IN-VIVO CLONING BY HOMOLOGOUS RECOMBINATION IN YEAST USING A 2-PLASMID-BASED SYSTEM
IN-VIVO CLONING BY HOMOLOGOUS RECOMBINATION IN YEAST USING A 2-PLASMID-BASED SYSTEM
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DOI：
10.1002/yea.320110704
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发表时间：
1995-06-15
期刊：
影响因子：
2.6
通讯作者：
ACHSTETTER, T
中科院分区：
文献类型：
--
作者：
DEGRYSE, E;DUMAS, B;ACHSTETTER, T
为了减少酵母表达质粒产生中的经典DNA操作和连接步骤的数量，描述了一系列载体，其通过更有效的“体内重组”技术促进此类质粒的组装。开发了两套载体。第一组，称为“表达载体”，包含一个表达盒和一个大肠杆菌复制子，该表达盒具有由多接头分开的酵母启动子和PGK终止子。在这些载体中亚克隆DNA片段产生与第二组E.大肠杆菌-酵母穿梭载体。这组“重组载体”含有用于补充宿主菌株营养缺陷型的基因或赋予携带质粒的宿主抗生素抗性的细菌基因的功能性拷贝的盒。质粒拷贝数可以通过使用URA 3或URA 3-d作为选择标记与ARS/CEN和2 μ m复制子一起调节。杆菌所有载体都含有噬菌体f1的复制起点，并允许在E.呈现的序列（图1a）已经以登录号Z48747输入EMBL数据库。
In order to reduce the number of classical DNA manipulation and ligation steps in the generation of yeast expression plasmids, a series of vectors is described which facilitate the assembly of such plasmids by the more efficient 'recombination in vivo' technique. Two sets of vectors were developed. The first set, called 'expression vectors', contains an expression cassette with a yeast promoter and the PGK terminator separated by a polylinker, and an Escherichia coli replicon. Subcloning in these vectors of a DNA fragment generates a 'transfer vector' which is compatible with the second set of E. coli-yeast shuttle vectors. This set of 'recombination vectors' contains a cassette for a functional copy of a gene complementing a host strain auxotrophy or a bacterial gene conferring an antibiotic resistance to the plasmid-bearing host. Plasmid copy numbers can be modulated through the use of URA3 or URA3-d as the selective marker together with an ARS/CEN and the 2 mu m replicon.Integration of the cloned DNAs into the yeast linearized replicative vectors occurs by recombination between homologous flanking sequences during transformation in yeast or E. coli. All the vectors contain the origin of replication of phage f1 and allow the generation of single-stranded DNA in E. coli for sequencing or site-directed mutagenesis.The sequence presented (Figure 1a) has been entered in the EMBL data library under Accession Number Z48747.