克隆差异表达基因的新策略

基因表达的变化有两种,即新出现的基因表达与表达量差异的基因表达.表达量差异的基因克隆技术主要有mRNA差异展示,此技术是目前筛选差异表达基因最有效的方法之一,但主要存在假阳性率高的不足,针对此缺点,近几年提出了新的策略与方法,如差异消减展示、基于PCR和减法杂交基础上的差异表达基因克隆技术,这些技术具有显著优势.     

新基因克隆技术进展

新基因克隆是当今生物学最热点的领域之一。本文从技术原理、适应性及使用例证等方面,综述和评价了已有的及发展中的新基因克隆技术,包括克隆已有多肽分子的基因、同源家族基因克隆、功能性筛选、差异及减数筛选等,并着重介绍了ＲＤＡ、ＥＤＳ、ＲＬＣＳ等差异筛选技术。     

一种基于核酸外切酶Ⅲ的PCR产物克隆方法

基因克隆作为一种常规技术被广泛应用于DNA及蛋白质的研究。在传统的基因克隆中,一般利用限制性内切酶对DNA片段进行消化,然后使用DNA连接酶完成连接,因此这种方法受到插入片段和载体酶切位点的限制。一些ligase-free克隆技术虽然克服了酶切位点的限制,但费时费力,成本较高。为了弥补其他ligase-free技术的不足,文中介绍了一种新的利用核酸外切酶Ⅲ进行ligase-free克隆的方法,反应时间仅需要30 min,提高了克隆效率并有效降低经济成本,有利于大规模基因克隆的应用。     

赤霉菌分子生物学研究进展

过去 1 0年中 ,由于基因克隆、遗传转化等分子生物学方法与技术的应用 ,对赤霉菌中赤霉素生物合成基因的克隆、鉴定、异源表达及其表达调控等分子生物学研究取得了很大进展。现从赤霉菌的转化系统、赤霉素生物合成基因克隆、合成机理及其基因表达调控等方面的研究进展进行综述     

研发动态

我国首例绿色荧光转基因克隆猪成功产下"荧光猪崽" 我国首例绿色荧光蛋白转基因克隆猪日前成功产下11头小猪,其中2头具有绿色荧光遗传特征.这次试验的成功标志着中国通过体细胞核移植技术生产转基因猪已经发展成熟.     

国内信息——五种基因克隆技术

经华中农业大学周国岭先生对国内外64篇有关基因克隆技术的论文概括和分析,当前基因克隆技术主要有5个类别,即体位克隆或状态克隆、转位或转DNA标记法、同系位候选基因法、快捷顺序标记法、差接式快捷基因克隆法等。     

基因克隆技术的研究进展

为能快速而准确地克隆目的基因,综述了一些基因克隆常用技术,包括差异表达基因分离技术、转座子标签技术、图位克隆技术、同源序列技术、表达序列标签技术的原理、应用及应用潜力,并对其作了简要的评价.这些技术有利有弊,应根据不同的实验目的和水平来选择相应的技术.     

抑制消减杂交技术(SSH)及其在植物基因克隆上的应用

介绍了抑制消减杂交技术（SSH）的主要原理、基本方法、优越性及主要缺陷,并就其在植物基因克隆上的应用作一综述。     

小麦、黑麦FISH技术特异序列探针的研究进展

荧光原位杂交(fluorescence in situ hybridization,FISH)技术在染色体定位、基因克隆、遗传标记以及染色体畸变研究中得到了广泛的应用.随着分子生物学的飞速发展,用于FISH技术的染色体特异重复序列探针的开发和利用有了巨大进展.就近年来小麦、黑麦等FISH技术中染色体特异重复序列探针的研究进展及应用作一综述.     

土壤宏基因组学技术及其应用

传统的基于培养的研究方法只能反映土壤中少数(0.1%～10 %)微生物的信息,而大部分微生物目前还不能培养,因而这部分微生物资源尚难以被有效地开发利用.宏基因组学是分子生物学技术应用于环境微生物生态学研究而形成的一个新概念,主要技术包括土壤DNA的提取、文库的构建和目标基因克隆的筛选.它可为揭示微生物生态功能及其分子基础提供更全面的遗传信息,并已在微生物新功能基因筛选、活性物质开发和微生物多样性研究等方面取得了显著成果.本文对土壤宏基因组学技术的方法和应用作了详细介绍.     

Entamoeba histolytica: alterations in EhRabB protein in a phagocytosis deficient mutant correlate with the Entamoeba dispar RabB sequence

We analyzed the expression and location of EhRabB in clone L-6, a phagocytosis-deficient mutant of Entamoeba histolytica, in comparison with the wild-type clone A. Intriguingly, trophozoites of clone L-6 express more EhRabB than those of clone A. However, the majority of EhRabB-containing vesicles remained in the cytoplasm of clone L-6 during phagocytosis. To investigate molecular alterations in EhRabB of clone L-6 we compared the EhrabB gene sequences from clones L-6 and A. We also isolated, sequenced and compared the RabB protein of Entamoeba dispar. Results showed that EhrabB gene of clone L-6 is 98.2 and 94.1% identical to rabB genes of E. dispar and clone A, respectively. The rabB genes from clone A and E. dispar have 92.2% identity. Four out of five amino acids changes in RabB proteins of clone L-6 and E. dispar are shared. These changes may alter the binding of effector proteins and the specific subcellular location of EhRabB.     

