U.S. patent application number 10/350799 was filed with the patent office on 2004-01-29 for inducible methods for repressing gene function.
Invention is credited to Moqtaderi, Zarmik, Struhl, Kevin.
Application Number | 20040018625 10/350799 |
Document ID | / |
Family ID | 22017025 |
Filed Date | 2004-01-29 |
United States Patent
Application |
20040018625 |
Kind Code |
A1 |
Struhl, Kevin ; et
al. |
January 29, 2004 |
Inducible methods for repressing gene function
Abstract
Methods for the rapid repression of gene function in eucaryotic
cells are disclosed including inducible means for both shutting
down a targeted gene's transcription and rapidly removing a
targeted gene's polypeptide product.
Inventors: |
Struhl, Kevin; (Weston,
MA) ; Moqtaderi, Zarmik; (Boston, MA) |
Correspondence
Address: |
FOLEY HOAG, LLP
PATENT GROUP, WORLD TRADE CENTER WEST
155 SEAPORT BLVD
BOSTON
MA
02110
US
|
Family ID: |
22017025 |
Appl. No.: |
10/350799 |
Filed: |
January 24, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10350799 |
Jan 24, 2003 |
|
|
|
09508400 |
Sep 19, 2000 |
|
|
|
6576469 |
|
|
|
|
09508400 |
Sep 19, 2000 |
|
|
|
PCT/US98/19026 |
Sep 10, 1998 |
|
|
|
60058474 |
Sep 10, 1997 |
|
|
|
Current U.S.
Class: |
435/455 ;
514/152 |
Current CPC
Class: |
C12N 15/635 20130101;
C07K 14/00 20130101; C07K 2319/00 20130101; C12N 2830/702 20130101;
C12N 15/67 20130101; C12N 2830/005 20130101; C12N 15/85 20130101;
C12N 2830/002 20130101 |
Class at
Publication: |
435/455 ;
514/152 |
International
Class: |
C12N 015/85; A61K
031/65 |
Claims
We claim:
1. A method for inducibly repressing the transcription of a target
gene expressed from a repressible promoter comprising: providing
said target gene expressed from said repressible promoter,
providing a transcriptional repressor expressed from an inducible
promoter, which transcriptional repressor represses said
repressible promoter, and providing an inducing agent that induces
said inducible promoter in order to express said transcriptional
repressor and repress said repressible promoter, thereby causing
the transcriptional repression of said target gene.
2. The method of claim 1, wherein the target gene encodes a
chimeric polypeptide comprising ubiquitin and a target
polypeptide.
3. The method of claim 1, wherein the repressible promoter is a
promoter from a gene selected from the group consisting of ANB1,
HEM13, ERG 11, OLE 1, GAL1, GAL10, and TET.sup.R.
4. The method of claim 1, wherein the transcriptional repressor is
selected from the group consisting of ROX1, Tet repressor, and lacI
repressor.
5. The method of claim 1, wherein the inducible promoter is a
copper-inducible promoter.
6. The method of claim 1, wherein the inducible promoter is induced
by Cu.sup.+2, tetracycline, or a gratuitous inducer of the lac
operon.
7. A method for inducibly degrading a target polypeptide wherein
said target polypeptide is provided as a ubiquitin-target
polypeptide fusion protein in which a specific amino terminal amino
acid residue of said target polypeptide is contiguously joined by a
peptide bond to a carboxyl terminal residue of a ubiquitin
polypeptide, said method comprising: providing said
ubiquitin-target polypeptide fusion protein, providing a ubiquitin
isopeptidase which endoproteolytically cleaves said peptide bond
thereby liberating said specific amino terminal amino acid residue
of said target polypeptide from said carboxyl terminal residue of
said ubiquitin polypeptide, providing an inducible means for the
proteolytic destruction of said target polypeptide possessing said
liberated amino terminal amino acid residue, and inducing said
inducible proteolytic means thereby causing the inducible
proteolytic destruction of said target polypeptide possessing said
liberated amino terminal amino acid residue.
8. The method of claim 7, wherein the target polypeptide is a
TAF.
9. The method of claim 7, wherein the specific amino terminal amino
acid residue of said target polypeptide is arginine.
