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. 1987 Jan;115(1):73–81. doi: 10.1093/genetics/115.1.73

Fine Structure Recombinational Analysis of Cloned Genes Using Yeast Transformation

Sam Kunes 1, Hong Ma 1, Karen Overbye 1, Maurice S Fox 1, David Botstein 1
PMCID: PMC1203065  PMID: 3549444

Abstract

We describe a general method for analyzing the genetic fine structure of plasmid-borne genes in yeast. Previously we 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, we 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, we 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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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