Note: Descriptions are shown in the official language in which they were submitted.
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PROTEIN INTERACTION AND TRANSCRIPTION FACTOR TRAP
Field of Invention
This invention relates to the use of gene trapping
methods for the identification of genes and two-hybrid
methodology for the identification of protein-protein
interactions.
Background of the Invention
Virtually all cellular responses, including growth and
differentiation, are stringently controlled by
physiological signals in the form of growth factors,
hormones, nutrients, and contact with neighbouring cells.
These various signals are processed and interpreted by
signal transduction mechanisms which ultimately induce the
cell to mount an appropriate response. Signalling pathways
stimulated by physiological signals involves a network of
specific protein-protein interactions which function to
transmit the signal to downstream effector molecules that
execute the response. Thus, specific interactions between
proteins are critical for signal transduction mechanisms as
well as regulation of cellular architecture and responses
to physiological signals. Given that specific
protein-protein interactions are involved in execution of
virtually all cellular functions, technologies which
simplify and facilitate detection and analysis of specific
protein-protein interactions will be valuable for the
discovery, design and testing of drugs that target highly
specific biological processes.
Eukaryotic gene expression is regulated by a class of
proteins variously known as transcriptional activators, or
enhancer binding proteins and are referred to herein as
"transcriptional regulatory proteins". These molecules,
bind to specific sequences on DNA within the promoters of
genes they regulate, and function by recruiting the general
transcriptional initiation complex to the site where
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transcription of DNA into messenger RNA (mRNA) begins. The
general eukaryotic transcriptional initiation complex may
consist of two large protein complexes represented by
transcription factor IID (TFIID), which contains the
TATA-element binding protein that functions to position the
general initiation complex at a precise location on the
promoter, and the RNA polymerase II holoenzyme, which
contains the catalytic function necessary to unwind the
double stranded DNA and transcribe a copy of the DNA
template into mRNA. Known transcriptional activators are
understood to function by forming direct protein-protein
interactions with parts of TFIID and/or the RNA polymerase
holoenzyme, and catalysing their assembly into an
initiation complex at TATA-element of the promoter.
Transcriptional regulatory proteins typically possess
two functional elements, a site-specific DNA-binding domain
and a transcriptional activation domain which can interact
with either TFIID or the RNA polymerase holoenzyme.
Eukaryotic transcriptional regulatory proteins are typified
by the Saccharomyces yeast GAL4 protein, which was one of
the first eukaryotic transcriptional activators on which
these functional elements were characterized. GAL4 is
responsible for regulation of genes which are necessary for
utilization of the six carbon sugar galactose. Galactose
must be converted into glucose prior to catabolism; in
Saccharomyces this process typically involves four
reactions which are catalysed by five different enzymes.
Each enzyme is encoded by a GAL gene (GAL 1, 2, 5, 7, and
10) which is regulated by the transactivator GAL4 in
response to the presence of galactose. Each GAL gene has
a cis-element within the promoter, termed the upstream
activating sequence for galactose (UAS~), which contains
17 base-pair sequences to which GAL4 specifically binds.
The GAL genes are repressed when galactose is absent, but
are strongly and rapidly induced by the presence of
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galactose. GAL4 is prevented from activating transcription
when galactose is absent by a regulatory protein GAL80.
GAL80 binds directly to GAL4 and likely functions
preventing interaction between GAL4's activation domains
and the general transcriptional initiation factors. When
yeast are given galactose, transcription of the GAL genes
is induced. Galactose causes a change in the interaction
between GAI14 and GAL80 such that GAL4's activation domains
become exposed to allow contact with the general
l0 transcription factors represented by TFIID and the RNA
polymerase II holoenzyme and catalyse their assembly at the
TATA-element which results in transcription of the GAL
genes. The functional regions of GAL4 have been defined by
a combination of biochemical and molecular genetic
strategies. GAL4 binds as a dimer to its specific
cis-element within the UASG of the GAL genes. The ability
to form tight dimers and bind specifically to DNA is
conferred by an N-terminal DNA-binding domain. This
fragment of GAL4 (amino acids 1-147) can bind efficiently
and specifically to DNA but cannot activate transcription.
Two parts of the GAL4 protein are necessary for activation
of transcription, called activating region 1 and activating
region 2. The activating regions are thought to function
by interacting with the general transcription factors . The
large central portion of GAL4 between the two activating
regions is required for inhibition of GAL4 in response to
the presence of glucose. The C-terminal amino acids of
GAL4 bind the negative regulatory protein GAL80; deletion
of this segment causes constitutive induction of GAL
transcription.
An important contribution towards development of
two-hybrid methodology was the discovery that a
transcriptional activator protein, the Herpes viral protein
16 (VP16), is indirectly recruited to DNA through
interaction with sequence specific DNA binding protein.
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VP16 activates transcription by forming a complex with the
cellular proteins Oct-1 and HCF; the Oct-1/HCF/VP16 complex
bind to enhancer elements of the Herpes immediate early
genes. It was subsequently shown that the negative
regulatory protein GAL80 could be converted into a
GAL4-dependent transactivator by fusion of a short
negatively-charged transcriptional activating sequence B17.
The GAL80-B17 fusion protein, when co-expressed with GAL4,
was found to cause activation of a GAL4-dependent reporter
gene to a greater extent than GAL4 alone.