10. The method of claim 7, wherein the specific amino terminal
amino acid residue of said target polypeptide is selected from the
group consisting of arginine, lysine and histidine.
11. The method of claim 7, wherein the specific amino terminal
amino acid residue of said target polypeptide is selected from the
group consisting of phenylalanine, tryptophan, tyrosine, leucine,
and isoleucine.
12. The method of claim 7, wherein the specific amino terminal
amino acid residue of said target polypeptide is selected from the
group consisting of aspartate, glutamate, cysteine, asparagine and
glutamine.
13. The method of claim 12, wherein the inducible means for the
proteolytic destruction of the target polypeptide is an inducible
transgene encoding an R-transferase.
14. The method of claim 12, wherein the specific amino terminal
amino acid residue of said target polypeptide is glutamine or
asparagine and the inducible means for the proteolytic destruction
of the target polypeptide is an inducible transgene encoding a
deamidase specific for an amino-terminal glutamine or
asparagine.
15. The method of claim 7, wherein said inducible proteolytic means
is an inducible transgene encoding a component of the N-end rule
system for ubiquitin dependent proteolytic destruction.
16. The method of claim 15, wherein said component of the N-end
rule system is selected from the group consisting of UBR1, UBC2,
NTA1, and ATE1.
17. The method of claim 15, wherein said component of the N-end
rule system is selected from the group consisting of mouse UBR1p
and human UBR1p.
18. A method for repressing the function of a target gene
expressing a target polypeptide by repressing the transcription of
said target gene and repressing the stability of said target
polypeptide, said method comprising: inducibly repressing the
transcription of said target gene by the method of claim 1, or
inducibly repressing the stability of said target polypeptide by
the method of claim 7 thereby repressing the function of said
target gene by repressing the transcription of said target gene and
repressing the stability of said target polypeptide.
19. A method for repressing the function of a target gene
expressing a target polypeptide by repressing the transcription of
said target gene and degrading said target polypeptide, said method
comprising: inducibly repressing the transcription of said target
gene, and inducibly degrading said target polypeptide by the method
of claim 7 thereby repressing the function of said target gene by
repressing the transcription of said target gene and degrading said
target polypeptide.
20. A method for repressing the function of a target gene
expressing a target polypeptide by repressing the transcription of
said target gene and degrading said target polypeptide, said method
comprising: inducibly repressing the transcription of said target
gene, and inducibly degrading said target polypeptide thereby
repressing the function of said target gene by repressing the
transcription of said target gene and degrading said target
polypeptide.
21. A eukaryotic cell containing: a target gene encoding a target
polypeptide an inducible transgene encoding proteolytic means for
the degradation of a target polypeptide encoded by a target
gene.
22. The eukaryotic cell of claim 21, wherein said inducible
transgene comprises a component of the N-end rule proteolytic
system.
23. The eukaryotic cell of claim 22, wherein said component of the
N-end rule proteolytic system is selected from the group consisting
of UBR1, UBC2 and NTA1.
24. The eukaryotic cell of claim 21, in which said target gene is
expressed from a repressible promoter.
25. The eukaryotic cell of claim 24, further containing an
inducible transcriptional repressor, which transcriptional
repressor represses said repressible promoter.
26. The eukaryotic cell of claim 25, wherein said repressible
promoter is a promoter from a gene selected from the group
consisting of ANB1, HEM13, ERG11, OLE1, GAL1, GAL10, and
TET.sup.R.
27. The eukaryotic cell of claim 25, wherein said transcriptional
repressor is selected from the group consisting of ROX1, Tet
repressor, and lacI repressor.
28. A target gene chimera comprising a ubiquitin polypeptide and a
target polypeptide.
29. The target gene chimera of claim 28, further comprising a
specific amino terminal amino acid residue of said target
polypeptide wherein said specific amino terminal amino acid residue
is contiguously joined by a peptide bond to a carboxyl terminal
reside of said ubiquitin polypeptide.
30. The target gene chimera of claim 29, in which said specific
amino terminal amino acid residue is arginine.
31. The target gene chimera of claim 29, in which said specific
amino terminal amino acid residue is selected from the group
consisting of arginine, lysine and histidine.
