NKX3.1基因沉默对LNCaP细胞基因表达谱的影响

目的探讨降低NKX3.1表达对雄激素依赖性前列腺癌细胞系（LNCaP）基因表达谱的影响。方法用表达NKX3.1 shRNA的质粒转染LNCaP细胞,经Northern blot和Western blot检测其干扰NKX3.1 mRNA和蛋白表达的效果,基因表达芯片法比较转染shRNA的细胞与转染空载体的对照细胞之间mRNA表达的差异。结果shRNA干扰可降低LNCaP细胞内NKX3.1蛋白的水平,可引起115种基因表达下调,114种基因表达上调;在严谨条件下进行功能聚类可得到与细胞凋亡相关、与糖代谢相关和与氨基酸代谢相关等共7个基因功能富集组。结论NKX3.1蛋白水平下调可引起LNCaP细胞基因表达谱的显著改变,其中一些基因表达的改变可能与NKX3.1的肿瘤调节活性密切相关。     

日本血吸虫Mago nashi基因RNA干扰系统的构建及鉴定

目的构建针对日本血吸虫Mago nashi样蛋白基因的RNA干扰的表达载体。方法设计及化学合成日本血吸虫Mago nashi样蛋白基因的短发夹结构的寡核苷酸,通过退火成双链DNA片段,将其与经限制性内切酶BglⅡ和HindⅢ双酶切的pSUPER质粒连接,构建表达shRNA的重组质粒。结果经酶切及测序证实针对日本血吸虫Mago nashi样蛋白基因的RNA干扰的表达载体pSUPER构建成功。结论成功构建针对日本血吸虫Mago nashi样蛋白基因的RNA干扰的表达载体,为进一步研究对日本血吸虫Mago nashi样蛋白基因RNA干扰作用及该基因功能奠定基础。     

RNAi-mediated immunity provides strong protection against the negative-strand RNA vesicular stomatitis virus in Drosophila

Activation of innate antiviral responses in multicellular organisms relies on the recognition of structural differences between viral and cellular RNAs. Double-stranded (ds)RNA, produced during viral replication, is a well-known activator of antiviral defenses and triggers interferon production in vertebrates and RNAi in invertebrates and plants. Previous work in mammalian cells indicates that negative-strand RNA viruses do not appear to generate dsRNA, and that activation of innate immunity is triggered by the recognition of the uncapped 5' ends of viral RNA. This finding raises the question whether antiviral RNAi, which is triggered by the presence of dsRNA in insects, represents an effective host-defense mechanism against negative-strand RNA viruses. Here, we show that the negative-strand RNA virus vesicular stomatitis virus (VSV) does not produce easily detectable amounts of dsRNA in Drosophila cells. Nevertheless, RNAi represents a potent response to VSV infection, as illustrated by the high susceptibility of RNAi-defective mutant flies to this virus. VSV-derived small RNAs produced in infected cells or flies uniformly cover the viral genome, and equally map the genome and antigenome RNAs, indicating that they derive from dsRNA. Our findings reveal that RNAi is not restricted to the defense against positive-strand or dsRNA viruses but can also be highly efficient against a negative-strand RNA virus. This result is of particular interest in view of the frequent transmission of medically relevant negative-strand RNA viruses to humans by insect vectors.     

Identification of genes that interact with the sina gene in Drosophila eye development.

The sina gene encodes a nuclear protein that is required for the correct development of R7 photoreceptor cells in the Drosophila eye. We conducted a genetic screen for mutations that reduce the activity of sina and found mutations that define nine genes whose products may be required for normal sina activity. Three of these genes also appear to be essential for signaling by the Sevenless-Ras pathway in R7 cells, of which one gene corresponds to the rolled locus (rl). The rl gene is known to encode a mitogen-activated protein kinase necessary for signaling by Ras. These results suggest that the products of these three genes may participate in a signaling pathway involving both Ras and Sina, possibly by functionally linking these two proteins.     

