Fine structure recombinational analysis of cloned genes using yeast transformation
The authors describe a general method for analyzing the genetic fine structure of plasmid-borne genes in yeast. Previously they had reported that a linearized plasmid is efficiently rescued by recombination with a homologous restriction fragment when these are co-introduced by DNA-mediated transformation of yeast. Here, they show that a mutation can be localized to a small DNA interval when members of a deletion series of wild-type restriction fragments are used in the rescue of a linearized mutant plasmid. The resolution of this method is to at least 30 base pairs and is limited by the loss of a wild-type marker with proximity to a free DNA end. As a means for establishing the nonidentity of two mutations, they determined the resolution of two-point crosses with a mutant linearized plasmid and a mutant homologous restriction fragment. Recombination between mutations separated by as little as 100 base pairs was detected. Moreover, the results indicate that exchange within a marked interval results primarily from one of two single crossovers that repair the linearized plasmid. These approaches to mapping the genetic fine structure of plasmids should join existing methods in a robust approach to the mutational analysis of gene structure in yeast.
- Research Organization:
- Massachusetts Institute of Technology, Cambridge
- OSTI ID:
- 6869792
- Journal Information:
- Genetics; (United States), Journal Name: Genetics; (United States) Vol. 115:1; ISSN GENTA
- Country of Publication:
- United States
- Language:
- English
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