The standard two-hybrid assay relies upon the fact
that many eukaryotic transcriptional regulatory systems
consist of the separate domains discussed above: the
DNA-binding domain (DNA-BD) that binds to a promoter or
other cis-transcriptional regulatory element; and, the
activation domain (AD) that directs RNA polymerase II to
transcribe a gene downstream from the site on the DNA where
the DNA-BD is bound. The DNA binding domain and the
activation domain may be separate proteins but will
function to activate transcription as long as the AD is in
proximity to a DNA-BD bound to the transcriptional
regulatory element. Where each of the AD and the DNA-BD is
fused to members of a pair of interacting proteins, the AD
will function via the link to the DNA-BD created by the
interacting proteins. Thus, the two-hybrid assay may be
used to investigate whether interaction occurs between two
proteins (termed "bait" and "prey") expressed as fusion
products with DNA-BD and AD peptides, respectively. A
positive event is identified by activation of a reporter
gene having an upstream promoter to which the DNA-BD binds .
The two-hybrid assay may be carried out in a variety
of eukaryotic cells including yeast (see: Fields, S. and
Song. O. 1989 A Novel Genetic System to Detect
Protein-Protein Interactions Nature 340:245-247; and
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Fields, S. 1993. The Two-hybrid System to Detect
Protein-Protein Interactions. Methods: A Companion to
Meth. Enzymol. 5:116-124.) and mammalian cells (see:
Luo, Y. et a1. 1997. Mammalian Two-Hybrid System: A
Complementary Approach to the Yeast Two-Hybrid System.
Biotechnics 22:350-52; and, Feron, E.R. et al. 1992.
Karyoplasmic Interaction Selection Strategy: A General
Strategy to Detect Protein-Protein Interactions in
Mammalian Cells Proc. Natl. Acad. Sci. U.S.A. 89:7958-62).
Commercial yeast and mammalian two-hybrid assay kits are
available from Clontech Laboratories, Inc., 1020 East
Meadow Circle, Palo Alto, California, 94303-4230, U.S.A.
A variant of the two-hybrid method, called the
"interaction trap" system, employs the principle of using
separate fusions with DNA-binding and transactivation
domains, except that the bait is fused to LexA, which is a
sequence-specific DNA binding protein from E. coli, and an
artificial transactivation domain known as B42 (31) is used
for the "prey" fusions. Interaction between the bait and
prey fusions is detected by expressed of a LexA-responsive
reporter gene.
A modification of the standard two-hybrid system known
as "Reverse Two-Hybrid" (Erickson et a1. U.S. Pat
No. 5,535,490; Vidal et al. International Application
Number PCT/US96/04995) has been described which is intended
for use in identifying specific inhibitors of a standard
two-hybrid protein-protein interaction. The reverse two-
hybrid system operates by driving the expression of relay
gene, such as the GAL80 gene, that encodes a protein that
bind to and masks the activation domain of a
transcriptional activator such as GAL4. Expression of the
reporter gene is made dependent upon the functioning of the
activation domain of the transcriptional activator. Only
when the level of the masking protein is reduced because a
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compound interferes with the two-hybrid interaction will
the activation domain of the transcriptional activator be
unmasked and allowed to function.
Specific protein-protein interactions are the basis
for many biological processes. Standard two-hybrid
techniques make use of specialized cDNA expression
libraries as a source of protein sequences used in
screening for specific interactions between proteins (for
example in drug screening programs). However, cDNA
expression libraries possess some intrinsic disadvantages.
For example, cDNA libraries produce a bias toward cloning
of highly expressed genes and rare gene transcripts are
unlikely to be discovered. The source of the mRNA for the
generation of the cDNA library is critical since many
tissue restricted genes and developmentally or temporally
regulated genes are not represented by a particular cDNA
library.
Gene trap vectors target the prevalent introns of the
eukaryotic genome. These vectors may consist of either a
splice-acceptor (SA) site upstream of a reporter sequence,
or an unpaired splice-donor (SD) site downstream from a
reporter sequence. Preferably, on the latter vector
comprising a SD, the reporter sequence is driven by an
appropriate transcriptional regulatory element
(eg. promoter). Integration of the above-described gene
trap vectors into an intron results in production of m-RNA
in which a transcript of the vector is joined to an
transcript of an adjacent exon. (seer Skarnes, W.C.
et al. 1992. A Gene Trap Approach in Mouse Embryonic Stem
Cells: The lacZ Reporter is Activated by Splicing, Reflex
Endogenous Gene Expression and is Mutagenic in Mice. Genes
Dev. 6:903-918; W.C. Skarnes 1993 The Identification of New
Genes: Gene Trapping in Transgenic Mice. Current Opinion
in Biotechnology 4:684-89; and, United States Patent
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No. 5,652,128 July 29, 1997.). A form of gene trapping
(termed "tagging") may also be accomplished by using a
vector comprising a peptide encoding segment and both an
upstream SA and a downstream SD (see United States Patent
No. 5,652,128 of Jarvik).
Features of gene trapping include:
(a) random integration into the genome;
(b) splice acceptor or splice donor containing
vectors result in fusion of a transcript of a
reporter gene from the vector with endogenous
gene transcripts;
(c) the full repertoire of genes are represented in
the genome without a bias towards highly
expressed genes;
(d) gene trapping can provide information about
coding regions of most genes that is independent
of their transcription status; and
(e) gene trapping is independent of the source of
mRNA (therefore, rare as well as tissue specific
genes and developmental temporally regulated
genes may be trapped).
