人X染色体35cMYAC重叠群的构建及物理图谱分析

人Ｘ染色体Ｘｐ１１．２－ｐ２１．３区域具有重要的基础遗传学和医学遗传学意义。为了对该区段编码的基因,尤其是疾病基因进行克隆与变异研究,对该区段染色体ＤＮＡ进行了ＹＡＣ克隆并将其依染色体排序,采用了一系列ＤＮＡ位标,尤其是多肽性微卫星序列位标筛选了３个ＹＡＣ方库,得到了１５１个ＹＡＣ克隆,对这些ＹＡＣ克隆进行了物理图谱分析,构建了这一区域的一系列ＹＡＣ重叠群,这些ＹＡＣ重叠群的总跨度约３５摩,基本覆     

人Xp21．1－p21．3上3．5MbYAC重叠群构建及物理图谱分析

用Ａｌｕ－ＰＣＲ指纹图谱法分析了人Ｘｐ２１．１－ｐ２１．３上一系列的酵母人工染色体（ｙｅａｓｔａｒｔｉｆｉｃｉａｌｃｈｒｏｍｏｓｏｍｅ,ＹＡＣ）克隆,发现其中的两个ＹＡＣ克隆构成包含ＤＸＳ１６６位点的重叠群,而且这一重叠群与以前构建的包含ＤＭＤ基因全序列的ＹＡＣ重叠群相连接,ＹＡＣ克隆末端探针交叉杂交证实了这一重叠,使这一ＹＡＣ重叠群至少延伸至ＤＸＳ１６６位点,形成一个跨度为３．５Ｍｂ的ＹＡＣ重叠群。基于这些重叠的ＹＡＣ克隆绘制了这一区域的大尺度限制酶切图谱,并在这一图谱上定位了ＤＸＳ１６６位点,从而确定了ＤＸＳ１６６位点与ＤＭＤ基因的物理关系。这一工作为ＤＭＤ基因的５＇远端调控作用研究及该区域未知基因的克隆奠定了基础。     

人类X染色体Xp11.3—21.3区部分YAC重叠群构建及大尺度物理图谱分析

人类Ｘ染色体短臂１１．３－２１．３区是含有视网膜色素奕性等种遗传基因位点的区域。我们对这个具有重要医学生物学意义的区段进行了ＹＡＣ重叠群构建及大尺度物理作图。用这一区域已知的探讨（ＯＴＣ２ｂＡ３、ＤＭＤｃＤＮＡ），以ＹＡＣ菌落原杂交法及ＰＣＲ法进行了ＹＡＣ的筛选；也采用了法国ＣＥＰＨ和英国ＩＣＲＦ的部分ＹＡＣ，总共得到了７７个阳性ＹＡＣ。对上述ＹＡＣ进行了长度测定。２６对微卫星ＳＴＳ图谱分析，单拷     

人基因组百万碱基级巨型YAC的构建

人基因组百万碱基级巨型ＹＡＣ的构建戴长虹,柴建华（复旦大学遗传学研究所,上海２００４３３）关键词人基因组;酵母人工染色体（ＹＡＣ）;百万碱基;巨型ＹＡＣ人类的全部遗传信息包含在３ｘ１０＇核着酸对（ｈｐ）中,共编码５００００～１０００００个基因。人类基...     

石貂的染色体研究

本文对分布在我国的石貂北方亚种染色体进行了较详细的研究。结果表明２ｎ＝３８,核型为１４（Ｍ）＋４（ＳＭ）＋１８（ＳＴ）,ＸＹ（Ｍ,Ａ）。Ｃ－带显示该亚种的一些染色体着丝粒区域结构异染色质弱化或消失。Ｎｏ,９染色体的短臂完全异染色质化;Ｘ染色体长臂丰出现插入杂色质带;Ｙ为完全结构异染色质组成。     

