19 cDNA Library Screening
293
cation makes such sequences selectively and directly accessible for subcloning and comparative sequence analysis (Watson et al. 1992).
DNA libraries themselves can serve as probes in screening of cDNA li- Differential
braries. Such strategies are used for isolation of differentially expressed hybridization
genes or extremely rare sequences. Libraries of DNA clones derived
from sequences that are differentially expressed can be prepared by subtractive hybridization (Lee et al. 1991) or differential display of mRNA
(Liang and Pardee 1992).
Screening based on antigenicity
In cases where no nucleic acid or protein sequences are available, expression vectors offer the alternative of looking for expression of specific eukaryotic cDNAs in host bacteria. Although both plasmid and phage vectors
are used, the most frequently used expression vector is probably A.gt11
(Young and Davis 1983a, Young and Davis 1983b). This and other widely
used expression vectors provide sequence elements that direct the synthesis
of the proper protein product from a cloned cDNA expressedunder the
control of a bacterial promoter. Usually, such proteins areexpressedas fusion proteins containing portions of prokaryotic proteins. Many expression
vectors permit insertion into the bacterial ß-galactosidase gene (lac Z). As a
consequence, cDNA inserts are expressed as ß-galactosidase fusion proteins. It has been shown that fusion of foreign sequences to the carboxy
terminus of ß-galactosidase gives rise to relatively stable fusion proteins
(Stanley 1983, Mierendorf et al. 1987). Proper expression of integrated
cDNA coding information depends on the reading frame and the orientation of the insert. Therefore, only one sixth of the recombinant clones will
have the potential to express the desired protein product. The host strains
used for expression (e.g. E. coli Y1090) are deficient in certain proteases
which prevents premature degradation of expressed proteins. These strains
also contain the lac repressor which prevents fusion protein synthesis until
isopropyl-ß-D-thiogalactopyranoside (IPTG) is added to the medium. This
allows the induction of potentially toxic foreign proteins to be delayed.
Manipulation of expression libraries is similar to conventionallibraries.
However, in addition to recombinant DNA bound to filter replicas, expressed fusion proteins will also bind to the filter membranes and can
be detected immunologically using an antibody directed against the peptide
product of the sequence to be identified. Some further points are worth
considering:
293
cation makes such sequences selectively and directly accessible for subcloning and comparative sequence analysis (Watson et al. 1992).
DNA libraries themselves can serve as probes in screening of cDNA li- Differential
braries. Such strategies are used for isolation of differentially expressed hybridization
genes or extremely rare sequences. Libraries of DNA clones derived
from sequences that are differentially expressed can be prepared by subtractive hybridization (Lee et al. 1991) or differential display of mRNA
(Liang and Pardee 1992).
Screening based on antigenicity
In cases where no nucleic acid or protein sequences are available, expression vectors offer the alternative of looking for expression of specific eukaryotic cDNAs in host bacteria. Although both plasmid and phage vectors
are used, the most frequently used expression vector is probably A.gt11
(Young and Davis 1983a, Young and Davis 1983b). This and other widely
used expression vectors provide sequence elements that direct the synthesis
of the proper protein product from a cloned cDNA expressedunder the
control of a bacterial promoter. Usually, such proteins areexpressedas fusion proteins containing portions of prokaryotic proteins. Many expression
vectors permit insertion into the bacterial ß-galactosidase gene (lac Z). As a
consequence, cDNA inserts are expressed as ß-galactosidase fusion proteins. It has been shown that fusion of foreign sequences to the carboxy
terminus of ß-galactosidase gives rise to relatively stable fusion proteins
(Stanley 1983, Mierendorf et al. 1987). Proper expression of integrated
cDNA coding information depends on the reading frame and the orientation of the insert. Therefore, only one sixth of the recombinant clones will
have the potential to express the desired protein product. The host strains
used for expression (e.g. E. coli Y1090) are deficient in certain proteases
which prevents premature degradation of expressed proteins. These strains
also contain the lac repressor which prevents fusion protein synthesis until
isopropyl-ß-D-thiogalactopyranoside (IPTG) is added to the medium. This
allows the induction of potentially toxic foreign proteins to be delayed.
Manipulation of expression libraries is similar to conventionallibraries.
However, in addition to recombinant DNA bound to filter replicas, expressed fusion proteins will also bind to the filter membranes and can
be detected immunologically using an antibody directed against the peptide
product of the sequence to be identified. Some further points are worth
considering: