ISOLATION OF TELOMERE-LIKE SEQUENCES FROM CRYPTOCOCCUS-NEOFORMANS AND THEIR USE IN HIGH-EFFICIENCY TRANSFORMATION
ISOLATION OF TELOMERE-LIKE SEQUENCES FROM CRYPTOCOCCUS-NEOFORMANS AND THEIR USE IN HIGH-EFFICIENCY TRANSFORMATION
复制标题
DOI：
10.1128/mcb.12.6.2777
复制
发表时间：
1992-06-01
影响因子：
5.3
通讯作者：
EDMAN, JC
中科院分区：
文献类型：
--
作者：
EDMAN, JC
开发人类病原体新型隐球菌的转化系统是鉴定毒力相关基因的重要先决条件。此前已有报道，利用克隆的新型隐球菌URA5基因和ura5突变体可以实现低效转化。将线性化 URA5 载体引入新型隐球菌中会产生不稳定的转化体，其显然含有线性染色体外 DNA 分子。在本文中，通过核酸外切酶消化证实这些分子的性质是线性的。大肠杆菌中染色体外 DNA 的回收和序列分析表明，端粒 DNA 的重复特征已添加到引入的 DNA 末端。回收的质粒能够以高得多的效率进行转化，无论是在超螺旋状态（每μg最多200个转化体）还是线性状态（每μg最多90,000个转化体）。
Development of a transformation system for the fungal human pathogen Cryptococcus neoformans is an important prerequisite for the identification of genes involved in virulence. It has previously been reported that low-efficiency transformation can be achieved by using the cloned C. neoformans URA5 gene and ura5 mutants. The introduction of linearized URA5 vectors into C. neoformans resulted in unstable transformants which apparently harbored linear extrachromosomal DNA molecules. In this paper, the nature of these molecules is confirmed to be linear by exonuclease digestion. Recovery of the extrachromosomal DNA in Escherichia coli and sequence analysis demonstrates that repeats characteristic of telomeric DNA have been added to the ends of the introduced DNA. The recovered plasmids are capable of transforming at much higher efficiencies either in the supercoiled state (up to 200 transformants per mu-g) or the linear state (up to 90,000 transformants per mu-g).