A full strategy for genome-wide analysis as well as
for drug discovery and assessment, should include a
systematic strategy for identification and characterization
of gene products according to their protein-protein
interaction characteristics.
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Summarv of Invention
Gene trap methodologies provide a repertoire of
protein domains encoded by exon sequences found within the
S genome. Two-hybrid techniques permit identification of
protein-protein interactions. This invention makes use of
a combination of gene trap and two-hybrid methodologies for
the identification and characterization of genes according
to protein-protein interactions of the gene product or for
the identification of genes encoding transcriptional
activator domains (AD). Interaction of an exon-encoded
protein domain with a given protein, or functioning of the
exon-encoded domain as an AD, is detected by reconstituting
the activity of a transcriptional activator.
This invention also provides gene trap vectors adapted
for use in a two-hybrid assay and methodologies for
identification of genes encoding proteins capable of
interacting with a selected protein. This invention also
provides gene trap vectors and methodologies for the
selective identification of genes encoding transcription
activator domains.
This invention provides a DNA construct comprising a
DNA sequence encoding a transcriptional regulatory protein
moiety selected from the group consisting of a DNA-BD and
a AD; and, a m-RNA splice site. The term ~~m-RNA" splice
site is defined herein as being a splice acceptor sequence
(SA), an unpaired splice donor sequence (SD).
This invention also provides a DNA construct
comprising a DNA sequence encoding a transcriptional
regulatory protein moiety selected from the group
consisting of a DNA-BD and an AD; and, a downstream SD.
This DNA construct preferably contains no nucleic acid
sequence which would encode a protein that will interact
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with a test protein employed in this invention.
Preferably, the only protein encoded by the construct or
the portion of the construct between the 5' end of the
sequence encoding the transcriptional regulatory protein
moiety and the 3' end of the SD is the transcriptional
regulatory protein moiety itself. Preferably, this
construct will have a transcriptional regulatory element
(eg. a promoter) operably linked to the sequence encoding
the transcriptional regulatory protein moiety.
This invention also provides a DNA construct
comprising a DNA sequence encoding a transcription
regulatory protein moiety selected from the group
consisting of a DNA-BD and an AD, together with an upstream
SA and a downstream SD. Alternatively, this DNA construct
may comprise an SA upstream of a transcriptional regulatory
protein moiety selected from the group consisting of a
DNA-BD and an AD; and, a downstream poly-adenylation
signal. Preferably, these DNA constructs will not encode
any protein which will interact with a test protein as used
on this invention. Preferably, the only protein encoded by
the construct or the portion of the construct between the
SA and the SD or the SA and the poly-adenylation signal,
will be the transcriptional regulatory protein moiety.
This invention also provides a method of making the
DNA constructs of this invention comprising the step of
joining a DNA sequence encoding a transcriptional
regulatory protein moiety as defined above with one or both
of a SA and a SD. Preferably, at least three such DNA
constructs are made in three different reading frames.
This invention also provides cells comprising the DNA
constructs of this invention obtainable by the method of
transforming eucaryotic cells with one or more DNA
constructs of this invention.
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This invention also provides kits that comprise the
above-described DNA construct of this invention. The DNA
constructs may be in the form of plasmids. The kits may
also comprise host cells, two-hybrid vectors or reporter
gene constructs as described herein. The two-hybrid
vectors of the kit may be plasmids constructed
(eg. presence, of suitable restriction sites) to permit
insertion of a test protein sequence to be part of a
two-hybrid vector as described herein. The kits may also
comprise materials and reagents useful for DNA insertions,
reporter gene activity assays, or sequencing of inserts
( eg . primers ) .
This invention also provides host cells whose genome
optionally comprises a reporter gene as described herein
and wherein the cell expresses a two-hybrid vector as
described herein. The two-hybrid vector may include a
sequence encoding a test protein.
This invention also provides a method for detecting
interaction between an endogenous protein of a cell and a
test protein, wherein said cell contains a first DNA
sequence encoding a reporter under transcriptional control
of a transcriptional regulatory element, and a second DNA
sequence that is expressed by the cell and which encodes a
first hybrid protein comprising:
(a) a first transcriptional regulatory protein moiety
selected from the group consisting of: a DNA-BD
that recognizes a binding site on the
transcriptional regulatory element controlling
the first DNA sequence and, a AD functional in
the cell; and
(b) a test protein;
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wherein the method comprises the steps of:
(a) placing into the cell or an ancestor of the cell,
a DNA construct comprising one or more m-RNA
splice sites, and a third DNA sequence encoding
a second transcriptional regulatory protein
moiety which, when combined with the first
transcriptional regulatory protein moiety will
reconstitute a transcriptional regulatory protein
capable of binding to and activating the
transcriptional regulatory element controlling
transcription of the first DNA sequence; and,
(b) determining whether the reporter is expressed by
the cell or a descendant of the cell, as an
indicator of expression of a second hybrid
protein comprising the second transcriptional
regulatory protein moiety and an endogenous
protein of the cell capable of interaction with
the test protein.
