YAC介导的天蚕丝素基因向家蚕的转移──天蚕丝素基因-YAC克隆在家蚕的表达

本文为天蚕Antheraeyamamai丝素基因在家蚕Bombyxmori成功表达的首次报道。我们构建了天蚕丝素基因的YAC克隆,然后把含有该克隆的DNA溶液导入家蚕受精卵。分子杂交实验证明天蚕丝素基因已整合到家蚕基因组中。通过丝心蛋白氨基酸组分分析以及茧丝的溶解性比较,发现有部分转基因家委表达了天蚕丝素基因。F2代的转基因家蚕蛾的染色体DNA中同时还存在YAC序列,说明YAC对丝素基因具有介导作用。天蚕丝素基因以单拷贝形式存在于转基因家蚕中。     

水稻核基因组DNA的YAC克隆和鉴定

将EcoRI部分消化的水稻(Oryza sativa L.)细胞核高分子量DNA电泳分部,回收大于200kb的片段,与内切酶消化过的酵母人工染色体(YAC)双质粒载体pJs97。pJS98DNA连接,转化酵母YPH252感受态原生质球,用ura^-,TrP^-双选择培养基直接筛选转化子,已获得2 000多个克隆。转化子DNA的southern杂交显示插入片段在200～820kb范围。     

YAC克隆系统

目前,利用质粒和噬菌体作为载体,已完成了许多基因与基因组DNA片段的克隆及作图.但是,质粒和噬菌体所能携带的外源DNA片段的大小是很有限的.一般来说,入噬菌体能携带的DNA片段不超过25kb;Cosmid质粒所能携带的DNA片段不超过45kb.而现在发现的一些大基因,如人类的Factor VIII基因(180kb)和Dystrophin基因(1800kb)都已远远超过上述载体的克隆容量.这些容量较小的克隆载体,在克隆真核基因组的染色体时,遇到了巨大的困难.为了克服这一难题,80     

第21人类染色体长臂DNA的YAC克隆及重叠排序

人类基因组的研究已在全世界主要发达国家展开。由于YAC(YeastArtificialChromosome)克隆技术的建立及广泛采用,已使人类及其他生物基因组的研究迈出了成功的第一步。以DanielCohen(CEPH,Paris)为首,联合法国、西班牙、美国和日本12家实验室的35名 合作者,已完成了人类第21染色体长臂DNA的YNC克隆及重叠排序(Nature359,380-387,1992)。     

酵母人工染色体克隆技术及其进展

酵母人工染色体克隆(YAC)是最近几年发展起来的大分子 DNA 克隆技术.文章综述了 YAC 克隆技术的发展,YAC 的分离、分析与鉴定,以及这一技术在分子生物学中的应用.     

用万能引物PCR法从YAC中分离制备荧光原位杂交探针的研究

报道了一种从微量且未知碱基顺序的YAC DNA中制备FISH探针的新方法——万能引物PCR(UP PCR),即把复性后的UP-Linker连接于PFGE分离后经Alu Ⅰ酶切的YAC DNA上,再用UP对其进行PCR扩增和标记。由于Alu Ⅰ的广谱性和UP与Linker长度不同等,使该法能根据PCR效率调节扩增产物的广泛性向特异性的转变,且产物能覆盖YAC嵌入DNA的全长。此法制备的人21号染色体FISH探针,特异性强,杂交信号明亮,21号染色体的检出率高。该法稳定简便。     

通过染色体步查建立水稻G200座位的重叠群

分别以G200及其旁侧区的Y2587L、Y4073L和L688片段为探针筛选水稻基因组YAC文库,共得到4个阳性克隆,均与G200、Y2587L和Y4073L座位重叠,插入片段的大小为240～650kb。用反向PCR法分离YAC克隆插入片段的末端序列,并利用这些末端序列确定克隆的方向以及进行染色体步查,共筛选到7个YAC克隆,建立了一个8厘摩(cM)的G200 YAC重叠群。     