Tiling genomes of pathogenic viruses identifies potent antiviral shRNAs and reveals a role for secondary structure in shRNA efficacy

shRNAs can trigger effective silencing of gene expression in mammalian cells, thereby providing powerful tools for genetic studies, as well as potential therapeutic strategies. Specific shRNAs can interfere with the replication of pathogenic viruses and are currently being tested as antiviral therapies in clinical trials. However, this effort is hindered by our inability to systematically and accurately identify potent shRNAs for viral genomes. Here we apply a recently developed highly parallel sensor assay to identify potent shRNAs for HIV, hepatitis C virus (HCV), and influenza. We observe known and previously unknown sequence features that dictate shRNAs efficiency. Validation using HIV and HCV cell culture models demonstrates very high potency of the top-scoring shRNAs. Comparing our data with the secondary structure of HIV shows that shRNA efficacy is strongly affected by the secondary structure at the target RNA site. Artificially introducing secondary structure to the target site markedly reduces shRNA silencing. In addition, we observe that HCV has distinct sequence features that bias HCV-targeting shRNAs toward lower efficacy. Our results facilitate further development of shRNA based antiviral therapies and improve our understanding and ability to predict efficient shRNAs.     

mu-2: mutator gene in Drosophila that potentiates the induction of terminal deficiencies.

An x-ray-dependent mutator on chromosome 3 of Drosophila melanogaster is described that specifically increases the recovery of deletions for chromosomal tip regions. Such deficiencies can be induced on any chromosome. More centromere proximal mutations, as assayed by the sex-linked recessive lethal test, are not increased over the wild-type control. As far as can be determined by genetic, cytological, and molecular assays, the deletions extend to the very end of the chromosome involved. In addition, the frequency of these deletions is directly proportional to x-ray dose, suggesting that they are one-break rearrangements. It is proposed that the mutator is blocked in a major pathway for the repair of DNA double-strand breaks, and that a minor repair pathway is responsible for the addition of new telomeres under these conditions.     

Ubiquitous internal gene duplication and intron creation in eukaryotes

Duplication of genomic segments provides a primary resource for the origin of evolutionary novelties. However, most previous studies have focused on duplications of complete protein-coding genes, whereas little is known about the significance of duplication segments that are entirely internal to genes. Our examination of six fully sequenced genomes reveals that internal duplications of gene segments occur at a high frequency (0.001–0.013 duplications/gene per million years), similar to that of complete gene duplications, such that 8–17% of the genes in a genome carry duplicated intronic and/or exonic regions. At least 7–30% of such genes have acquired novel introns, either because a prior intron in the same gene has been duplicated, or more commonly, because a spatial change has activated a latent splice site. These results strongly suggest a major evolutionary role for internal gene duplications in the origin of genomic novelties, particularly as a mechanism for intron gain.     

肾素(前体)受体miRNA干扰质粒的构建及鉴定

目的 设计及构建肾素(前体)受体微小干扰核糖核酸(miRNA)质粒,并鉴定出有效干扰质粒.方法 设计及构建4对肾素(前体)受体的pcDNATM6·2-GW/EmGFPmiR miRNA及1对阴性对照miRNA干扰质粒,通过测序鉴定.将干扰质粒用LipofectamineTM 2000转染大鼠血管平滑肌细胞,通过顺时转染获得细胞系,倒置荧光显微镜下观察绿色荧光及流式细胞术确定转染效率,Real-time PCR和Western blot检测4对干扰质粒、阴性对照质粒肾素(前体)受体的mRNA及蛋白表达水平.结果 测序显示肾素(前体)受体干扰序列及读框完全正确,倒置荧光显微镜及流式细胞术结果显示干扰质粒瞬时转染的大鼠血管平滑肌细胞系的转染效率均在60%以上.Real-time PCR和Western blot结果显示X2-2-3、X2-3-3干扰质粒对肾素(前体)受体 mRNA及蛋白有较好的抑制效果.结论 成功构建了肾素(前体)受体干扰真核表达载体,筛选出有效干扰质粒,为进一步研究肾素(前体)受体在大鼠血管平滑肌细胞中的作用提供参考.     