The DNA construct comprising a third DNA sequence
described in the method above may be selected from the
following group, in which it is preferable that the only
protein encoded by the construct or the portion of the
construct described above, be the transcriptional
regulatory moiety itself:
(I) a gene trap vector comprising the third DNA
sequence to reconstitute a transcriptional
regulatory protein, followed by a SD; and
preferably, a transcriptional regulatory element
operably linked to the third DNA sequence;
(II) a gene trap vector without a transcriptional
regulatory element and comprising a SA upstream
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of the third DNA sequence to reconstitute a
transcriptional regulatory protein; and
preferably, the third DNA sequence is followed by
a poly-adenylation signal; and
(III) a gene trap vector comprising the third DNA
sequence to reconstitute a transcriptional
regulatory protein, with an upstream SA and a
downstream SD.
In embodiments of the method described above in which
the second DNA sequence encodes a DNA-BD that recognizes a
binding site on the transcriptional regulatory element
controlling the reporter, the DNA construct comprising the
third DNA sequence will encode an AD. Where the second DNA
sequence encodes an AD, the DNA construct will comprise a
DNA-BD capable of binding to the transcriptional regulatory
element controlling the reporter. When the second
nucleotide sequence is expressed in a cell in which the
third DNA sequence is also expressed (resulting in a hybrid
protein containing an endogenous portion that interacts
with the test protein) reconstitution of the
transcriptional regulatory protein occurs. Binding of the
latter protein by means of the DNA-BD to the
transcriptional regulatory element controlling the reporter
results in expression of the reporter.
In the method described above, the third DNA sequence
will preferably encode an AD, not a DNA-BD. This may
minimize false positives resulting from reconstitution of
a transcriptional regulatory protein when the third DNA
sequence is expressed with an exon that encodes an
endogenous protein that itself is capable of functioning as
an AD.
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This invention also provides a method for detecting an
endogenous transcription activator domain (AD) of a cell,
wherein the cell contains a first DNA sequence encoding a
reporter under transcriptional control of a transcriptional
regulatory element, wherein the method comprises the steps
of
(a) placing into the cell or an ancestor of the cell,
a DNA construct comprising a m-RNA splice site
and a second DNA sequence encoding a DNA-BD that
recognizes a binding site on the transcriptional
regulatory element controlling transcription of
the first DNA sequence; and
(b) detecting expression of the reporter in the cell
or a descendant of the cell, as an indicator of
expression of a hybrid protein comprising the
DNA-BD and an endogenous protein of the cell
capable of functioning as an activator domain.
The DNA construct comprising a second DNA sequence as
used in the above-described method for detecting an
endogenous transcription activator domain may be selected
from the following group in which it is preferred that the
only protein encoded by the construct or the portion of the
construct described above, be the transcriptional
regulatory moiety itself:
(IV) a gene trap vector comprising the second DNA
sequence, followed by a SD; and preferably, a
transcriptional regulatory element is operably
linked to the second DNA sequence;
(V) a gene trap vector without a transcriptional
regulatory element and comprising a SA upstream
of the second DNA sequence; and preferably, the
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second DNA sequence is followed by a
poly-adenylation signal; and
(VI) a gene trap vector comprising the second DNA
sequence, with an upstream SA and a downstream
SD.
Detailed Description of the Invention
The terms "host cell" and "cell" are used
interchangeably herein. It is understood that such terms
refer not only to the particular subject cell but to the
progeny or potential progeny of such a cell. Because
certain modifications may occur in succeeding generations
due to either mutation or environmental influences, such
progeny may not, in fact, be identical to the parent or
ancestor cell, but are still included within the scope of
the terms as used herein. A cell as used in the method of
this invention is a eukaryotic cell.
A "DNA construct" is a deoxynucleic acid (DNA)
molecule, either single- or double-stranded, that has been
modified through human intervention to contain segments of
DNA combined and juxtaposed in an arrangement not existing
in nature.
A "reporter" as used herein, may refer to a
polynucleotide sequence (structural sequence) encoding a
reporter protein or the term may refer to the reporter
protein itself, depending upon the context.
The term "operably linked" is intended to mean that a
DNA sequence is linked to a regulatory sequence in a manner
which allows expression of the DNA sequence. Such a
regulatory sequence includes promoters, enhancers and other
expression control elements.
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The terms "polypeptide", "peptide" and "protein" as
used herein refer to a polymer of amino acid residues.
The term "endogenous" refers to that which is produced
or arises from within a cell or organism.
The term "plasmid" refers to a circular, double
stranded, extrachromosomal bacterial DNA into which
additional DNA segments may be ligated and which replicates
automatically. Methodologies for selection and
construction of vectors, plasmids and DNA constructs may be
found, for example in: Molecular Clonina: A Laboratory
Manual; (2d), Sambrook et al. 1989, Cold Spring Harbor
Laboratory Press. Suitable host cells are discussed
further in Goeddel; "Gene Expression Technology" in:
Methods in Enzymology 185, Academic Press, San Diego,
California (1990) .
In the present invention, DNA constructs are
introduced into a host cell and expressed in the host cell
in sufficient quantities for a reporter gene to be
activated. The host cell may be any eukaryotic cell,
including yeast, zebrafish, C. elegans, Drosophila and
mammalian cells having a genome one would like to screen
for interactive protein encoding exons or AD encoding
exons.
