Heterologous Expression in Budding Yeast as a Tool for Studying the Plant Cell Morphogenesis Machinery - Plant Cell Morphogenesis: Methods and Protocols - page 259

Viable recessive mutations, in particular those causing
temperature-sensitive growth defects, are obvious candidates for
complementation studies. We shall use complementation of a
temperature-sensitive mutation as an example throughout, albeit
also mutations causing, e.g., increased sensitivity to various chemi-
cals may be complemented as well (adjust media composition
appropriately for such cases).
Use preferentially haploid strains (often referred to as mating
type MATa or MAT ) and obtain also isogenic (parental) strains

for control purposes together with the mutants. Pay attention to
the metabolic requirements of your yeast strains. Most laboratory
strains are polyauxotrophic. Make sure that the strains are compat-
ible with the selection marker(s) of the vectors you will be using
and with your inducible gene expression system, if you intend to
use one ( see Note 2 ).

α

2.2 Expression
Vectors and Libraries

For heterologous expression, the gene of interest must be cloned
into a suitable binary (shuttle) Escherichia coli -yeast vector that
allows DNA amplifi cation in bacteria, as well as maintenance, selec-
tion, and expression in S. cerevisiae ( see Note 3 ) . Many such vec-
tors can be obtained from public repositories such as the ATCC
( http://www.atcc.org ), where a large collection of yeast expres-
sion vectors [ 8 ] is available (order No. 87669). Addgene ( http://
www.addgene.org ) provides another very large collection of plas-
mids [ 9 ] designed for use with the Gateway cloning system
(Invitrogen).
Complementation of a host yeast strains' auxotrophic muta-
tion is usually used to select for yeast transformants. Among the
commonly used markers, the URA3 gene complementing the
ura3 uracil auxotrophy provides an extra advantage of possible
selection not only for plasmid acquisition (leading to uracil-
independent growth) but also for plasmid loss, resulting in resis-
tance towards 5-fl uoroorotic acid, or 5-FOA [ 10 ].
Both constitutive and inducible promoters can be used for het-
erologous protein expression in yeast. Among the later, the most
common is the galactose-inducible “two-headed” promoter of the
GAL1 / GAL10 genes and its derivatives. If preparing constructs
based on existing cDNAs, avoid introducing extraneous sequences
into the 5 UTR. If possible, locate the start of transcription at the

beginning of the original plant 5

′

UTR, or, alternatively, maintain
as much of the yeast-derived vector sequence upstream of the ATG
as possible.
For screening experiments, a good plant cDNA library in a
binary vector fulfi lling the above requirements is essential. Besides
constructing the library de novo (which is out of scope of this
chapter), some libraries, such as the “Davis library” of Arabidopsis
thaliana seedling aboveground organ cDNAs in the Lambda YES
vector featuring the GAL1 promoter and the URA3 markers [ 11 ],

′