>>Frederick Cross have developed the swap plasmids for one-step gene >disruption of the most commonly used nutritional markers HIS3, LEU2, >TRP1 and URA3. >Is there any swap plasmids available for one-step gene disruption of a >kanamycin cassette ? >>David Keszenman-Pereyra >University of Sheffield >Department of Molecular Biology & Biotechnology >D38, Firth Court, Western Bank >Sheffield, S10 2TN, UK
Yes, there are several plasmids that can be used to disrupt a KanMX
cassette.
Plasmid pFA6a:His3MX6 contains the S. pombe his5+ gene (complements S.
cerevisiae his3, but without DNA sequence homology). The promoter and
terminator of the His3MX6 cassette are identical to the KanMX cassette, and
thus can be used for KanMX:His3MX marker swap. We have successfully used
this plasmid to convert a KanMX disruption to a His3MX disruption. The
pFA6a:His3MX6 plasmid is described in Longtine et al. 1998. "Additional
modules for versatile and economical PCR-based gene deletion and
modification in Saccharomyces cerevisiae." Yeast 14:953-961.
Two other plasmids that can be used analogously are pAG60 and pAG61
described in Goldstein et al. 1999. "Heterologous URA3MX cassettes for gene
replacement in Saccharomyces cerevisiae." Yeast 15:507-511. pAG60 contains
the Ura3MX4 cassette and pAG61 contains Ura3MX3. The URA3 sequences are from
the C. albicans URA3 gene, and lack homology to S. cerevisiae. The promoter
and terminator are the same as in the KanMX and His3MX cassettes. Ura3MX4
contains just the Ura3 gene, while in Ura3MX3 the Ura3 gene is flanked by
direct repeats, allowing one to use FOA to select for loss of the Ura3
marker as for the hisG-URA3-hisG cassettes.
I am curious, and I have a question for the yeast community. Does anyone
know what the "MX" stands for in "KanMX?"
David
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David Stillman
Division of Molecular Biology and Genetics
Dept. of Oncological Sciences
Univ. of Utah Health Sciences Center
50 N. Medical Drive, Room 5C334 SOM
Salt Lake City, UT 84132
stillman at hci.utah.edu
phone: 801-581-5429
fax: 801-581-3607
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