酿酒酵母5号染色体精细物理图谱绘制

Fine Physical Mapping of Yeast Chromosome V

利用脉冲电泳（ＰｕｌｓｅｄＦｉｅｌｄＧｅｌＥｌｅｃｔｒｏｐｈｏｒｅｓｉｓ，ＰＦＧＥ）分析了酵母菌Ａ３６４ａ的电泳核型，以５号染色体专一探针确定了该染色体在电泳核型中的位置，以内切酶ＢａｍＨＩ对该染色体ＤＮＡ进行部分酶切后，与整合型载体ＹＩｐ５连接获得了一个染色体专一的基因文库，其转化子数目超过了理论要求值。从文库中筛选与已知探针有同源性的片段并用内切酶ＢａｍＨＩ，ＥｃｏＲＩ，ＨｉｎｄＩＩ，ＰｓｔＩ和ＳａｌＩ分析这些插入片段，获得了一个覆盖Ａ３６４ａ５号染色体（其长度估计为６２０ｋｂ）９．４％的精细物理图谱。利用边界克隆和菌落杂交将使我们能够对整条染色体进行进一步的“步查”

lectrophoretic karyotype of yeast strain A364a was obtained by pulsed field gel electrophoresis and the position of chromosome V on such karyotype was determined by means of dot hybridization with chromosome V-specific probe URA3By cloning partially digested BamHI fragments of this chromosome DNA into integreti vevector Yip5,a gene library specific to this chromosome was constructedThe number of the recombinants was much more than theoretically requiredAfter screening probe-homologous fragments from this library and analysing such fragments with restriction enzymes BamHI,EcoRI,HindIII,PstI,and SalI,a fine physical map covering about 94% of A364a chromosome V(which was estimated as 620kb)was constructedFurther colony hybridization with boundary clones will enable us to“walk”throughout the whole chromosome