32. The target gene chimera of claim 29, in which said specific
amino terminal amino acid residue is selected from the group
consisting of phenylalanine, tryptophan, tyrosine, leucine, and
isoleucine.
33. The target gene chimera of claim 29, in which said specific
amino terminal amino acid residue is selected from the group
consisting of aspartate, glutamate, cysteine, asparagine and
glutamine.
34. The target gene chimera of claim 29, further comprising an
epitope tag.
35. The target gene chimera of claim 29, further comprising a
segment of the lacI repressor carboxyl to said specific amino
terminal amino acid residue.
36. The target gene chimera of claim 29, further comprising a
lysine residue carboxyl to said specific amino terminal amino acid
residue.
37. A eukaryotic cell expressing the target gene chimera of claim
29.
38. The target gene chimera of claim 29, wherein said target
polypeptide is a transcriptional repressor which represses an
inducible promoter.
39. The method of claim 1, wherein the inducible promoter is
repressed by a derepressible repressor and wherein said
derepressible repressor is further subject to targeted proteolysis
by the N-end rule system.
Description
1. BACKGROUND OF THE INVENTION
[0001] Genetics is essentially an approach to understanding
biological processes through the systematic elimination of gene
function. Historically, the genetic approach has involved the
development of "screens" for mutations in genes which affect a
specific phenotypic trait of an organism. The great advantage of
this approach has been that no prior knowledge of the molecular
nature of the genes involved is required because the "screen"
identifies the affected genes by marking them with mutations. The
mutation involved is frequently a change in the gene's sequence
which results in a loss-of-function of the encoded gene product.
Unfortunately the genetic approach has many limitations. Indeed the
study of essential genes, required for cell viability, is
exceedingly difficult using a purely genetic approach. As the
famous molecular biologist David Botstein once put it, "death is
not a phenotype;" an expression which encapsulates the frustration
of attempting to study the function of essential genes. The
implementation of "reverse genetics" in yeast (see e.g. Winston et
al. (1983) Methods Enzymol 101: 211-28) and, later, in mammals (see
e.g. Capecchi (1989) Science 244: 1288-92), has allowed the
positive identification of a gene as essential through the
inability to recover viable yeast haploid gene "knockout" spores or
homozygous recessive "knockout" mice. Nevertheless, the exact
biological processes in which the essential gene is involved are
difficult to determine due to the inability to isolate and/or study
the doomed knockout yeast spore or the inviable homozygous mouse
zygote. Thus the downstream effects on specific aspects of cell
function following removal of the essential gene product cannot be
readily determined using these traditional "knockout" studies.
Furthermore, while traditional gene "knockout" experiments may be
useful in demonstrating that a given gene is essential for the life
of the organism, they provide no data on precisely how important
the gene is or to what extent so-called "second-site suppressing"
mutations can arise which restore cell viability following the
removal of the essential gene. These considerations are important
in the selection of targets for the rational design of, for
example, antibiotic or chemotherapeutic pharmaceutical agents.
[0002] Others have endeavored to devise systems for the directed
inactivation of a specific target gene in a host eucaryotic cell.
For example, in an attempt to provide for a systematic means of
deriving temperature-sensitive conditional alleles of a given gene
target, Dohmen et al. have devised a temperature-sensitive "degron"
cassette that can be appended to any gene of interest and used to
render it thermosensitive (Dohmen et al. (1994) Science 263:
1273-6). This approach could thus be applied in theory to any
essential gene of interest. However, the generality with which the
thermosensitive degron can be successfully applied to specific gene
targets has yet to be determined and the necessity of relying upon
thermal induction for the resulting system is a major drawback.
Indeed, eucaryotic cells experience a transient heat-shock response
which can have profound effects on some cellular processes such as
transcription. Furthermore, the requirement for induction by heat
shock precludes useful application to mammalian transgenic animal
systems. Still other systems have been developed for the specific
targeted removal of a host gene. Notably the Cre/lox system (see
e.g. Sauer (1998) Mehods 14: 381-92) allows for the inducible
deletion of a specific target gene through the action of the Cre
site-specific DNA recombinase. Using this system, genetic switches
can be designed to target ablation of a target gene in a specific
tissue and at a specific time during development. One shortcoming
of this method is that, following recombinational deletion of the
targeted gene from the chromosome, the remaining mRNA and
polypeptide products of the gene may only slowly be titrated out of
the host cell through consecutive mitotic cell divisions and/or the
eventual turnover of the mRNA and polypeptide by cellular
ribonucleases and proteases. Thus it would be desirable to have a
more rapid means for directly inactivating specific target genes in
a host eucaryotic cell.