白颊长臂猿染色体的研究

本文对我国云南南部的白须长臂猿（Ｈ．ｌｅｕｃｏｇｅｎｙｓ）染色体的Ｇ带、Ｃ带、晚复制带及Ａｇ－ＮＯＲｓ进行了较为详细的研究。它的２ｎ＝５２,核型公式为４４（Ｍ或ＳＭ）＋６（Ａ）,ＸＹ（Ｍ,Ａ）。Ｃ带表明一些染色体着丝点Ｃ带弱化;有的染色体出现插入的和端位的Ｃ带;Ｘ染色体两臂有端位Ｃ带,Ｙ染色体是Ｃ带阳性和晚复制的。Ａｇ－ＮＯＲｓ的数目,雌体有４个,雄体有５个,Ｙ染色体上具ＮＯＲ。本文对白颊长臂猿与其它长臂猿间的亲缘关系、核型进化的可能途径进行了讨论。     

人X染色体YAC图谱分析及DMD基因研究──在第二届联合国教科文组织南北人类基因组学术会议上的报告

人Ｘ染色体ＹＡＣ图谱分析及ＤＭＤ基因研究──在第二届联合国教科文组织南北人类基因组学术会议上的报告柴建华（复旦大学遗传学研究所人类基因组实验室上海２００４３３）我们实验室从１９８７年在国家高技术发展计划（８６３）的资助下开始人类基因组研究。现在还同时得到国家自然科学基金重点及重大项目的支持。     

人基因组YAC克隆DNA的Alu—PCR反应条件的系统研究

在人基因组ＹＡＣ克隆的Ａｌｕ－ＰＣＲ指纹分析中要求ＤＮＡ扩增带具有ＹＡＣ特征性;在Ａｌｕ－ＰＣＲ方法对ＹＡＣ克隆中的人类基因组ＤＮＡ片段进行特异的同位素标记时则要求被增标记的ＤＮＡ序列在插入片段中具有一定弥散性,我们建立了两种不同的Ａｌｕ－ＰＣＲ反应体系以满足这一不同要求,并已得到较为满意的结果,根据不同条件下的Ａｌｕ－ＰＣＲ结果,分析了多引发位点ＰＣＲ中的一些现象,并作了解释。     

人基因组YAC克隆DNA的Alu－PCR反应条件的系统研究

在人基因组ＹＡＣ克隆的Ａｌｕ－ＰＣＲ指纹分析中要求ＤＮＡ且扩增带具有ＹＡＣ特征性;在用ＡｌＵ－ＰＣＲ方法对ＹＡＣ克隆中的人类基因组ＤＮＡ片段进行特异的同位素标记时则要求被扩增标记的ＤＮＡ序列在插入片段中具有一定的弥散性。我们建立了两种不同的Ａｌｕ－ＰＣＲ反应体系以满足这一不同要求,并已得到较为满意的结果。根据不同条件下的Ａｌｕ－ＰＣＲ结果,分析了多引发位点ＰＣＲ中的一些现象,并作出了解释。     

kar交配法转移YACs至新宿主的研究

利用改进的ｋａｒ交配法,将一个含有３４０ｋｂ人基因组ＤＮＡ的ＹＡＧ片段的供体酵母菌株ＹＡＣ２３与受体菌株ＹＬＢ５０４进行交本,以选择平板对所形成的侯选ＹＡＣ导入菌进行筛选,经ＰＣＲ分析,候选ＹＡＣ导入菌在４０４ｂｐ处有一个扩增带,即具有受体菌株的交配型（ＭＡＴα）。地一步用脉冲电沪进行核型鉴定。     

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.     

A 2-Mb YAC Contig and Physical Map Covering the Chromosome 8q12 Breakpoint Cluster Region in Pleomorphic Adenomas of the Salivary Glands

Pleomorphic adenomas are benign epithelial tumors originating from the major and minor salivary glands. Extensive cytogenetic studies have demonstrated that they frequently show chromosome abnormalities involving chromosome 8, with consistent breakpoints at 8q12. In previous studies, we have shown that these breakpoints are located in a 9-cM interval betweenMOS/D8S285 and D8S260. Here, we describe directional chromosome walking studies starting from D8S260 as well as D8S285. Using the CEPH and ICRF YAC libraries, these studies resulted in the construction of two nonoverlapping YAC contigs of about 2 and 5 Mb, respectively. Initial fluorescencein situhybridization (FISH) analysis suggested that the majority of 8q12 breakpoints clustered within the 2-Mb contig, which was mapped to the centromeric part of chromosome band 8q12. This contig has at least double coverage and consists of 34 overlapping YAC clones. The localization of the YACs was confirmed by FISH analysis. On the basis of mapping data of landmarks with an average spacing of 65 kb as well as restriction enzyme analysis, a long-range physical map was established for the chromosome region spanned by the 2-Mb contig. The relative positions of various known genes and expressed sequence tags within this contig were also determined. Subsequent FISH analyses of pleomorphic adenomas using YACs as well as cosmids revealed that all but two of the 8q12 breakpoints in the primary tumors tested mapped within a 300-kb interval between theMOSproto-oncogene and STS EM156. The target gene affected by the chromosome aberrations mapping within this interval was recently shown to be thePLAG1gene, which encodes a novel zinc finger protein.     