The host cell is constructed to contain and ultimately
express a reporter gene having a transcription regulatory
element known to include a binding site for the DNA-BD to
be employed. The reporter gene product produces a
detectable signal when the reporter gene is
transcriptionally activated. Thus, a reporter is a moiety
whose transcription is detectable, or which expresses a
detectable protein or a protein the expression of which may
otherwise be determined by monitoring an effect of
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expression of the protein. Examples of reporter gene
products that are readily detectable are well-known and
include: ~i-galactosidase, green fluorescent protein,
luciferase, alkaline phosphatase, and chloramphenicol
acetyl transferase (CAT) as well as other enzymes and
proteins that are also known as selectable markers. Other
examples of detectable signals include cell surface markers
such as CD4. In the exemplified embodiment, the reporter
gene used is the pac gene which encodes the puromycin
resistance marker.
In yeast cells, the reporter gene may be homologous
the yeast URA3 gene, the yeast CAN1 gene, the yeast GAL1
gene, the yeast HIS3 gene, or the E_. coli LacZ gene . In
mammalian cells, the reporter gene may be homologous to the
CAT gene, the LacZ gene, the SEAP gene, the Luciferase
gene, the GFP gene, the BFP gene, the CD2 gene, the Flu HA
gene, or the tPA gene.
The reporter gene in the host cell will be driven by
a transcriptional regulatory element (including promoters
and enhancers) that is capable of binding the DNA-BD
employed in the assay and is functional in the host cell.
Many examples of suitable regulatory elements are well-
known, particularly, promoters including those described
below.
The assay may make use of host cells in which the
reporter gene has been previously incorporated, or a
construct containing the reporter gene may be introduced to
the cell at the same time as other vectors used in the
assay.
Other vectors used in the assay include a gene trap
vector and a two-hybrid vector. The gene-trap vector is
employed for random insertion of a transcriptional
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regulatory protein moiety into the genome of the host cell
and may comprise DNA encoding either a AD or a DNA-BD and
either: an upstream splice acceptor (SA); or, an upstream
transcriptional regulatory element (eg. a promoter) capable
of functioning in the host cell for transcription of the
downstream AD or DNA-BD which in turn is followed by an
unpaired splice donor sequence (SD). In an alternate
embodiment, the gene trap vector has both an upstream SA
and a downstream SD.
Incorporation of the gene trap vector within an intron
will permit processing of a chimeric message comprising a
transcript of a flanking endogenous exon joined to the
transcript for the DNA-BD or AD. Use of a gene trap vector
having a downstream SD and an upstream promoter is
preferred since transcription of the chimeric message will
not be dependent upon endogenous expression of the host
cell gene.
A splice donor (SD) is defined as a nucleotide moiety
having an ability to effect m-RNA splicing to a splice
acceptor site. Conversely, a splice acceptor (SA) is
defined by its ability to effect mRNA splicing to a splice
donor site. Generally, an unpaired splice donor includes
the 3' end of an exon and the 5' end of an intron, and a
splice acceptor includes the 3' end of an intron and the 5'
end of an exon (eg. as defined by Alberts, B. et al.,
at page 373 of Molecular Biolocrv of the Cell (1994) , (3d)
Garland Publishing, N.Y. Sequences that may be used as
splice acceptors and donors are known and include the
examples of SA and SD sequences as set out in the Examples
herein.
The two-hybrid vector will comprise an upstream
transcriptional regulatory element (eg. a promoter) capable
of a functioning in the host cell and driving transcription
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of a sequence intended to reconstitute the transcriptional
regulatory protein. Thus, the two-hybrid vector will
express either a DNA-BD or a AD as the case may be,
depending upon the makeup of the gene trap vector.
Preferably, the two-hybrid vector will express DNA-BD. The
two-hybrid vector also contains a nucleotide sequence which
is under the control of the regulatory element and which
encodes a selected protein (including a peptide or a
polypeptide) of interest (test protein) in respect of which
protein-protein interactions are to be determined.
Expression of the two-hybrid vector in the host cell
results in the translation of a chimeric protein comprising
the transcriptional regulatory protein moiety (eg. DNA-BD)
fused with the test protein. Incorporation of the gene
trap vector into a gene encoding a protein capable of
interaction with the selected protein will result in
production in the cell of .a reconstituted transcription
regulatory protein via interaction of the test protein and
the protein product of the trapped gene. Activation of the
reporter gene occurs as a result of binding of the DNA-BD
to the reporter gene promoter.
Reference herein to "interaction" of proteins (such as
an endogenous protein with a test protein) means any
interaction whereby proteins tend to be associated in
proximity. Such interaction includes any known form of
chemical bonding occurring between proteins that are found
to be interacting.
In an alternate embodiment used for detecting exons
encoding endogenous transcription activator domains
(protein capable of functioning as an AD) , the gene trap
vector comprising a DNA-BD is used without a two-hybrid
vector. When the gene trap vector integrates into a gene
containing an exon that encodes a protein capable of
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functioning as an AD in the cell, the resulting gene
product is a chimeric protein that j oins both the DNA-BD
coded for by the vector DNA and the AD coded for by the
endogenous exon. Thus, a transcriptional regulatory
protein is constituted, capable of activating the reporter
gene in the cell.
A DNA-BD and a AD employed in DNA constructs for use
in this invention may be derived from a single known
transcriptional regulatory protein having separate
DNA-binding and transcriptional activation domains (for
example, the yeast GAL4 and GEN4 proteins). Alternatively,
the DNA-BD and AD moieties may be derived from separate
known sources. For example, the DNA-BD may be derived from
LexA in E_. coli. The DNA-BD may be from DNA binding
proteins other than activators (eg. repressers). The AD
could be derived from amino acids 147-238 of GAL4. The
moieties may also be synthetic, such as the B42 activation
domain. Preferably, the DNA-BD and the AD are from
different proteins. In any case, the DNA-BD should not be
capable of functioning significantly as an activator domain
on its own and the AD should not be capable of binding to
the promoter of the reporter gene.