2. SUMMARY OF THE INVENTION
[0003] In general, the present invention provides a rapid and
effective means for inactivating target genes, including target
genes involved in important biological pathways. The invention also
provides a system for the rapid and reliable repression of gene
function regardless of whether the gene of interest is known or
suspected of being an essential gene.
[0004] In one aspect, the present invention provides multiple means
for the rapid and inducible elimination of gene function in a
controlled and reproducible manner in a population of otherwise
mitotically viable eucaryotic cell. The methods described include a
method for rapidly repressing the transcription of a target gene
through the action of an inducible repressor, a method for rapidly
removing the polypeptide product of a target gene through directed
proteolysis, and an integrated method in which both transcriptional
repression and directed proteolysis occurs. In one embodiment, the
method provides an inducible means for the passive removal of an
mRNA product of a target gene (i.e. new target gene mRNA synthesis
is blocked and the existing target gene mRNA is allowed to degrade
through the natural turnover of the remaining target mRNA). In
another embodiment, the method provides an inducible means for the
active removal of a polypeptide product of a target gene (i.e.
while new target gene polypeptide synthesis, or translation, is not
blocked per se, the existing target gene polypeptide product is
actively degraded by proteolysis). In yet another, preferred
embodiment, the first and second embodiments are "integrated"
thereby allowing an optimal rate at which gene function can be
eliminated by the simultaneous removal of both mRNA and polypeptide
products of the gene.
[0005] The present invention thus provides a method of determining
which genes represent effective targets for the design of
antibiotic and/or chemotherapeutic agents. In particular, an array
of essential genes can be screened to determine which are most
vital to cell viability using the method of the invention. For
example, essential genes which, when targeted for destruction by
this two-pronged inducible repression system, result in the
immediate death of the host cell, are likely to be effective
targets for antibiotic or chemotherapeutic agents designed to stop
cell growth. The present invention further provides a means of
genetically modifying a population of cells so as to render them
subject to killing by a normally benign inducing agent. This
modification provides a convenient way to terminate or attenuate
the physiological effects on a host organism of a population of
bioengineered cells which have been delivered to the host. In this
application, virtually any essential gene can be targeted for the
inducible repressional shut-off of the present invention. In still
other applications, a bioengineered cell population which produces
a specific physiologically active gene product could be designed so
that the gene product itself is subject to the inducible
repressional shut-off system. When such a bioengineered cell
population is introduced into a host, the delivery of the
physiologically active gene product produced by the bioengineered
cells can be adjusted throughout the lifetime of the host/cell
combination by administration of a benign inducing agent.
3. BRIEF DESCRIPTION OF THE FIGURES
[0006] FIG. 1 provides a generalized illustration of the essential
components of the method for inducible repression of a target gene.
The two prongs of the method are illustrated separately here for
clarity. Panel A diagrams the essential elements of the first prong
of the method--the inducible transcriptional shut-off of a target
gene. Panel B diagrams the essential elements of the second prong
of the method--the inducible degradation of the target
polypeptide.
[0007] FIG. 2 illustrates the manner in which a target gene is
modified to become newly susceptible to the two prongs of the
repression method. FIG. 2A illustrates the replacement of a native
target gene promoter (naPr) with a repressible promoter (rePr)
which is responsive to an inducible repressor. FIG. 2A thus
illustrates the modifications essential to make the target gene
susceptible to the inducible transcriptional shut-off prong. FIG.
2B illustrates the insertion of a ubiquitin coding sequence (Ub)
and a unique codon (X), destined to become the amino-terminal amino
acid residue of the target polypeptide, upstream of the target
polypeptide-encoding sequence, or target "ORF." An optional epitope
tag (Ep) marker for the target gene is also indicated in the
figure. FIG. 2B thus illustrates the modifications essential to
make the target gene susceptible to the inducible N-end rule
proteolytic effector.
