同源重组探针在暂时转化的烟草原生质体内的重组和缺失

通过电激法(electroporation)将同源重组探针pBC 7导入烟草原生质体,再从转化的烟草原生质体中提取DNA,并将其转化到E.coli DH 1细胞.结果在E.codi DH 1细胞中获得了一种新的质粒分子,该质粒含有功能的neo基因,与重组探针pBC 7相比有较大的缺失、倒位,并出现了新的限制酶切部位,重组探针pBC 7的主要特征为含有Tn5的neo基因两个截短的不等部份,只有通过重排才可能产生完整的neo基因,这表明了暂时转化的原生质体在外源DNA整合以前具有这样的基因重排功能。     

Programmed gene rearrangements altering gene expression

Programmed gene rearrangements are used in nature to to alter gene copy number (gene amplification and deletion), to create diversity by reassorting gene segments (as in the formation of mammalian immunoglobulin genes), or to control the expression of a set of genes that code for the same function (such as surface antigens). Two major mechanisms for expression control are DNA inversion and DNA transposition. In DNA inversion a DNA segment flips around and is rejoined by site-specific recombination, disconnecting or connecting a gene to sequences required for its expression. In DNA transposition a gene moves into an expression site where it displaces its predecessor by gene conversion. Gene rearrangements altering gene expression have mainly been found in some unicellular organisms. They allow a fraction of the organisms to preadapt to sudden changes in environment, that is, to alter properties such as surface antigens in the absence of an inducing stimulus. The antigenic variation that helps the causative agents of African trypanosomiasis, gonorrhea, and relapsing fever to elude host defense is controlled in this way.     

Stable integration and expression of a bacterial gene in the mosquito Anopheles gambiae

Foreign DNA was successfully introduced into the germline of the African mosquito vector of malaria Anopheles gambiae. Stable integration of genes into the germlines of insects had been achieved previously only in Drosophila melanogaster and related species and required the use of the P element transposon. In these experiments with Anopheles gambiae, the plasmid pUChsneo was used, which contains the selectable marker neo gene flanked by P element inverted repeats. Mosquitoes injected with this plasmid were screened for resistance to the neomycin analog G-418. A single event of plasmid insertion was recovered. Integration appears to be stable and, thus far, resistance to G-418 has been expressed for eight generations. The transformation event appears to be independent of P.     

Rearrangement of the bacterial chromosome: forbidden inversions

The order of genes in the chromosome of enteric bacteria has been evolutionarily conserved despite the existence of mechanisms for rearrangement. Homologous chromosomal sequences in the same orientation recombine to form deletions or duplications. When homologous sequences in inverse orientation recombine, one expects to form an inversion of the intervening chromosomal segment. This expectation was tested by placing pairs of homologous sequences in inverse order at various points in the chromosome. Sequences at many pairs of sites (permissive) do recombine to generate the expected inversion, while the same sequences placed at other pairs of sites (nonpermissive) do not form an inversion. For the one nonpermissive interval tested, the missing inversion type can be constructed by an alternative transductional method; strains with this inversion are viable. Thus mechanistic limitations must prevent sequences at particular sites from undergoing the recombination event required to form an inversion.     

转座子沉默与DNA甲基化

转座子(transposable elements,TEs)在生物体基因组可以通过转座或逆转座移动,它拷贝数的大规模增加是基因组不稳定的重要因素,因此,维持TEs沉默是宿主进化的方向。DNA甲基化被认为是沉默TEs的可遗传表观遗传修饰方式,同时也在维持基因组稳定、基因印迹、调节基因表达中发挥作用。本研究综述了TEs对生物基因组进化和基因表达的影响,重点总结了以DNA甲基化为主的转座子沉默机制的最新研究进展,归纳了环境因素通过DNA去甲基化调控转座子跳跃的机理。图4参82     

Dependence of heterochromatic histone H3 methylation patterns on the Arabidopsis gene DDM1

The Arabidopsis gene DDM1 is required to maintain DNA methylation levels and is responsible for transposon and transgene silencing. However, rather than encoding a DNA methyltransferase, DDM1 has similarity to the SWI/SNF family of adenosine triphosphate-dependent chromatin remodeling genes, suggesting an indirect role in DNA methylation. Here we show that DDM1 is also required to maintain histone H3 methylation patterns. In wild-type heterochromatin, transposons and silent genes are associated with histone H3 methylated at lysine 9, whereas known genes are preferentially associated with methylated lysine 4. In ddm1 heterochromatin, DNA methylation is lost, and methylation of lysine 9 is largely replaced by methylation of lysine 4. Because DNA methylation has recently been shown to depend on histone H3 lysine 9 methylation, our results suggest that transposon methylation may be guided by histone H3 methylation in plant genomes. This would account for the epigenetic inheritance of hypomethylated DNA once histone H3 methylation patterns are altered.     