Molecular cloning of a Drosophila diacylglycerol kinase gene that is expressed in the nervous system and muscle.

We have isolated a Drosophila melanogaster diacylglycerol kinase (DGK, EC 2.7.1.107) homologue by using a porcine DGK cDNA probe and we have characterized its structure and expression. The DGK cDNA has a single open reading frame that encodes 791 amino acids. The Drosophila and porcine DGKs share a similar carboxyl-terminal region, a putative catalytic domain, which is divided into two separate domains in Drosophila. The DGK gene was mapped to the cytogenetic position 43F1, and its DGK mRNA is abundant both in embryo and in adult fly. By in situ hybridization to sections of adult flies, we demonstrated that the mRNA is present predominantly in the nervous system and muscles, including compound eyes, brain cortex, fibrillar muscle, and tubular muscle. In a 10- to 11-hr embryo, the DGK gene is expressed abundantly in a limited number of cells in the procephalic region and in the ventral nerve cord. The pattern of temporal and spatial expression suggests that the DGK protein has an important function in the adult nervous system and muscle and during the development of the embryonic nervous system.     

Locus control region elements HS1 and HS4 enhance the therapeutic efficacy of globin gene transfer in beta-thalassemic mice

Globin gene transfer in autologous hematopoietic stem cells is a promising therapeutic option for subjects with beta-thalassemia major. In this approach, high level, erythroid-specific globin transgene expression should correct ineffective erythropoiesis and hemolytic anemia following the delivery of only 1 to 2 vector copies per cell. The generation of vectors that provide high-level globin expression and require low vector copy (VC) integration is therefore essential for both safety and efficacy. We show here the major roles played by 2 lesser-known locus control region elements, termed HS1 and HS4. Partial deletions within HS4 markedly reduce in vivo globin expression requiring multiple VC per cell to correct the anemia. Most strikingly, addition of HS1 to HS2-3-4 increases globin expression by 52%, yielding 9 g Hb/VC in beta-thalassemic mice. Thus, while vectors encoding HS2-3-4 provide curative levels of hemoglobin at 1 to 2 copies per cell, adding HS1 is a promising alternative strategy if upcoming clinical trials prove higher levels of expression to be necessary.     

Development of a transgenic mouse that overexpresses a novel product of the growth hormone-releasing hormone gene

The GH-releasing hormone (GHRH) precursor molecule contains a 30-amino acid C-terminal region that has been designated GHRH-related peptide (GHRH-RP). To begin to understand the physiological role of GHRH-RP, transgenic (Tg) mice that constituitively express this peptide were developed. To generate these mice, a transgene (SS-RP) was constructed by overlap primer extension PCR. This transgene, under the control of the mouse phosphoglycerate kinase gene, selectively expresses GHRH-RP, but not GHRH. Western blot analysis confirmed that the transgene produces GHRH-RP. Animals were evaluated for the effect of excess GHRH-RP on growth, fertility, behavior, stem cell factor (SCF) expression, and hematopoiesis. Northern blot and RT-PCR were used to demonstrate ubiquitous expression of the transgene in tissues from GHRH-RP Tg animals. These tissues also had marked overexpression of SCF messenger RNA compared with controls. Tg animals had significantly increased cell cycling for granulocyte-macrophage, erythroid, and multilineage progenitor cells. Transgenic animals did not differ from control mice in their growth, fertility, or behavior. These findings demonstrate, for the first time, that in vivo the C-terminal peptide of the pro-GHRH molecule is a biologically active peptide that is capable of stimulating the expression of SCF and hematopoiesis in vivo and suggests that GHRH-RP may play a role in normal blood cell development.     