大片段克隆载体研究进展

DNA克隆技术是分子生物学研究中一项重要的技术手段。自第一个质粒载体pSC1 0 1作为克隆载体以来 ,随着分子生物学技术的发展 ,克隆载体的整体结构、容载能力和转化效率都有了很大的改善。尤其是人类基因组计划的实施 ,产生了YAC和BAC克隆体系。随着植物基因组计划的进行 ,又产生了既能够克隆大片段DNA又能够将候选克隆直接通过农杆菌介导进行功能互补实验的载体。综述了几种常用大片段克隆载体YAC、BAC、BIBAC、PAC和TAC的特点及其应用 ,并对克隆载体的发展作了展望。     

基因组YAC文库构建方法的几点改进

以pJS97、pJS98为载体, 对中国人基因组YAC文库的构建进行了尝试.建立了一种对小片段DNA“原位电泳筛除”的方案, 采用多胺溶液处理以减少大片段DNA的降解, 使整个建库工作更加简便、有效. 此外, 改进了转化子的挑取和保存的方法, 既简化了操作, 又减少了杂菌污染和交叉污染. 这些改进的方案, 可以推广到其他高等生物基因组YAC文库的构建和应用工作.     

利用酵母人工染色体(YAC)减数分裂同源重组构建人免疫球蛋白κ链基因组大片段

具有同源重叠区的酵母人工染色体(YAC)可以利用酵母细胞减数分裂进行同源重组,从而构建更大的人工染色体基因组,这对生命科学基础研究和生物技术应用研究有着非常重要的意义。本实验以两个含人免疫球蛋白κ链基因簇片段的YAC克隆为材料,通过酵母改型、异型接合、二倍体发孢、单孢子筛选和分子生物学鉴定等技术和方法,利用酵母菌减数分裂同源重组机制,构建了一条包含人的免疫球蛋白κ轻链32个Vκ基因、5个Jκ基因、Cκ基因、Eκ基因和κde基因的YAC重组体,长度约400kb。同时,本实验利用溶壁酶消化法获取单孢子重组体,代替了传统的显微分孢操作。使得利用酵母人工染色体减数分裂同源重组的技术更加简便可行。     

Construction of a human chromosome 4 YAC pool and analysis of artificial chromosome stability.

In order to construct a human chromosome 4-specific YAC library, we have utilized pYAC4 and a mouse/human hybrid cell line HA(4)A in which the only human chromosome present is chromosome 4. From this cell line, approximately 8Mb of chromosome 4 have been cloned. The library includes 65 human-specific clones that range in size from 30kb to 290kb, the average size being 108kb. In order to optimize the manipulation of YAC libraries, we have begun to investigate the stability of YACs containing human DNA in yeast cells; these studies will also determine if there are intrinsic differences in the properties of chromosomes containing higher eukaryotic DNAs. We are examining two kinds of stability: 1] mitotic stability, the ability of the YAC to replicate and segregate properly during mitosis, and 2] structural stability, the tendency of the YAC to rearrange. We have found that the majority of YACs examined are one to two orders of magnitude less stable than authentic yeast chromosomes. Interestingly, the largest YAC analyzed displayed a loss rate typical for natural yeast chromosomes. Our results also suggest that increasing the length of an artificial chromosome improves its mitotic stability. One YAC that showed a very high frequency of rearrangement by mitotic recombination proved to be a mouse/human chimera. In contrast to studies using total human DNA, the frequency of chimeras (i.e., mouse/human) in the YAC pool appeared to be low.     

The telomeric 60 kb of chromosome arm 4p is homologous to telomeric regions on 13p, 15p, 21p, and 22p.

A telomere YAC clone containing the most distal 115 kb of chromosome arm 4p has been previously isolated. This clone is of particular interest as it spans a potential candidate region for the Huntington disease gene. The YAC was subcloned into a phage vector, and a high-resolution restriction map extending to within 13 kb of the telomere was constructed. In situ hybridization of the YAC to human metaphase spreads gives a peak of hybridization on 4pter but also an increase in the number of signals close to several other telomeres. Where possible, these results were investigated further by the hybridization of probes from the YAC to somatic cell hybrids containing single human chromosomes. This analysis indicates that the most telomeric 60 kb of chromosome arm 4p is homologous to telomeric regions on 13p, 15p, 21p, and 22p. The extent of this homology makes it less likely that the mutation for Huntington's disease is located within the telomere YAC clone.     

