Technology Development |
Our
laboratory has been involved in developing technologies that allow the identification
of genes using selections carried out in yeast and bacteria. We have also
most recently devised a cloning method called the Univector plasmid fusion
system (UPS) that allows extremely rapid subcloning and epitope tagging
of protiens.
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The  YES cDNA Expression System |
We
have developed a multifunctional lambda expression vector system, lYES,
designed to facilitate gene isolation from eukaryotes by complementation
of E. coli and S. cerevisiae mutations. lYES vectors have a selection for
cDNA inserts using an oligo adaptor strategy and are capable of expressing
genes in both E. coli and S. cerevisiae . They are also allow conversion
from phage l to plasmid clones using the cre-lox site-specific recombination
system, referred to as automatic subcloning. lYES vectors utilize an adaptor
selection for inserts and can generate libraries with 109 recombinants per
mg of cDNA insert. cDNA libraries constructed in these vectors have been
used to isolate genes from humans by complementation of yeast. Using this
technology we isolated the human Cdk2 gene by complementation of a yeast
cdc28 ts mutation.
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Advances in the Two-hybrid System |
The
two hybrid cloning system is a powerful genetic method to identify genes
via protein-protein interactions. We have made a number of improvements
to this method which are designed to increase the number of potential protein-protein
interactions detectable and to streamline the labor intensive process of
authenticating clones identified in the primary screen. The first improvement
was the design of genetic selections instead of screens to detect interacting
proteins. We developed the strain Y190 using HIS3 as a reporter and Y166
which employed a URA3 reporter for this purpose. A selection vastly increases
the numbers of library clones that can be searched. Secondly, through the
use of negative selections for plasmid loss coupled with a replica-mating
strategy, we have developed a rapid method to distinguish between the genuinely
interacting clones and the nonspecific background that is inherent to the
system. Third, a l phage vector, lACT2, was made that allows construction
of highly complex, HA epitope-tagged, directional libraries of GAL4 activation
domain-fused cDNAs that can be converted to plasmid form by in vivo cre-lox
mediated site-specific recombination. We used these methods to identify
the human p21 and p57 Cdk inhibitors.
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A Novel Membrane-based Two-hybrid System, the SOS-recruitment System
(SRS) |
In
collaboration with Michael Karin's lab, we have developed a novel two hybrid
system for the cloning of genes involved in protein-protein interactions.
This system, called the SOS Recruitment System, SRS, relies on the activation
of the ras signalling pathway when a fusion protein to the ras guanine nucleotide
exchange factor, SOS, is recruited to the inner surface of the plasma membrane
by physical association with a second protein targetted to the same membrane
by a myristylation sequence. When SOS is successfully recruited to the plasma
membrane, it activates ras and bypasses the need for the endogenous exchange
factor, Cdc25, an essential protein. This system allows protein-protein
interactions to be detected in the cytoplasm. Furthermore, proteins that
activate transcription can be used in this system giving it an advantage
over the transcriptionally based two hybrid system. We have developed a
lambda-based expression vector, lMS-TRP for expression of myristylation-fused
cDNA libraries an other useful vectors for this method. Visit
our protocols page for more information.
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The Univector Plasmid Fusion System (UPS) |
We
have developed a series of novel cloning methods that facilitate the rapid
construction of recombinant DNA molecules. The central method is called
the Univector Plasmid-fusion System (UPS). The UPS employs cre-lox site-specific
recombination in vitro to catalyze plasmid fusion between the Univector,
a plasmid containing the gene of interest, and host vectors containing regulatory
information. Plasmid fusion events are genetically selected and result in
placement of the gene of interest under the control of novel regulatory
elements. A second UPS-related method, POT, allows for the precise transfer
of coding sequences alone from a Univector into a host vector. The UPS eliminates
the need for restriction enzymes, DNA ligases, and many in vitro manipulations
required for subcloning and allows the rapid construction of multiple constructs
with minimal effort. In one afternoon a given gene could be cloned into
dozens of vectors. We have demonstrated that the UPS can generate constructs
for expression in multiple organisms and that this method can be used to
transfer whole libraries into new vectors. We have also developed new methods
for directional subcloning of PCR fragments into the Univector that rely
upon reconstitution of lacO sites. We have also devised methods for the
generation of epitope tags and other fusions at the 3' end of open reading
frames using homologous recombination in E. coli. Together, these methods
constitute a comprehensive recombinational strategy for the generation and
manipulation of recombinant DNA that can be used for the parallel processing
of gene sets. We have currently developed a collection of approximately
35 expression vectors that allow expression of cDNAs cloned into the Univector
to be expressed in bacteria, yeast, insect cells, or mammalian cells as
native proteins or a variety of fusions such as Gst, His6, HA, FLAG or MYC
tagged. Visit our protocols page for more information.
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