Vernalization-induced flowering in cereals is associated with changes in histone methylation at the VERNALIZATION1 gene

Prolonged exposure to low temperatures (vernalization) accelerates the transition to reproductive growth in many plant species, including the model plant Arabidopsis thaliana and the economically important cereal crops, wheat and barley. Vernalization-induced flowering is an epigenetic phenomenon. In Arabidopsis, stable down-regulation of FLOWERING LOCUS C (FLC) by vernalization is associated with changes in histone modifications at FLC chromatin. In cereals, the vernalization response is mediated by stable induction of the floral promoter VERNALIZATION1 (VRN1), which initiates reproductive development at the shoot apex. We show that in barley (Hordeum vulgare), repression of HvVRN1 before vernalization is associated with high levels of histone 3 lysine 27 trimethylation (H3K27me3) at HvVRN1 chromatin. Vernalization caused increased levels of histone 3 lysine 4 trimethylation (H3K4me3) and a loss of H3K27me3 at HvVRN1, suggesting that vernalization promotes an active chromatin state at VRN1. Levels of these histone modifications at 2 other flowering-time genes, VERNALIZATION2 and FLOWERING LOCUS T, were not altered by vernalization. Our study suggests that maintenance of an active chromatin state at VRN1 is likely to be the basis for epigenetic memory of vernalization in cereals. Thus, regulation of chromatin state is a feature of epigenetic memory of vernalization in Arabidopsis and the cereals; however, whereas vernalization-induced flowering in Arabidopsis is mediated by epigenetic regulation of the floral repressor FLC, this phenomenon in cereals is mediated by epigenetic regulation of the floral activator, VRN1.     

Evidence from disruption of the lmcpb gene array of Leishmania mexicana that cysteine proteinases are virulence factors.

The mammalian form of the protozoan parasite Leishmania mexicana contains high activity of a cysteine proteinase (LmCPb) encoded on a tandem array of 19 genes (lmcpb). Homozygous null mutants for lmcpb have been produced by targeted gene disruption. All life-cycle stages of the mutant can be cultured in vitro, demonstrating that the gene is not essential for growth or differentiation of the parasite. However, the mutant exhibits a marked phenotype affecting virulence-- its infectivity to macrophages is reduced by 80%. The mutants are as efficient as wild-type parasites in invading macrophages but they only survive in a small proportion of the cells. However, those parasites that successfully infect these macrophages grow normally. Despite their reduced virulence, the mutants are still able to produce subcutaneous lesions in mice, albeit at a slower rate than wild-type parasites. The product of a single copy of lmcpb re-expressed in the null mutant was enzymatically active and restored infectivity toward macrophages to wild-type levels. Double null mutants created for lmcpb and lmcpa (another cathepsin L-like cysteine proteinase) have a similar phenotype to the lmcpb null mutant, showing that LmCPa does not compensate for the loss of LmCPb.     

Application of intron 9 and intron 25 dinucleotide repeats of the factor VIII gene for carrier diagnosis in haemophilia A

Summary.  We describe the usefulness of two dinucleotide repeats located in intron 9 and in intron 25 of the factor VIII gene for carrier diagnosis of haemophilia A. We analyzed 100 unrelated Spanish women and 34 women from haemophilia A (HA) families in whom known intragenic markers were unhelpful in determining their carrier status. The heterozygosity rate of intron 9 and intron 25 markers in the 100 control women was lower (0.28 and 0.38, respectively) than the values obtained with common markers routinely used in our laboratory. However, the application of intron 9 and intron 25 markers was effective in identifying the at-risk X chromosome in 11 of 34 (32%) of the uninformative women from HA families. The combined use of these repeats with current markers may facilitate the identification of the X chromosome in HA families for application in carrier, prenatal and pre-implantation diagnoses.     

Expression in transgenic plants of a viral gene product that mediates insect transmission of potyviruses.

Helper component (HC) is a virus-encoded nonstructural protein that is required for transmission of potyviruses by their aphid vectors. As a prelude to studies on the molecular basis of HC activity, a cDNA clone (pPB-3) was constructed that contained the first three cistrons (34 kDa-HC-42 kDa) of the RNA genome of the potyvirus tobacco vein mottling virus, the first six nucleotides of the adjacent cylindrical inclusion body protein cistron, and a synthetic translation termination codon. This construction was introduced into tobacco cells via a Ti plasmid-based vector. Northern blot analysis of transgenic plants demonstrated the presence of an RNA of the size expected from the construction of pPB-3, and Western blot analysis revealed the presence of a protein that comigrated with authentic HC, indicating that the proteolytic activity necessary to produce mature-sized HC was encoded by pPB-3. The HC produced in the transgenic plants was demonstrated to be active in a virus transmission bioassay with aphids.     