In the exemplified embodiment, the DNA-binding domain
is derived from the N-terminal region of the yeast GAL4
protein (eg. amino acids 1-147) and the transcriptional
activation domain is derived from the transcriptional
activator of Herpes Simplex Virus VP16 (eg. amino
acids 411-455 of VP16) which is known not bind to DNA but
will function as a transcriptional activator.
The reporter gene may be present in the genome of the
host cell at the time of introduction of the first and/or
second DNA constructs. Alternatively, a construct
comprising the reporter gene may be introduced into the
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host cell genome at the same time as the first and/or
second DNA constructs. Also, further DNA constructs to be
used in this invention may be introduced to the cell and
made part of the host cell genome before further constructs
are introduced, or such constructs may be introduced at the
same time.
DNA constructs, plasmids and the like, as used in this
invention can be delivered or placed in cells in vivo using
methods known in the art and the methods referred to in the
Examples herein. Such methods include direct injection of
DNA, receptor-mediated DNA uptake or viral-mediated
transfection. Direct injection has been used to introduce
named DNA into cells in vivo (see eg. Acsadi et al. (1991)
Nature 332:815-818; Wolff et al. (1990) Science 247:1465-
1468). A delivery apparatus (eg. a "gene gun") for
injecting DNA into cells in vivo can be used. Such an
apparatus is commercially available (eg. from BioRad).
Naked DNA can also be introduced into cells by complexing
the DNA to a cation, such as polylysine, which is coupled
to a ligand for a cell-surface receptor (see for example
Wu, G. and Wu, C.H. (1998) J. Biol. Chem. 263:14621; Wilson
et al. (1992) J. Biol. Chem. 267:963-967; and U.S. Pat.
No. 5,166,320). Binding of the DNA-ligand complex to the
receptor facilitates uptake of the DNA by receptor-mediated
endocytosis. Additionally, a DNA-ligand complex linked to
adenovirus capsids which naturally disrupt endosomes,
thereby releasing material into the cytoplasm can be used
to avoid degradation of the complex by intracellular
lysosomes (see for example Curiel et a1. (1991) Proc. Natl.
Acad. Sci. USA 88:8850; Cristiano et al. (1993) Proc Natl.
Acad. Sci. USA 90:2122-2126).
Endogenous genes into which the gene trap vector has
integrated may be cloned and sequenced, for example by the
5' RACE method of PCR (polymerase chain reaction).
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Furthermore, undifferentiated embryonic stem (ES) cells can
be further used to generate mice mutated from the
endogenous gene. Heterologous DNA can be inserted into the
site of the endogenous gene by known methods including
homologous recombination and site directed to
recombination.
5' Rapid PCR amplification of cDNA ends (RACE) may be
carried out (for example, as described by Skarnes, et a1.
at (1992) Genes and Development 6, 903-918) to clone a
portion of the endogenous gene flanking a gene trap vector
insertion. This provides fragments for sequencing and to
probe for genes. The source of reagents may be the 5' RACE
kit commercially available from Gibco-BRL.
Examples of ES cell lines which may be used in this
invention are: porcine (eg. U.S. Patent 5523226 Transgenic
Swine Compositions and Methods); murine (eg. D3, R1, CGRB,
AB1 ES cell lines); primate (eg. rhesus monkey); rodent;
marmoset; avian (eg. chicken); bovine; rabbit; sheep; and
horse.
Murine R1 ES cells from A. Nagy [Proc. Nat. Acad. Sci.
U.S.A. (1993) 90, 8424-8428] may be grown on Primary
Embryonic Fibroblast feeder layers or on gelatinized dishes
in the presence of 1000 U/ml murine leukemia inhibitory
factor (LIF), ESGRO~" (GIBCO BRL). Selection conditions can
be: 150 ~,g/ml 6418, 1.0 ~g/ml puromycin, 110 ~.g/ml
Hygromycin B. R1 cells (eg. 2 x 10' cells) may be
electroporated with, for example, 100 ~,g linearized DNA in
0.8 ml PBS at 500 ~.F and 240 V with a BioRad Gene Pulserr"'
at room temperature.
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Examt~le I: Protein Interaction Trap
This aspect of the invention may be conveniently
practiced by modification of standard commercial two-hybrid
assay components. In the following example, the Clontech
Mammalian MatchmakerT"" two-hybrid assay kit is modified and
supplemented to provide a reporter gene as a selectable
marker (pac) for puromycin resistance; a DNA-BD from GAL4
(as provided in the commercial kit); and an AD from Herpes
Simplex Virus VP16 (as provided in the kit). In this
example, all DNA constructs, including the reporter gene
are introduced into a murine R1 ES cell line host cell.
The first DNA construct (two-hybrid vector) comprises
a sequence encoding a GAL4 DNA-BD which recognizes a
binding site on the reporter gene and further comprises a
sequence encoding p53 protein (Clontech, pM-53 plasmid).
The second DNA construct (gene trap vector) is novel
and comprises a promoter capable of operation in the host
cell, driving a VP16 AD upstream of a splice donor
sequence. In an alternate embodiment, the novel gene trap
vector does not contain a promoter and has a splice
acceptor sequence upstream of the VP16 AD followed by a
poly-adenylation signal.
