Southern blot分析表明,HC－Pro基因及其突变体已经整合到烟草染色体中,Western blot分析证明,正义HC－Pro基因及其缺失突变体在转基因烟草中有表达产物,攻毒试验结果表明,转正义,HC－Pro基因及其缺失突变体不仅能够提高T1转基因烟草中PVY－C的病毒积累和致病,而且对异源病毒PVX具有同样的作用,而转反义HC－Pro基因烟草对PVY－C和PVX的致病性无影响,因此,PVY－C HC－Pro基因介导PVX／PVY的协作作用。     

生精细胞凋亡相关基因

细胞凋亡(apoptosis)是一种基因控制的细胞生理性自杀行为,用以维持细胞数量的相对恒定,可由某种刺激或抑制剂的移除而激活。在哺乳动物精子发生过程中,各级生精细胞都会发生相应的凋亡,通过严格调控以确保成熟精子生成的数量和质量。生精细胞的凋亡是一个许多基因参与的复杂的不可逆过程,其中Bcl-2/Bax基因族、p53基因、Fas-Fasl基因、C-myc基因、CREM基因、HSP基因族、c-Kit/SCF基因、Insl3基因、iNOS基因、BMP8B基因、TR基因和存活蛋白(survivin)基因等发挥了重要作用。研究哺乳动物睾丸生精细胞凋亡相关基因,有利于了解生精细胞凋亡机制,为进一步阐明精子发生的调控机制,预防和治疗精子发生相关疾病提供重要的理论依据。     

The freshwater planarian Dugesia (G.) tigrina contains a great diversity of homeobox genes

To identify potential pattern control and cell determination and/or differentiation genes in the freshwater planarian Dugesial (G.) tigrina, we searched for homeobox genes of different types in the genome of this primitive metazoan. We applied two basic approaches: 1) Screening the cDNA library with degenerate oligonucleotides corresponding to the most conserved amino acid sequence from helix-3 of the homeodomain of each family; and 2) PCR amplification of genomic DNA or cDNA, using two sets of degenerated oligonucleotides corresponding to helices 1 and 3 of the homeodomain or two specific domains of the POU family. Using the first strategy we have identified and characterized two tissue-specific cell determination and/or differentiation NK-type homeobox genes. Using the second strategy we have identified several homeobox genes that belong to the HOM/Hox, paired (prd) or POU families.     

Biosynthesis of dothistromin

Dothistromin is a mycotoxin that is remarkably similar in structure to versicolorin B, a precursor of both aflatoxin and sterigmatocystin. Dothistromin-producing fungi also produce related compounds, including some aflatoxin precursors as well as alternative forms of dothistromin. Dothistromin is synthesized by pathogenic species of Dothistroma in the red bands of pine needles associated with needle blight, but is also made in culture where it is strongly secreted into the surrounding medium. Orthologs of aflatoxin and sterigmatocystin biosynthetic genes have been found that are required for the biosynthesis of dothistromin, along with others that are speculated to be involved in the same pathway on the basis of their sequence similarity to aflatoxin genes. An epoxide hydrolase gene that has no homolog in the aflatoxin or sterigmatocystin gene clusters is also clustered with the dothistromin genes, and all these genes appear to be located on a minichromosome in Dothistroma septosporum. The dothistromin genes are expressed at an early stage of growth, suggesting a role in the first stages of plant invasion by the fungus. Future studies are expected to reveal more about the role of dothistromin in needle blight and about the genomic organization and expression of dothistromin genes: these studies will provide for interesting comparisons with these aspects of aflatoxin and sterigmatocystin biosynthesis.     

PhiC31 integrase mediates integration in cultured synovial cells and enhances gene expression in rabbit joints

BACKGROUND: Gene transfer to synovium in joints has been shown to be an effective approach for treating pathologies associated with rheumatoid arthritis (RA) and related joint disorders. However, the efficiency and duration of gene delivery has been limiting for successful gene therapy for arthritis. The transient gene expression that often accompanies non-viral gene delivery can be prolonged by integration of vector DNA into the host genome. We report a novel approach for non-viral gene therapy to joints that utilizes phage phiC31 integrase to bring about unidirectional genomic integration. METHODS: Rabbit and human synovial cells were co-transfected with a plasmid expressing phiC31 integrase and a plasmid containing the transgene and an attB site. Cells were cultured with or without G418 selection and the number of neo-resistant colonies or eGFP cells determined, respectively. Plasmid rescue, PCR query, and DNA sequence analysis were performed to reveal integration sites in the rabbit and human genomes. For in vivo studies, attB-reporter gene plasmids and a plasmid expressing phiC31 integrase were intra-articularly injected into rabbit knees. Joint sections were used for histological analysis of beta-gal expression, and synovial cells were isolated to measure luciferase expression. RESULTS: We demonstrated that co-transfection of a plasmid expressing phiC31 integrase with a plasmid containing the transgene and attB increased the frequency of transgene expression in rabbit synovial fibroblasts and primary human RA synoviocytes. Plasmid rescue and DNA sequence analysis of plasmid-chromosome junctions revealed integration at endogenous pseudo attP sequences in the rabbit genome, and PCR query detected integration at previously characterized integration sites in the human genome. Significantly higher levels of transgene expression were detected in vivo in rabbit knees after intra-articular injection of attB-reporter gene plasmids and a plasmid expressing phiC31 integrase. CONCLUSION: The ability of phiC31 integrase to facilitate genomic integration in synovial cells and increase transgene expression in the rabbit synovium suggests that, in combination with more efficient DNA delivery methods, this integrase system could be beneficial for treatment of rheumatoid arthritis and other joint disorders.     

基因表达系列分析法(SAGE)的改进及其在植物功能基因组研究中的应用

基因表达系列分析方法(SAGE)是一种新的基因表达分析方法,与基因芯片技术一样具有高通量的特点,可测定特定组织的基因表达水平,在全基因组水平上同时定量检测数万个基因表达模式;可在未知目的基因的前提下,分析来自一个细胞的全部转录本信息;对已知或未知基因表达进行定性和定量分析.目前,虽然在疾病、发育、细胞凋亡、药物筛选等多个领域已有利用SAGE方法进行的研究,但该方法在植物功能基因组研究中的应用相对较少.本文主要综述了该方法在RNA用量、PCR循环次数、SAGE效能和可靠性、标签长度和未知标签分析等方面的改进及其在植物中构建SAGE文库、筛选新基因、基因表达图谱分析等方面的应用,从而为其在植物功能基因组研究中的进一步应用提供理论参考.     

长白蝮蛇类凝血酶基因的克隆及分析

从长白蝮蛇（Agkistrodon halys Ussuriensis)毒腺中抽提总RNA,采用RT－PCR扩增其类凝血酶基因,经全序列测定,类凝血酶基因Ussurin全长为708个核苷酸,即编码236个氨基酸;根据同源性,推测它的活性中心为His^43,Asp^88和Ser^182;二硫键为Cys^7-Cys^141,Cys^28-Cys^44,Cys^76-Cys^234,Cys^120-Cys^188,Cys^152-Cys^167和Cys^178-Cys^203。该蛇毒类凝血酶cDNA序列及推导的氨基酸序列均为首次报道。