沙门菌鞭毛基因表达的相变换研究进展

沙门菌是目前研究较多的细菌之一,广泛分布于脊椎动物的肠道和很多种类的节肢动物中。大多数沙门菌为Ⅱ相菌株,具有鞭毛相变换的特征,这与其毒力和逃避宿主免疫监视有关。综述了沙门菌鞭毛相变换的研究进展,为进一步研究沙门菌的致病机制提供了理论依据。     

The unusual varl gene of yeast mitochondrial DNA

The var1 gene specifies the only mitochondrial ribosomal protein known to be encoded by yeast mitochondrial DNA. The gene is unusual in that its base composition is nearly 90 percent adenine plus thymine. It and its expression product show a strain-dependent variation in size of up to 7 percent; this variation does not detectably interfere with function. Furthermore, var1 is an expandable gene that participates in a novel recombinational event resembling gene conversion whereby shorter alleles are preferentially converted to longer ones. The remarkable features of var1 indicate that it may have evolved by a mechanism analogous to exon shuffling, although no introns are actually present.     

Global transposon mutagenesis and a minimal Mycoplasma genome

Mycoplasma genitalium with 517 genes has the smallest gene complement of any independently replicating cell so far identified. Global transposon mutagenesis was used to identify nonessential genes in an effort to learn whether the naturally occurring gene complement is a true minimal genome under laboratory growth conditions. The positions of 2209 transposon insertions in the completely sequenced genomes of M. genitalium and its close relative M. pneumoniae were determined by sequencing across the junction of the transposon and the genomic DNA. These junctions defined 1354 distinct sites of insertion that were not lethal. The analysis suggests that 265 to 350 of the 480 protein-coding genes of M. genitalium are essential under laboratory growth conditions, including about 100 genes of unknown function.     

Establishment of a Gene Expression System in Rice Chloroplast and Obtainment of PPT-Resistant Rice Plants

In contrast to the situation of random integration of foreign genes in nuclear transformation, the introduction of genes via chloroplast genetic engineering is characterized by site-specific pattern via homologous recombination. To establish an expression system for alien genes in rice chloroplast, the intergenic region of ndhF and trnL was selected as target for sitespecific integration of PPT-resistant bar gene in this study. Two DNA fragments suitable for homologous recombination were cloned from rice chloroplast genome DNA using PCR technique, and the chloroplast-specific expression vector pRB was constructed by fusing a modified 16S rRNA gene promoter to bar gene together with terminator ofpsbA gene 3 sequence. Chloroplast transformation was carried out by biolistic bombardment of sterile rice calli with the pRB construct. Subsequently, the regenerated plantlets and seeds of progeny arising from reciprocal cross to the wild-type lines were obtained. Molecular analysis suggested that the bar gene has been integrated into rice chloroplast genome. Genetic analysis revealed that bar gene could be transmitted and expressed normally in chloroplast genome. Thus, the bar gene conferred not only selection pressure for the transformation of rice chloroplast genome, but PPT-resistant trait for rice plants as well. It is suggested that an efficient gene expression system in the rice chloroplast has been established by chloroplast transformation technique.     

反刍月形单胞菌乙酸生成缺陷株的构建及相关酶活性分析(英文)

[目的]构建反刍月形单胞菌乙酸生成的缺陷株并分析其发酵特性。[方法]应用转座子标签法,通过转座子供体菌E.coliS17-1/pZJ25∷Tn5对受体菌反刍月形单胞菌进行转座子诱变,采用含卡那霉素和氟乙酸纳的选择性培养基筛选接合子。[结果]共筛选出稳定的对卡那霉素和氟乙酸具有抗性的转座工程菌7株。对反刍月形单胞菌的突变株进行16SrRNA鉴定和Tn5的PCR鉴定,及乙酸激酶(AK)和磷酸乙酰转移酶(PTA)酶比活力分析,确定突变株属于pta基因缺失型氟乙酸抗性菌株。[结论]为进一步研究反刍兽瘤胃微生物乙酸的细胞代谢网络和调控奠定基础。     

