Biology Reference
In-Depth Information
Agarose
10 mM dNTP mix
Taq polymerase and buffer
Zymolyase (Zymoresearch, CA)
Valap (equal weights of Vaseline (petrolatum), lanolin, and paraffin wax)
Polyclonal anti-GFP antibody (Applied Biological Materials, Canada)
22.2
INTRODUCING TUBULIN MUTATIONS INTO YEAST
Themost commonmethodused for structure-function studies of yeast tubulin leverages
homologous recombination-mediated gene replacement to introduce desired mutations
into tubulin genes (
Fig. 22.1
). Directedmutagenesis is performed
in vitro
using standard
molecular biology techniques. Following gene replacement, the modified tubulin is
expressed as a single copyunder control of the native promoter and regulatory elements.
Another useful approach is the “plasmid shuffle” in which a plasmid containing
mutated tubulin is exchanged in the cell for a plasmid carrying a wild-type copy:
described in
Sikorski and Boeke (1991)
. This approach offers increased throughput
relative to gene replacement. However, the number of gene copies cannot be tightly
controlled using plasmids, which may confound interpretation of cell-based assays.
Thus, we focus here on the strategy of direct gene replacement.
22.2.1
Preparation of modified tubulin DNA
Specific mutations are introduced into a plasmid containing the tubulin open reading
frame (ORF) flanked by upstream and downstream genomic sequences and a proto-
trophic marker cassette. The prototrophy gene is inserted several hundred bases
downstream from the stop codon and positioned not to interfere with neighboring
genes. Site-directed mutagenesis can be accomplished with commercially available
systems such as QuikChange (Agilent Technologies). Three commonly used starting
plasmids are described in
Table 22.1
. In pMG1,
TUB2
has been tagged with 6
His
to aid in purification of mutated tubulin (
Gupta et al., 2002; Johnson, Ayaz,
Huddleston, & Rice, 2011
).
Note:
It is essential to sequence the entire tubulin gene and promoter region after
mutagenesis to ensure additional, unwanted mutations did not occur during
in
vitro
manipulation.
To generate linear DNA for yeast transformation, digest
g plasmid as described
in
Table 22.1
, verify complete digestion by agarose gel electrophoresis, and gel-
purify the appropriate fragment (
Table 22.1
).
5
m
>
22.2.2
Transformation of modified tubulin DNA into yeast
Tubulin is an essential gene. Thus, introducing a nonfunctional mutant into haploid
cells may select for unwanted suppressor mutations. Thus, unless a mutation has
been previously verified as nonlethal, the mutated gene should be transformed into
a diploid strain and then sporulated to generate a haploid.
