Chimeric plasmids have been constructed containing two heteroallelic mutant copies of the yeast HIS3 gene as an inverted repetition. Intramolecular exchange events between these two allelic mutant copies are capable of generating a wild-type allele. Plasmids containing two mutant heteroalleles have been transformed into appropriate his3- yeast strains, and the frequency of exchange events generating His+ prototrophs has been measured during mitotic division. After 20 generations of growth under nonselective conditions, between 0.1 and 1 % of the transformed yeast cells become His+ prototrophs. This percentage decreases at least ten-fold in a strain with a rad52 mutation. Plasmid molecules having undergone exchange events have been isolated from yeast cells and have been examined after transfer to Escherichia coli. Physical examination shows that less than 10 % of the plasmids having undergone genetic exchange have also undergone an internal reciprocal recombination event as evidenced by reorientation of linked restriction sites. The remainder of the plasmids having undergone genetic exchange do not exhibit reciprocal recombination. Characterization of the individual allelic copies within a plasmid having undergone exchange reveals that in 24 of 25 examples only one of the two HIS3 copies has become wild type, and that either copy is equally likely to become wild type. We conclude that the model plasmid we have constructed undergoes intramolecular genetic exchange events and will be useful for studying genetic recombination.
Bibliographical noteFunding Information:
We are gratefult o Kevin Struhl for supplying clonedc opieso f mutantH IS3 genesa ndto Thomas Petes and Gerald Fink for bacterial and yeast strains.W e thank James Fuchs for designingt he transductions chemeu sedf or transferringth e hisB mutationb etweenb acteriaa nd Sara Buchsteinf or carryingo ut someo f thesem anipulationsW. e also thankS eishiM urakamif or encouragemenatn dh elp throughoutth e courseo f this work. Andrew Strand, Neil Palosaari,S cott Knee, Byung-Yoon Ahn and Mary Harkins providedt echnicala ssistanced uring partso f the investigationT.h e work was supported by NIH grantG M 28709.
- HIS3 heteroalleles
- Saccharomyces cerevisiae
- gene conversion
- mitotic recombination
- reciprocal recombination