Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation and analysis of the CEN1-ADE1-CDC15 region
Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation and analysis of the CEN1-ADE1-CDC15 region
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酿酒酵母 I 号染色体 DNA 的分子克隆：CEN1-ADE1-CDC15 区域的分离和分析
DOI：
10.1128/mcb.7.1.410-419.1987
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发表时间：
1987
影响因子：
5.3
通讯作者：
D. Kaback
中科院分区：
文献类型：
--
作者：
H. Y. Steensma;J. Crowley;D. Kaback
为了继续对整个酿酒酵母染色体的物理和遗传组织进行系统检查，对 I 号染色体 CEN1-ADE1-CDC15 区域的 DNA 进行了分离和表征。从先前克隆的 ADE1 基因（J. C. Crowley 和 D. B. Kaback, J. Bacteriol. 159:413-417, 1984）开始，通过重叠杂交获得了一系列含有来自染色体 I 的 82 KB 连续 DNA 的重组 lambda 噬菌体。克隆的序列用限制性内切核酸酶绘制图谱，并通过确定 CDC15 基因和着丝粒 DNA (CEN1) 的物理位置来相对于遗传图谱进行定向。通过从 YCp50 酿酒酵母基因组文库中分离与 cdc15-1 突变互补的质粒来定位 CDC15 基因。发现来自这些质粒的酿酒酵母序列是通过重叠杂交已经获得的序列中的代表。 cdc15-1 互补质粒均仅共享一个完整的转录区域，通过体外基因破坏和一步替换以删除该基因的染色体副本，显示该区域包含真正的 CDC15 基因。这种缺失产生了隐性致死表型，该表型对于 cdc15-1 也是隐性的。通过从克隆区域的适当部分找到稳定自主复制酿酒酵母质粒载体遗传的序列来定位 CEN1。最后，使用 RNA 印迹杂交和含有 R 环的 DNA 的电子显微镜来绘制从 CEN1 到 CDC15 的 23 KB DNA 中的转录区域。除了对应于 ADE1 和 ADC15 基因的转录区域外，该 DNA 还包含五个产生聚腺苷酸化 RNA 的区域、至少两个与 4S RNA 物种互补的区域以及一个 Ty1 转座元件。值得注意的是，所检测的 DNA 中被转录成 RNA 的比例高于平均水平。
To continue the systematic examination of the physical and genetic organization of an entire Saccharomyces cerevisiae chromosome, the DNA from the CEN1-ADE1-CDC15 region from chromosome I was isolated and characterized. Starting with the previously cloned ADE1 gene (J. C. Crowley and D. B. Kaback, J. Bacteriol. 159:413-417, 1984), a series of recombinant lambda bacteriophages containing 82 kilobases of contiguous DNA from chromosome I were obtained by overlap hybridization. The cloned sequences were mapped with restriction endonucleases and oriented with respect to the genetic map by determining the physical positions of the CDC15 gene and the centromeric DNA (CEN1). The CDC15 gene was located by isolating plasmids from a YCp50 S. cerevisiae genomic library that complemented the cdc15-1 mutation. S. cerevisiae sequences from these plasmids were found to be represented among those already obtained by overlap hybridization. The cdc15-1-complementing plasmids all shared only one intact transcribed region that was shown to contain the bona fide CDC15 gene by in vitro gene disruption and one-step replacement to delete the chromosomal copy of this gene. This deletion produced a recessive lethal phenotype that was also recessive to cdc15-1. CEN1 was located by finding a sequence from the appropriate part of the cloned region that stabilized the inheritance of autonomously replicating S. cerevisiae plasmid vectors. Finally, RNA blot hybridization and electron microscopy of R-loop-containing DNA were used to map transcribed regions in the 23 kilobases of DNA that went from CEN1 to CDC15. In addition to the transcribed regions corresponding to the ADE1 and ADC15 genes, this DNA contained five regions that gave rise to polyadenylated RNA, at least two regions complementary to 4S RNA species, and a Ty1 transposable element. Notably, a higher than average proportion of the DNA examined was transcribed into RNA.
用于分离功能性着丝粒 DNA 的直接选择程序。
DOI：
10.1073/pnas.78.6.3760
发表时间：
1981
影响因子：
11.1
作者：
Hsiao,CL;Carbon,J
通讯作者：
Carbon,J
酵母染色体 I 上的温度敏感致死突变似乎只定义了少数基因。
影响因子：
3.3
作者：
Kaback,DB;Oeller,PW;YdeSteensma,H;Hirschman,J;Ruezinsky,D;Coleman,KG;Pringle,JR
通讯作者：
Pringle,JR
影响因子：
3.3
作者：
L. Hartwell;David M. Smith
通讯作者：
L. Hartwell;David M. Smith