刚地弓形虫醛缩酶基因及其内含子的分析

目的刚地弓形虫醛缩酶是其速殖子入侵宿主细胞过程中的一个重要分子,本研究对该酶基因序列及其内含子进行实验和分析。方法提取刚地弓形虫RH株速殖子基因组DNA和总RNA,采用PCR技术分别从基因组DNA和cDNA中扩增编码果糖1,6-二磷酸醛缩酶的基因,并克隆到T载体上,经测序鉴定后对其基因组序列和cDNA序列进行比较分析。通过NCBI数据库资源对醛缩酶内含子的分布及大小进行比较分析。结果根据醛缩酶基因设计的引物从基因组DNA扩增得到的片段长度为1315bp,从cDNA扩增得到的片段为993bp。比对结果显示来源于基因组DNA的序列在对应于编码序列起始密码子下游109bp后有一个322bp的内含子。通过在线工具收集得到NCBI及EMBL数据库中来源于不同物种的32种醛缩酶数据,分析显示推断氨基酸序列具有保守性。结论编码刚地弓形虫(Toxoplasmagondii)RH株果糖1,6-二磷酸醛缩酶基因编码区内存在一个322bp的内含子。醛缩酶氨基酸序列保守性较高。内含子在不同进化等级之间插入位置数目及大小两方面均存在明显的增加趋势。     

shRNA干扰Rab9 GTPase表达对麻疹病毒野生株体外增殖的抑制作用

目的构建Rab9 GTPase短发夹RNA（shRNA）表达载体,观察其对Rab9 GTPase基因表达和麻疹病毒野生株体外增殖的抑制作用。方法参照GenBank中Rab9 GTPase基因序列设计合成2对Rab9 GTPase基因特异性shRNA,定向克隆人表达载体,构建重组表达载体,酶切鉴定和序列分析证实后脂质体法转染U937细胞,然后感染麻疹病毒野生株,逆转录聚合酶链反应（RT-PCR）和免疫印迹技术（Western blot）检测转染细胞内Rab9 GTPase mRNA和蛋白质的表达水平;标准蚀斑试验测定病毒滴度;流式细胞仪检测细胞凋亡率的变化;RT-PCR检测转染细胞内双链RNA依赖蛋白激酶（PKR）和2′-5′寡腺甙酸合成酶（OAS-1）的mRNA水平。结果酶切和序列分析证实,成功构建了靶向Rab9 GTPase基因的shRNA表达载体。2个shRNAs均可特异性抑制U937细胞内Rab9 GTPase mRNA和蛋白质的表达,最高抑制率分别为（90.5±0.2）％和（92.1±0.3）％;蚀斑试验结果表明,shRNAs可以有效抑制麻疹病毒野生株体外增殖,其抑制率可达到90％以上;流式细胞仪检测转染后细胞的凋亡率无明显变化;RT-PCR检测PKR和OAs-1的mRNA水平转染前后无明显变化。结论成功构建Rab9 GTPase特异性shRNA表达载体。shRNAs通过特异性抑制Rab9 GTPase基因表达抑制麻疹病毒野生株体外增殖。     

Fecundity and life span in transgenic Drosophila melanogaster overexpressing hsp70

In the present study, we investigated the effect of hsp70 over expression on some life history components in transgenic fruit flies. We measured life span in mated flies and fecundity in flies subjected or not subjected to a heat shock inducing hsp70. Heat shock increased life span of the parental line, but not of the transgenic lines. Genetic manipulation of the flies altered their fecundity, but the heat shock had no effect on fecundity. To conclude, we have observed some costs of genetic manipulation by itself on life span and fecundity. However, the over expression of thehsp70 extra copies by the exposure to heat did not alter the studied variables. This revised version was published online in July 2006 with corrections to the Cover Date.