• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Molecular and Cellular Biology >Preliminary characterization of the transcriptional and translational products of the Saccharomyces cerevisiae cell division cycle gene CDC28.
【24h】

Preliminary characterization of the transcriptional and translational products of the Saccharomyces cerevisiae cell division cycle gene CDC28.

机译：酿酒酵母细胞分裂周期基因CDC28的转录和翻译产物的初步表征。

获取原文
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

The CDC28 gene was subcloned from a plasmid containing a 6.5-kilobase-pair segment of Saccharomyces cerevisiae DNA YRp7(CDC28-3) by partial digestion with Sau3A and insertion of the resulting fragments into the BamHI sites of YRp7 and pRC1. Recombinant plasmids were obtained containing inserts of 4.4 and 3.1 kilobase pairs which were capable of complementing a cdc28(ts) mutation. R-loop analysis indicated that each yeast insert contained two RNA coding regions of about 0.8 and 1.0 kilobase pairs, respectively. In vitro mutagenesis experiments suggested that the smaller coding region corresponded to the CDC28 gene. When cellular polyadenylic acid-containing RNA, separated by agarose gel electrophoresis after denaturation with glyoxal and transferred to nitrocellulose membrane, was reacted with labeled DNA from the smaller coding region, and RNA species of about 1 kilobase in length was detected. Presumably, the discrepancy in size between the R-loop and electrophoretic determinations is due to a segment of polyadenylic acid which is excluded from the R-loops. By using hybridization of the histone H2B mRNAs to an appropriate probe as a previously determined standards, it was possible to estimate the number of CDC28 mRNA copies per haploid cell as between 6 and 12 molecules. Hybrid release translation performed on the CDC29 mRNA directed the synthesis of a polypeptide of 27,000 daltons, as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. This polypeptide was not synthesized when mRNA prepared from a cdc28 nonsense mutant was translated in a parallel fashion. However, if the RNA from a cell containing the CDC28 gene on a plasmid maintained at a high copy number was translated, the amount of in vitro product was amplified fivefold.
机译：通过用Sau3A部分消化并将所得片段插入YRp7和pRC1的BamHI位点，从含有酿酒酵母DNA YRp7（CDC28-3）的6.5碱基对片段的质粒亚克隆CDC28基因。获得了重组质粒，该质粒含有能够互补cdc28（ts）突变的4.4和3.1碱基对的插入片段。 R-环分析表明，每个酵母插入片段分别包含两个约0.8和1.0千碱基对的RNA编码区。体外诱变实验表明较小的编码区对应于CDC28基因。当含乙二醛变性后通过琼脂糖凝胶电泳分离的含细胞多腺苷酸的RNA与转移到硝化纤维素膜上的RNA发生反应后，与较小编码区的标记DNA反应，检测到长度约为1千碱基的RNA。据推测，R-环与电泳测定之间的尺寸差异是由于聚腺苷酸的一部分被排除在R-环之外。通过使用组蛋白H2B mRNA与适当探针的杂交作为先前确定的标准，可以估计每个单倍体细胞CDC28 mRNA的拷贝数为6至12个分子。通过在十二烷基硫酸钠中的聚丙烯酰胺凝胶电泳确定，对CDC29 mRNA进行的杂交释放翻译指导27,000道尔顿的多肽合成。当将从cdc28无义突变体制备的mRNA进行平行翻译时，无法合成该多肽。但是，如果翻译了维持高拷贝数的质粒中含有CDC28基因的细胞的RNA，则体外产物的量会扩增五倍。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号