Construction of YAC Contigs on Rice Chromosome 5

A physical map of rice chromosome 5 was constructed with yeastartificial chromosome (YAC) clones along a high-resolution molecularlinkage map carrying 118 DNA markers distributed over 123.7cM of genomic DNA. YAC clones have been identified by colonyand Southern hybridization for 105 restriction fragment lengthpolymorphism (RFLP) markers and by polymerase chain reaction(PCR) screening for 8 sequence-tagged site (STS) markers and5 randomly amplified polymorphic DNA (RAPD) markers. Of 458YACs, 235 individual YACs with an average insert length of 350kb were selected and ordered on chromosome 5 from the YAC library.Forty-eight contigs covering nearly 21 Mb were formed on thechromosome 5; the longest one was 6 cM and covered 1.5 Mb. Thelength covered with YAC clones corresponded to 62% of the totallength of chromosome 5. There were many multicopy sequencesof expressed genes on chromosome 5. The distribution of manycopies of these expressed gene sequences was determined by YACSouthern hybridization and is discussed. A physical map withthese characteristics provides a powerful tool for elucidationof genome structure and extraction of useful genetic informationin rice.     

Isolation of the human sex determining region from a Y-enriched yeast artificial chromosome library

We describe the isolation and characterization of yeast artificial chromosome (YAC) clones spanning the male sex determining region on the short arm of the human Y chromosome. The clones were isolated by hybridizing probes in the interval between the genes MIC2 and ZFY to a Y chromosome-enriched YAC library. The YAC clones were consistent with the order of probes established for this interval and may be useful for functional studies of the region in male sex determination. However, many of the YAC clones from this library carried only one arm of the vector ("half-YACs"), deleted sequences from one end, and contained much smaller inserts (148 kb average) than the size of ligated fragments selected by pulsed-field gel electrophoresis (greater than 440 kb). These problems were overcome by protecting DNA with polyamines during YAC library construction and a second Y-enriched YAC library was constructed with an average insert size of 627 kb.     

PCR amplification and analysis of yeast artificial chromosomes

A strategy for the analysis of yeast artificial chromosome (YAC) clones that relies on polymerase chain reaction (PCR) amplification of small restriction fragments from isolated YACs following adapter ligation was developed. Using this method, termed YACadapt, we have amplified several YACs from a human Xq24-qter library and have used the PCR products for physical mapping by somatic cell hybrid deletion analysis and fluorescent in situ hybridization. One YAC, RS46, was mapped to band Xq27.3, near the fragile X mutation. The PCR product is an excellent renewable source of YAC DNA for analyses involving hybridization of YAC inserts to a variety of DNA/RNA sources.     

A yeast artificial chromosome contig containing the complete Duchenne muscular dystrophy gene.

A contig of 36 overlapping yeast artificial chromosome (YAC) clones has been constructed for the complete Duchenne muscular dystrophy (DMD) gene in Xp21. The YACs were isolated from a human 48,XXXX YAC library using the DMD cDNA and brain promoter fragments as hybridization probes. The YAC clones were characterized for exon content using HindIII or EcoRI digests, hybridization of individual DMD cDNA probes, and polymerase chain reaction (PCR) amplification of specific exons near the 5' end of the gene. For comparison to the known long-range restriction map of the DMD gene, YAC clones were digested with SfiI and hybridized with DMD cDNA probes. The combined analysis of the exon content and the SfiI map allowed an approximately 3.2-Mb YAC contig to be constructed. The complete 2.4-Mb DMD gene could be represented in a minimum set of 7 overlapping YAC clones.