Characterization of four human YAC libraries for clone size, chimerism and X chromosome sequence representation.

Four collections of human X-specific YACs, derived from human cells containing supernumerary X chromosomes or from somatic cell hybrids containing only X human DNA were characterized. In each collection, 80-85% of YAC strains contained a single X YAC. Five thousand YACs from the various libraries were sized, and cocloning was assessed in subsets by the fraction of YAC insert-ends with non-X sequences. Cocloning was substantial, ranging up to 50% for different collections; and in agreement with previous indications, in all libraries the larger the YACs, the higher the level of cocloning. In libraries made from human-hamster hybrid cells, expected numbers of clones were recovered by STS-based screening; but unexpectedly, the two collections from cells with 4 or 5 X chromosomes yielded numbers of YACs corresponding to an apparent content of only about two X equivalents. Thus it is possible that the DNA of inactive X chromosomes is poorly cloned into YACs, speculatively perhaps because of its specialized chromatin structure.     

Microinjection of Intact 200- to 500-kb Fragments of YAC DNA into Mammalian Cells

DNA of yeast artificial chromosomes (YACs) was prepared for microinjection by separation from most of the natural yeast chromosomes on a pulsed-field gel, treatment with agarase, and centrifugation. A salt concentration of 100 mM NaCl was necessary to protect the DNA from shear during these procedures. Injection of a 590-kb YAC, yGART2, into Chinese hamster ovary cells gave rise to cells expressing the 40-kb human GART gene carried on the YAC. Nine of 12 cell lines analyzed contained an intact stretch of at least 110 kb of YAC DNA surrounding the GART gene, and one cell line contained at least 480 kb, but not the entire 590 kb, intact. Mouse L A-9 cells were similarly injected with DNA of a 230-kb YAC containing the human β-globin gene cluster and a mammalian selectable marker. Seven of 10 of the resulting cell lines contained both YAC vector arms plus the intact 140-kb SfiI fragment spanning the β-globin gene. Three cell lines were analyzed by Rec A-assisted restriction endonuclease (RARE) cleavage and found to contain the entire intact 210-kb YAC insert. Introduction of similarly prepared DNA into mammalian cells by lipofection gave rise to cell lines with multiple YAC fragments that were generally shorter than the YAC fragments found in microinjected cell lines. The results show that microinjection of gel-purified YAC DNA into mammalian cells is an efficient method of transferring DNA fragments several hundred kilobase pairs in size into mammalian cells.     

Cloning and in vivo expression of the human GART gene using yeast artificial chromosomes.

Two Yeast Artificial Chromosomes (YACs) were isolated each with a full-length copy of the human gene that encodes the trifunctional protein containing phosphoribosylglycinamide synthetase (GARS), phosphoribosylglycinamide formyltransferase (GART) and phosphoribosylaminoimidazole synthetase (AIRS). The YACs were characterized by restriction mapping and by in situ hybridization of cosmid subclones containing the YAC ends to human metaphase chromosomes. One of the YACs contains co-cloned non-contiguous DNA whereas the other appears to have a single 600 kbp insert from 21q22.1, the location of the GART gene. A restriction map of the gene was obtained from two cosmid subclones which together span the 40 kb gene. The gene is functional when YAC DNA is transferred into GARS- or GARS-and-AIRS-deficient Chinese Hamster Ovary cells. The gene transfer was carried out both by lipofection using purified yeast DNA and by fusion between yeast spheroplasts and the hamster cells. Restriction analysis of DNA from cell lines whose purine auxotrophy was complemented by the YAC showed that with either method a complete and unrearranged copy of the gene can be transferred. The majority of the fusion cell lines appear to contain at least 80% of the YAC.     

利用酵母人工染色体（YAC）减数分裂同源重组构建人免疫球蛋白κ链基因组大片段

具有同源重叠区的酵母人工染色体(YAC)可以利用酵母细胞减数分裂进行同源重组,从而构建更大的人工染色体基因组,这对生命科学基础研究和生物技术应用研究有着非常重要的意义。本实验以两个含人免疫球蛋白κ链基因簇片段的YAC克隆为材料,通过酵母改型、异型接合、二倍体发孢、单孢子筛选和分子生物学鉴定等技术和方法,利用酵母菌减数分裂同源重组机制,构建了一条包含人的免疫球蛋白κ轻链32个Vκ基因、5个Jκ基因、Cκ基因、Eκ基因和κde基因的YAC重组体,长度约400kb。同时,本实验利用溶壁酶消化法获取单孢子重组体,代替了传统的显微分孢操作。使得利用酵母人工染色体减数分裂同源重组的技术更加简便可行。     

Modification and transfer into an embryonal carcinoma cell line of a 360-kilobase human-derived yeast artificial chromosome.

A neomycin resistance cassette was integrated into the human-derived insert of a 360-kilobase yeast artificial chromosome (YAC) by targeting homologous recombination to Alu repeat sequences. The modified YAC was transferred into an embryonal carcinoma cell line by using polyethylene glycol-mediated spheroplast fusion. A single copy of the human sequence was introduced intact and stably maintained in the absence of selection for over 40 generations. A substantial portion of the yeast genome was retained in hybrids in addition to the YAC. Hybrid cells containing the YAC retained the ability to differentiate when treated with retinoic acid. This approach provides a powerful tool for in vitro analysis because it can be used to modify any human DNA cloned as a YAC and to transfer large fragments of DNA intact into cultured mammalian cells, thereby facilitating functional studies of genes in the context of extensive flanking DNA sequences.     

Fluorescence in situ hybridization of YAC clones after Alu-PCR amplification.

Alu-PCR protocols were optimized for the generation of human DNA probes from yeast strains containing yeast artificial chromosomes (YACs) with human inserts between 100 and 800 kb in size. The resulting DNA probes were used in chromosome in situ suppression (CISS) hybridization experiments. Strong fluorescent signals on both chromatids indicated the localization of specific YAC clones, while two clearly distinguishable signals were observed in greater than or equal to 90% of diploid nuclei. Signal intensities were generally comparable to those observed using chromosome-specific alphoid DNA probes. This approach will facilitate the rapid mapping of YAC clones and their use in chromosome analysis at all stages of the cell cycle.     

Distribution of moderately repetitive sequences pTR5 and LF1 in Xq24-q28 human DNA and their use in assembling YAC contigs.

Xq24-q28 DNA, from a hamster/human hybrid cell containing only that portion of the human X chromosome, was found to contain 56 TaqI restriction fragments that hybridized to the moderately repetitive sequence pTR5. Using the pTR5 sequence as a probe in colony hybridization, 136 cognate yeast artificial chromosome (YAC) clones were detected among a collection of 820 containing about three genomic equivalents of the Xq24-q28 DNA. The YACs were then grouped into 48 contigs and single clones containing one or more of the TaqI fragments. Overlaps were confirmed both by fingerprinting YACs with AluI and L1 probes and by additional information. A less complete analysis was also carried out with a second moderately repetitive sequence, LF1, and some smaller contigs were merged into larger ones. Moderately repetitive sequences can thus be used as probes for multiple loci in single hybridization experiments and can help to organize and confirm YAC overlaps during the development of maps with long-range contiguity.     

Rescue of end fragments of yeast artificial chromosomes by homologous recombination in yeast.

Yeast artificial chromosomes (YACs) provide a powerful tool for the isolation and mapping of large regions of mammalian chromosomes. We developed a rapid and efficient method for the isolation of DNA fragments representing the extreme ends of YAC clones by the insertion of a rescue plasmid into the YAC vector by homologous recombination. Two rescue vectors were constructed containing a yeast LYS2 selectable gene, a bacterial origin of replication, an antibiotic resistance gene, a polylinker containing multiple restriction sites, and a fragment homologous to one arm of the pYAC4 vector. The 'end-cloning' procedure involves transformation of the rescue vector into yeast cells carrying a YAC clone, followed by preparation of yeast DNA and transformation into bacterial cells. The resulting plasmids carry end-specific DNA fragments up to 20 kb in length, which are suitable for use as hybridization probes, as templates for direct DNA sequencing, and as probes for mapping by fluorescence in situ hybridization. These vectors are suitable for the rescue of end-clones from any YAC constructed using a pYAC-derived vector. We demonstrate the utility of these plasmids by rescuing YAC-end fragments from a human YAC library.