When the gene trap is integrated into an intron
adjacent to an exon of the host cell encoding a protein
domain capable of interaction with p53 protein, a
transcriptional regulatory protein comprising GAL4 BD and
the VP16 AD is constituted. Expression of the reporter
gene in a host cell as a result of binding by the DNA-BD is
detected by culturing the transformed cells in the presence
of puromycin. Cells in which the reporter gene has been
activated will survive. Alternatively, the reporter used
in the assay could remain as CAT and determination of
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reporter gene activity may be carried out according to
standard assay procedures, for example as taught in the
Clontech kit instructions.
Host cells are transformed by any of the well-known
methods, selected as being suitable for the particular cell
type. Electroporation or calcium phosphate mediated
transfection are suitable for mammalian cells.
Transfection procedures as taught in the Clontech kit
instructions may be used. A preferred method known for ES
cells is electroporation.
The following plasmids are constructed and/or employed
in this example. The reporter (pGSPuro) is a modified
version of the GAL4 responsive CAT reporter construct from
the Clontech MatchmakerT"" kit (pG5CAT) . In this example,
the CAT reporter gene is replaced by the selectable marker
pac, generating a reporter construct containing the
puromycin resistance gene under the control of the
adenovirus Elb minimal promoter used in the Clontech
plasmid. Upstream, are five copies of the 17 nucleotide
consensus GAL4 binding site (galactose upstream activating
sequence : UAS~) .
The second plasmid is the pM-53 vector from the
Matchmaker'"' kit which is an expression plasmid containing
the SV40 promoter driving a GAL4 DNA-BD. The commercial
construct encodes p53 protein, but the multiple cloning
site downstream from the DNA-BD may be used to insert
different bait proteins. This functions as the two-hybrid
vector.
A gene trap vector plasmid is constructed by inserting
an oligomer sequence encoding a consensus SD sequence in
frame into a SalI/BspMI digested pVPl6 plasmid (Clontech)
simultaneously deleting the stop codons and
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poly-adenylation signal. Thus, a gene trap vector is
generated comprising an SV40 promoter driving expression of
the AD. Three versions of this vector were created
resulting in splicing in all three potential reading
frames. The following are examples of consensus SD
sequences:
AGGTAAGT (SEQ ID N0:1)
AGGTGAGT (SEQ ID N0:2)
each of which may be preceded by C or A.
An alternate gene trap vector plasmid may be
constructed containing the VP16 AD downstream of a SA
sequence. Three constructs should be generated, each
resulting in splicing in each of three possible reading
frames. SA sequences comprise a polypyrimidine tract
followed by a nucleotide, T or C, AG, and at least G or A.
Examples are the murine En-2 splice acceptor and the splice
acceptors from human (3-globin and rabbit b-globulin.
The following methods may be used for construction of
VP16 gene trap vectors:
(I) To construct the gene trap vector consisting of
the SV40 promoter driving the expression of VP16
fused to an unpaired splice donor sequence:
(a) Digest pVPl6 (Clontech) with SalI and BspMI;
(b) Isolate and purify the 3.0 kb fragment;
(c) Ligate the 3.0 kb pVPl6 fragment with each
of the following pairs of oligomers to
create fusions of VP16 with unpaired splice
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donor sequences in all three possible
reading frames:
Pair #l: 5' tcgacaggtaagt 3' (SEQ ID N0:3)
5' tcatacttacctg 3' (SEQ ID N0:4)
Pair #2 5' tcgaccaggtaagt 3' (SEQ ID N0:5)
5' tcatacttacctgg 3' (SEQ ID N0:6)
Pair #3 5' tcgacccaggtaagt 3' (SEQ ID N0:7)
5' tcatacttacctggg 3' (SEQ ID N0:8)
(II) To construct an alternate gene trap vector
comprising the En-2 SA sequence fused 5' of the
VP16 transcriptional activator:
(A) (1) digest pGT4SA vector (Gossler et al.
1989 Science 244:463-465) with Xbal;
(2) fill in ends with T4 DNA polymerise to
generate blunt ends;
(3) digest with NdeI; and
(4 ) Isolated and purify the 2 . 0 kb fragment
encoding the En-2 splice acceptor
sequence.
(B) (1) digest pVPl6 (Clontech) with NheI;
(2) fill in ends with T4 DNA polymerise to
generate blunt end;
(3) digest with NdeI; and
(4) isolated and purify the 2.8 kb fragment
encoding the VP16 transcriptional
activator sequence.
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(C) Ligate 2.0 kb En-2 splice acceptor fragment
to 2.8 kb VP16 containing vector.
(D) To generate SA-VP16 in the other two
potential reading frames:
(1) digest the above vector with SexAI and
BglII;
(2) ligate the following pairs of oligomers
to generate fusions in the other two
possible reading frames:
Pair #1 5' ccaggtcgca 3' (SEQ ID N0:9)
5' gatctgcga 3' (SEQ ID NO:10)
Pair #2 5' ccaggtgca 3' (SEQ ID NO:11)
5' gatctgca 3' (SEQ ID N0:12)
The three forms of the gene trap vector representing
all three potential reading frames are placed in a head to
tail tandem array allowing the use of alternate promoters
to generate three hybrid mRNAs fusing the VP16 domain in
all three possible reading frames to a adjacent exon upon
integration into a gene within the host cell genome.
