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LUIZ F. ONUCHIC AND GREGORY G. GERMINO
scriptional activators such as GAL4 into two modular domains: a DNAbinding domain (BD) and a transcriptional activation domain (AD). While
the first localizes the transcription factor to specific DNA sequences positioned in regulatory sequences immediately 5' of a reporter gene, the second
domain contacts other transcription-related elements necessary for the initiation of transcription. Both domains must be in close proximity for normal transactivation to occur. Normally, this occurs because the two domains are segments of the same gene product. In the yeast two hybrid technique, the domains are separately encoded as portions of hybrid proteins
containing other protein domains. Transactivation only occurs when the
AD and BD domain are brought together because of an interaction between
the other domains of their respective hybrid proteins. Proteins that interact
with the bait protein are identified by generating hybrids of the BD and the
bait protein (Hl), and the AD and a library ofproteins (H2). A specific interaction between Hl and H2localizes both the BD and the AD to a reporter
gene, leading to its transcriptional activation and identifying a candidate
binding partner for the target protein. This system is sensitive enough
to detect weak and transient protein-protein interactions, generally observed in large native complexes.
Expression Libraries. Libraries that are constructed to optimize expression of their inserted cDNA sequences are called expression libraries. The
vectors used in construction of cDNA expression libraries have special features. One essential element is an RNA promoter sequence adjacent to the
cloning site. This is used to transcribe RNA from the cDNA insert in a bacterial host. Expression must be regulated, however, since the products of
many cloned genes are toxic to the hast cell. Ifleft unregulated, clones expressing deleterious products would be at a growth disadvantage and would
be rapidly lost from a library. Most vectors used in constructing expression
libraries contain inducible promoters that allow propagation of a clone before inducing expression of its insert. Expressed proteins can be fixed to
nitrocellulose membranes in a manner similar to that used to make
DNA filters and then screened with labeled antibodies, ligands, etc. An
added feature of expression libraries is that they also can be plated so as
to generate DNA ftlters suitable for hybridization with nucleic acid probes.
Thus, expression libraries are the most versatile since they can be used for
multiple purposes.
There are, however, several drawbacks to their use. First, expression libraries without full-length copies of mRNA molecules are the most useful.
The extreme 5' end of mRNA molecules is non-coding and its inclusion
could disrupt expression of the remainder of the message. Furthermore,
smaller foreign polypeptides are more stably expressed in prokaryotic
LUIZ F. ONUCHIC AND GREGORY G. GERMINO
scriptional activators such as GAL4 into two modular domains: a DNAbinding domain (BD) and a transcriptional activation domain (AD). While
the first localizes the transcription factor to specific DNA sequences positioned in regulatory sequences immediately 5' of a reporter gene, the second
domain contacts other transcription-related elements necessary for the initiation of transcription. Both domains must be in close proximity for normal transactivation to occur. Normally, this occurs because the two domains are segments of the same gene product. In the yeast two hybrid technique, the domains are separately encoded as portions of hybrid proteins
containing other protein domains. Transactivation only occurs when the
AD and BD domain are brought together because of an interaction between
the other domains of their respective hybrid proteins. Proteins that interact
with the bait protein are identified by generating hybrids of the BD and the
bait protein (Hl), and the AD and a library ofproteins (H2). A specific interaction between Hl and H2localizes both the BD and the AD to a reporter
gene, leading to its transcriptional activation and identifying a candidate
binding partner for the target protein. This system is sensitive enough
to detect weak and transient protein-protein interactions, generally observed in large native complexes.
Expression Libraries. Libraries that are constructed to optimize expression of their inserted cDNA sequences are called expression libraries. The
vectors used in construction of cDNA expression libraries have special features. One essential element is an RNA promoter sequence adjacent to the
cloning site. This is used to transcribe RNA from the cDNA insert in a bacterial host. Expression must be regulated, however, since the products of
many cloned genes are toxic to the hast cell. Ifleft unregulated, clones expressing deleterious products would be at a growth disadvantage and would
be rapidly lost from a library. Most vectors used in constructing expression
libraries contain inducible promoters that allow propagation of a clone before inducing expression of its insert. Expressed proteins can be fixed to
nitrocellulose membranes in a manner similar to that used to make
DNA filters and then screened with labeled antibodies, ligands, etc. An
added feature of expression libraries is that they also can be plated so as
to generate DNA ftlters suitable for hybridization with nucleic acid probes.
Thus, expression libraries are the most versatile since they can be used for
multiple purposes.
There are, however, several drawbacks to their use. First, expression libraries without full-length copies of mRNA molecules are the most useful.
The extreme 5' end of mRNA molecules is non-coding and its inclusion
could disrupt expression of the remainder of the message. Furthermore,
smaller foreign polypeptides are more stably expressed in prokaryotic