Dear yeast colleagues:
Our lab wants to make a Chlamydomonas expression library in a yeast:e.
coli shuttle vector. Our goal is to clone a Chlamy adenylyl cyclase by
rescuing an adenylyl cyclase deficient strain of Saccharomyces
cerevisiae. We are posting this note to ask if anyone could help us
identify and locate an appropriate yeast:e. coli shuttle vector. We have
on hand an excellent Chlamy cDNA library in Lambda Zap. We are
considering excising the cDNA's from this library and inserting them into
a yeast vector. To do this the vector should have a multiple cloning site
that contains an EcoR1 and an Xho1 site. In addition, whether we use
our existing Lambda Zap library or make new cDNA's, we do not want to
have to rely on the presence of a translation initiation ATG in our inserts.
Transmembrane adenylyl cyclases that have been cloned from
unicellular and mutlicellular eucaryotes are encoded in mRNA's that range
from 3.5 to 11.5 kb.
Thus, the vector we want needs to have translation initiation ATG's
downstream of a promoter (preferably a regulatable promoter, we think)
and upstream of our inserts. The adenylyl cyclase-deficient yeast strain
we intend to use is also leu- and trp-. We have considered both the
pYES2 and pVT101-L yeast expression vectors, but neither has a
translation initiation ATG downstream of the promoter and upstream of
the MCS. In addition pVT101-L does not have an EcoR1 site in the MCS,
but does have an EcoR1 site at an inappropriate site.
(We almost were misled by conflicting descriptions of the pVT series
of vectors. When we looked up the vector map for the pVT series in a
reference book on vectors (Cloning Vectors: A Laboratory Manual by
P.H. Pouwels, B.E. Enger-Valk and W.J. Brammar; 1985;Elsevier
Science), the description indicated that the pVT vectors were designed
for expression of proteins whose cDNA's lack their promoter and
translation initiation signals. On the other hand when we consulted the
original publication describing the construction of the pVT vectors
(T.Vernet et al, 1987;Gene 52:225-233) we learned that that the
inserted DNA needs to contain its own ATG translation initiation codon.)
In addition to information about locating an appropriate vector, we
welcome insights about the wisdom of using an existing Lamba Zap
library as a source of cDNA's, whether to use random primers or polydT
primers to make the cDNA library, and any other ideas about this
approach. As best we can tell, although the Arabidopsis folks have
used this approach to clone Arabidopsis cDNA's, no one has done it yet
in Chlamydomonas. Thus, your input may be useful not only to our lab,
but to an entire unicellular community.
Bill Snell and Kiran Kaur
University of Texas Southwestern Medical Center
Dallas, TX 75235-9039
T 214-648-2332
F 214-648-8694
email Snell03 at UTSW.SWMED.edu