The following protocol may be followed:
1. Construct a reporter murine embryonic stem (ES) cell
line using standard methods by co-electroporation of
linearized pGSPuro, pM-53 and pPGKHyg into the murine RI ES
cell line. Hygromycin resistance is used to monitor
transfection efficiency.
2. Characterize the reporter cell lines for its ability
to detect protein-protein interactions by electroporating
with pVPl6T (Clontech) as a positive control and pVPl6-CP
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(Clontech) as a negative control for protein-protein
interaction. pVPl6T expresses a fusion of the VP16
activation domain to the SV40 large T antigen, which is
known to interact with p53. The pVPl6-CP negative control
plasmid expresses a fusion of the VP16 activation domain to
a viral coat protein, which does not interact with p53.
3. Upon electroporation of positive or negative control
plasmids, cells are then placed under 1.0 ug/ml puromycin
selection.
4. Select appropriate reporter cell clones that confer
puromycin resistance in the presence of VP16T but not with
pVPl6-CP (cells express pGSPuro and pM-53).
5. Electroporate gene trap vectors into reporter cell
line and select for puromycin resistance with 1.0 ug/ml
puromycin.
6. Pick individual puromycin resistant colonies and
isolate RNA from each clone.
7. Isolate and sequence trapped exon/gene by rapid
amplification of cDNA end (RACE) PCR (eg. see: Skarnes,
et a1. 1992. Genes and Development 6:903-18). Clontech
sequencing primers for VP16 may be used.
Example II: Transcriptional Activator Domain Trap
In this example, the methods employed in the preceding
example are used in an assay employing the ES host cell,
the same reporter gene construct (pGSCAT) employed in the
preceding example, and a gene trap vector plasmid designed
to trap genes expressing endogenous protein capable of
functioning as a transcriptional activator domain (AD) in
conjunction with the DNA-BD expressed by the gene trap
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vector. Expression of chimeric proteins comprising the
DNA-BD fused to an endogenous protein capable of
functioning as a AD will result in activation of the
reporter gene which comprises a binding site for the
DNA-BD.
The gene trap vector plasmid is constructed by
inserting an oligomer sequence encoding the consensus SD
sequence in frame into the SalI/BspMI digested pM plasmid
(Clontech) resulting a vector comprising of the SV40
promoter driving the GAL4 DNA-binding domain linked to a SD
sequence. Three versions of this vector are created
resulting in splicing in each of the three potential
reading frames, respectively. A consensus splice donor
sequence domain contains the following:
Exon....AGGTAAGT...Intron (SEQ ID NO: l)
To construct the vector consisting of the SV40
promoter driving the expression of the GAL4 DNA binding
domain fused to an unpaired splice donor sequence:
(a) digest pM (Clontech) with SalI and BspMI;
(b) isolate and purify the 3.2kb fragment; and
(c) ligate the 3.2 kb pVPl6 fragment with each of the
following pairs of oligomers to create fusions of
VP16 with SD sequences in all three possible
reading frames:
Pair #1 5' tcgacaggtaagt 3' (SEQ ID N0:3)
5' tcatacttacctg 3' (SEQ ID N0:4)
Pair #2 5' tcgaccaggtaagt 3'(SEQ ID N0:5)
5' tcatacttacctgg 3'(SEQ ID N0:6)
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Pair ##3 5' tcgacccaggtaagt 3' (SEQ ID N0:7}
5' tcatacttacctggg 3' (SEQ ID N0:8)
The three forms of the gene trap are then placed in a
head-to-tail tandem array allowing the use of alternative
promoters to generate three hybrid mRNAs fusing the GAL4
DNA domain in all three possible reading frames to the next
endogenous exon upon integration into a gene within the
genome.
io
The following protocol may be used:
1. Construct a reporter murine embryonic stem (ES) cell
line using standard methods by co-electroporation of
linearized pGSPuro, and pPGKHyg into the murine Rl ED cell
line.
2. Select, and expand several clones which contain
pGSPuro.
3. Characterize the reporter cell line for ability to
express transcriptional activator domains by
electroporating with pM3-VP16 (Clontech) as a positive
control and pM-53 (Clontech) as a negative control for
transcriptional activator domains. pM3-VP16 expresses a
fusion of the VP16 activation domain to the GAL4 DNA
binding domain which is known transactivate the GAL4
responsive promoter in pGSPuro. The pm-53 negative control
plasmid expresses a fusion of the VP16 activation domain to
p53, which does not transactivate the GAL4 responsive
promoter in pGSPuro.
4. Upon electroporation of positive or negative control
plasmids, cells are then placed under 1.0 ug/ml puromycin
selection.
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5. Select appropriate reporter cell clones that confer
puromycin resistance in the presence of pM3-VP16 but not
with pM-53.
6. Electroporate gene trap vector into reporter cell line
and select puromycin resistance with 1.0 ug/ml puromycin.
7. Pick individual puromycin resistant colonies and
isolate RNA from each clone.
8. Isolate and sequence trapped exon/gene by rapid
amplification of cDNA ends (RACE-PCR).
All publications and patents cited in this
specification are incorporated herein by reference.
Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of
clarity of understanding, it will be readily apparent to
those of ordinary skill in the art in light of the
teachings of this invention that changes and modification
may be made thereto without departing from the spirit or
scope of the appended claims.
