Abstract
We have developed a system which facilitates the rapid modification of yeast artificial chromosome (YAC) insert DNA. Specific modifications, such as deletions, insertions and point mutations, can be generated by a two-step allele replacement method using the yeast translational suppressor, SUP4-o, as both a positive and negative selection. The introduction of the SUP4-o gene was successful in 4 out of 24 selected transformant colonies, while the subsequent homologous elimination occurred in 2 out of 30 colonies. The use of a simple, short-range PCR assay rapidly identified the correct events among the genetically selected isolates and should be generally applicable to YAC modifications.
Full Text
The Full Text of this article is available as a PDF (34.1 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Burke D. T., Carle G. F., Olson M. V. Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science. 1987 May 15;236(4803):806–812. doi: 10.1126/science.3033825. [DOI] [PubMed] [Google Scholar]
- Chernoff Y. O., Lindquist S. L., Ono B., Inge-Vechtomov S. G., Liebman S. W. Role of the chaperone protein Hsp104 in propagation of the yeast prion-like factor [psi+]. Science. 1995 May 12;268(5212):880–884. doi: 10.1126/science.7754373. [DOI] [PubMed] [Google Scholar]
- Cox B. S. A recessive lethal super-suppressor mutation in yeast and other psi phenomena. Heredity (Edinb) 1971 Apr;26(2):211–232. doi: 10.1038/hdy.1971.28. [DOI] [PubMed] [Google Scholar]
- Hawthorne D. C., Mortimer R. K. Genetic mapping of nonsense suppressors in yeast. Genetics. 1968 Dec;60(4):735–742. doi: 10.1093/genetics/60.4.735. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Huxley C. Transfer of YACs to mammalian cells and transgenic mice. Genet Eng (N Y) 1994;16:65–91. [PubMed] [Google Scholar]
- Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Käufer N. F., Fried H. M., Schwindinger W. F., Jasin M., Warner J. R. Cycloheximide resistance in yeast: the gene and its protein. Nucleic Acids Res. 1983 May 25;11(10):3123–3135. doi: 10.1093/nar/11.10.3123. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nasmyth K. At least 1400 base pairs of 5'-flanking DNA is required for the correct expression of the HO gene in yeast. Cell. 1985 Aug;42(1):213–223. doi: 10.1016/s0092-8674(85)80117-3. [DOI] [PubMed] [Google Scholar]
- Rothstein R. Deletions of a tyrosine tRNA gene in S. cerevisiae. Cell. 1979 May;17(1):185–190. doi: 10.1016/0092-8674(79)90306-4. [DOI] [PubMed] [Google Scholar]
- Rothstein R. Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. Methods Enzymol. 1991;194:281–301. doi: 10.1016/0076-6879(91)94022-5. [DOI] [PubMed] [Google Scholar]
- Scherer S. E., Veres G., Caskey C. T. The genetic structure of mouse ornithine transcarbamylase. Nucleic Acids Res. 1988 Feb 25;16(4):1593–1601. doi: 10.1093/nar/16.4.1593. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Scherer S., Davis R. W. Replacement of chromosome segments with altered DNA sequences constructed in vitro. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4951–4955. doi: 10.1073/pnas.76.10.4951. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tuite M. F., Cox B. S., McLaughlin C. S. In vitro nonsense suppression in [psi+] and [psi-] cell-free lysates of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1983 May;80(10):2824–2828. doi: 10.1073/pnas.80.10.2824. [DOI] [PMC free article] [PubMed] [Google Scholar]