PCR扩增葡萄球菌肠毒素A全长基因方法的建立及意义

为了分析和克隆葡萄球菌肠毒素（ＳＥ）Ａ全长基因,设计了一对针对ＳＥＡ全长基因的特异性引物,成功地用ＰＣＲ反应从标准产ＳＥＡ的葡萄球菌基因组中扩增出了一条约７７０ｂｐ的条带,为进一步用ＰＣＲ法克隆ＳＥＡ基因,并把它用于抗肿瘤研究奠定了实验基础     

A Drosophila ribosomal protein gene is located near repeated sequences including rDNA sequences.

We have isolated a cloned segment of Drosophila genomic DNA containing a ribosomal protein gene. Hybridization analysis of the DNA in this clone indicates a complex organization of repeated elements within this cloned segment. At least one of these repeated elements is homologous to regions of rDNA. Restriction analysis of the clone shows that some of the repeated elements are present as tandem duplications and in scattered locations within the cloned DNA segment. There are also three non-ribosomal protein genes contained in this clone, each of which is expressed along with the ribosomal protein gene into RNA species present in Drosophila embryos.     

Haploinsufficiency of ALX4 as a potential cause of parietal foramina in the 11p11.2 contiguous gene-deletion syndrome

Heterozygous mutations in MSX2 are responsible for an autosomal dominant form of parietal foramina (PFM). PFM are oval defects of the parietal bones that are also a characteristic feature of a contiguous gene-deletion syndrome caused by a proximal deletion in the short arm of chromosome 11 (Potocki-Shaffer syndrome). We have identified a human bacterial artificial chromosome (BAC) clone mapping to chromosome 11, containing a region homologous to the human homeobox gene MSX2. Further sequence analysis demonstrated that the human orthologue (ALX4) of the mouse Aristaless-like 4 gene (Alx4) is contained within this 11p clone. We used FISH to test for the presence-or for the heterozygous deletion-of this clone in two patients with the 11p11.2-deletion syndrome and showed that this clone is deleted in these patients. ALX4 and Alx4 were shown to be expressed in bone and to be absent from all other tissues tested. The involvement of Alx4 in murine skull development, its bone-specific expression pattern, the fact that Alx4 is a dosage-sensitive gene in mice, and the localization of a human genomic clone containing ALX4 to 11p11.2, with hemizygosity in patients with deletion of 11p11.2 who have biparietal foramina, support the contention that ALX4 is a candidate gene for the PFM in the 11p11.2-deletion syndrome.     

百合基因工程研究进展(综述)

本文从百合基因克隆、外源基因转化方法及转基因应用等方面,介绍近年来百合基因工程的研究进展,并讨论了百合基因工程研究中存在的问题和应用前景。     

Sequence and tissue-specific expression of a putative peroxidase gene from wheat (Triticum aestivum L.)

We have used a cDNA clone encoding a pathogen-induced putative wheat peroxidase to screen a genomic libary of wheat (Triticum aestivum L. cv. Cheyenne) and isolated one positive clone, lambda POX1. Sequence analysis revealed that this clone contains a gene encoding a putative peroxidase with a calculated pI of 8.1 which exhibits 58% and 83% sequence identity to the amino acid sequence of the turnip (Brassica rapa) peroxidase and a pathogen-induced putative wheat peroxidase, respectively. The two introns in the wheat gene are at the same positions as introns in the peroxidase genes of tomato and horseradish. Results of S1-mapping experiments suggest that this gene is neither pathogen-nor wound-induced in leaves but is constitutively expressed in roots.     

Heteroduplex analysis of the RNA of clone 3 Moloney murine sarcoma virus.

Heteroduplex analysis of the RNA isolated from purified virions of clone 3 Moloney murine sarcoma virus (M-MSV) hybridized to cDNA's from Moloney murine leukemia virus (M-MLV) and clone 124 M-MSV shows that the main physical component of clone 3 RNA is missing all or most of the 1.5-kilobase (kb) clone 124 M-MSV specific sequence denoted beta s (S. Hu et al. Cell 10:469--477, 1977). This sequence is either deleted in clone 3 RNA or substituted by a very short (0.3-kilobase) sequence. In other respects, clone 3 and clone 124 RNAs show the same heteroduplex structure relative to M-MLV. Since beta s is believed to contain the src gene(s) of clone 124 RNA, this result leaves as an unresolved question the nature of the src gene(s) of the clone 3 M-MSV RNA complex.