Chloroplast transformation in Chlamydomonas with high velocity microprojectiles

Bombardment of three mutants of the chloroplast atpB gene of Chlamydomonas reinhardtii with high-velocity tungsten microprojectiles that were coated with cloned chloroplast DNA carrying the wild-type gene permanently restored the photosynthetic capacity of the algae. In most transformants of one of the mutants, a fragment with a 2.5-kilobase deletion was restored to normal size by a homologous replacement event; in about 25 percent of the transformants the restored restriction fragment was 50 to 100 base pairs smaller or larger than that of wild type. About one-fourth of the transformants of this mutant contained unintegrated donor plasmid when first examined. This plasmid persisted in four different transformants after 65 cell generations of continuous liquid culture but was lost from all transformants maintained on plates of selective medium. The restored wild-type atpB gene remains in all transformants as an integral part of the chloroplast genome and is expressed and inherited normally.     

西安地区一次大雾天气过程的数值模拟研究

利用非静力平衡中尺度模式WRFV3.2、NCEP 1°×1°再分析资料和常规观测资料,对2002年12月12～13日西安地区一次持续大雾天气进行阶段性数值模拟研究。结果表明,利用WRFV3.2模式能较好地模拟出大雾的范围、强度和生消过程,但模拟白天雾的强度较弱。这次大雾是平流辐射雾过程,在夜间的辐射降温和弱冷空气的入侵共同作用下,西安地区形成冷气垫,造成逆温产生,逆温的存在为大雾的产生和维持发展提供了有利的层结条件;地面湿度大,逆温层结稳定又有效地抑制了水汽向高空传输和能量交换,为大雾产生提供了充沛的水汽条件;在大雾产生和维持期间,微风有利于近地面的湍流交换,使辐射冷却作用扩散到适当高度,也可使水汽垂直输送到一定高度,有利于雾的产生和发展。而白天太阳短波辐射造成的逆温层抬升是引起大雾减弱和日变化明显的主要原因。     

RNA干涉及其应用

在各种各样的真核生物当中,双链RNA(dsRNA)促使了有效的同源mRNA分子降解,这个过程被称为RNA干扰(RNAi)。RNAi现象通过调控RNA抑制基因表达,它在植物、动物、真菌中广泛存在,被认为是一种抑制病毒性复制和转座子活动的抗御机制,而在此过程中,侵染型病毒为了能够不被这种机制干扰抑制,继续在寄主体内生存、繁殖,则产生了一种反抗御机制———即产生抑制蛋白来对付转录后基因沉默(PTGS)。对RNAi以及RNAi抑制物的作用机制、抑制物类型、特点及相关应用方面作了比较详尽的综述。dsRNA介导的PTGS将是分子生物学领域的研究热点之一,在研究基因功能,基因治疗,抗病毒基因工程,解剖和调控次生代谢途径等方面有着广泛的应用前景。     

Mutations in a protein kinase C homolog confer phorbol ester resistance on Caenorhabditis elegans

The tpa-1 gene mediates the action of tumor-promoting phorbol esters in the nematode Caenorhabditis elegans. A genomic fragment that constitutes a portion of the tpa-1 gene was cloned by Tc1 transposon tagging and was used as a probe to screen a nematode complementary DNA library. One of the isolated complementary DNA clones had a nucleotide sequence that predicts a polypeptide of 526 amino acids. The predicted amino acid sequence revealed that the predicted tpa-1 protein sequence is highly similar to protein kinase C molecules from various animals, including man.     

高等植物基因克隆的策略

基因克隆是进行植物基因功能研究的先决条件。随着拟南芥、水稻、杨树等植物全基因组序列的公布,标志着基因组进入后基因组研究时代是分子生物学发展的必然趋势,同时为进一步克隆植物中控制重要性状的主效基因提供强大的基因组数据支撑。目前有较多的基因克隆传统及衍生技术,文章综述了图位克隆、同源克隆、转座子标记法、表达序列标签法、差异表达基因克隆等5种应用比较广泛的基因克隆技术,并就其发展方向和策略作了展望。