Site-directed, recombination-mediated mutagenesis of a complex gene locus

Michelle Craig Barton, Merl F. Hoekstra, Beverly M. Emerson

Research output: Contribution to journalArticle

21 Scopus citations

Abstract

We have generated a site-specific 17 bp insertion within a 38 kb chick globin gene cluster by employing the recombination abilities of Saccharomyces cerevisiae. This gene cluster contains four β-type globin genes which share a high degree of sequence homology. In this procedure, a small fragment of βA-globin DNA containing a 17 bp insertion is subcloned into a URA3-based yeast integrating vector (YIp). This mutated globin subclone is introduced into cells that carry the 38 kb globin cluster clone on a single-copy, circular vector derived from a yeast artificial chromosome (YAC). Insertion of the 17 bp oligomer is achieved by targeted integration of the YIp subclone. The recombinant contains the normal βA-globin gene, the mutant gene and YIp vector sequences between the two copies. Excision of the vector sequences and one copy of the duplicated globin sequences by homologous recombination is required for cell survival when exposed to the selective agent 5-fluoroorotic acid, which is toxic to ura+ yeast cells. Depending upon the point of the cross-over, a ura- yeast cell bearing either a wild-type globin gene or a 17 bp insertion mutation will result. By restriction mapping and in vitro transcription analysis, the βA-globin gene containing the 17 bp insert has no nonspecific mutations generated during the recombination and selection procedures. Specific mutations of regulatory regions, including protein-DNA binding sites, can be accurately targeted within extensive DNA clones by this method.

Original languageEnglish (US)
Pages (from-to)7349-7355
Number of pages7
JournalNucleic acids research
Volume18
Issue number24
DOIs
StatePublished - 1990

ASJC Scopus subject areas

  • Genetics

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