[0008] FIG. 3 illustrates the generalized structure of a target
gene which has been modified so as to made susceptible to the
action of both the transcriptional shut-off prong and the directed
proteolysis prong of the method. This preferred embodiment of the
modified target gene can be readily transplaced into the eucaryotic
genome at the site of the native target gene by standard "knock-in"
technology as shown in Panel B.
4. DETAILED DESCRIPTION OF THE INVENTION
[0009] 4.1 General
[0010] The two prongs of the method are illustrated separately in
FIG. 1 for clarity, but it is understood that, in a preferred
embodiment, the two prongs are employed simultaneously to maximize
the speed and efficacy of the method. Panel A diagrams the
essential elements of the first prong of the method--the inducible
transcriptional shut-off of a target gene. Panel B diagrams the
essential elements of the second prong of the method--the inducible
degradation of the target polypeptide. Both prongs rely on the use
of an inducible promoter (inPr) to drive the inducible expression
of an effector of suppression. In Panel A, the effector of
suppression is a transcriptional repressor which is capable of
repressing transcription from a repressible promoter (rePr). In
panel B, the effector of suppression is a component of the N-end
rule pathway which effects the proteolytic destruction of
polypeptides possessing certain amino-terminal amino acid residues.
Both prongs further rely on the construction of a synthetic version
of the target gene which has been engineered from its native form
so as to be uniquely sensitized to the effectors of suppression
described above. As shown in FIG. 2A, the target gene can be
modified so that its native promoter is replaced by a natural or
synthetic promoter (rePr) which is subject to repression by the
inducible repressor. This alteration of the target gene potentiates
repression by the transcriptional shut-off prong of the method. As
shown in FIG. 2B, the target gene can also be modified so that its
encoded polypeptide (the target ORF--target open reading frame) is
fused in frame to the carboxy-terminal codon of a
ubiquitin-encoding sequence. Furthermore a unique amino-terminal
amino acid encoding codon is engineered at the point of fusion of
the two coding sequences--i.e. just downstream of ubiquitin's final
glycine codon and just before the target ORF.
[0011] 4.2 Definitions
[0012] The term "agonist", as used herein, is meant to refer to an
agent that mimics or upregulates (e.g. potentiates or supplements)
bioactivity of the protein of interest. An agonist can be a
wild-type protein or derivative thereof having at least one
bioactivity of the wild-type protein. An agonist can also be a
compound that upregulates expression of a gene or which increases
at least one bioactivity of a protein. An agonist can also be a
compound which increases the interaction of a polypeptide of
interest with another molecule, e.g, a target peptide or nucleic
acid.
[0013] "Antagonist" as used herein is meant to refer to an agent
that downregulates (e.g. suppresses or inhibits) bioactivity of the
protein of interest. An antagonist can be a compound which inhibits
or decreases the interaction between a protein and another
molecule, e.g., a target peptide, such as interaction between
ubiquitin and its substrate. An antagonist can also be a compound
that downregulates expression of the gene of interest or which
reduces the amount of the wild type protein present.
[0014] The term "allele", which is used interchangeably herein with
"allelic variant" refers to alternative forms of a gene or portions
thereof. Alleles occupy the same locus or position on homologous
chromosomes. When a subject has two identical alleles of a gene,
the subject is said to be homozygous for that gene or allele. When
a subject has two different alleles of a gene, the subject is said
to be heterozygous for the gene. Alleles of a specific gene can
differ from each other in a single nucleotide, or several
nucleotides, and can include substitutions, deletions, and/or
insertions of nucleotides. An allele of a gene can also be a form
of a gene containing mutations.
[0015] The term "cell death" or "necrosis", is a phenomenon when
cells die as a result of being killed by a toxic material, or other
extrinsically imposed loss of function of a particular essential
gene function.
[0016] "Biological activity" or "bioactivity" or "activity" or
"biological function", which are used interchangeably, for the
purposes herein means a catalytic, effector, antigenic or molecular
tagging function that is directly or indirectly performed by the
polypeptides of this invention (whether in its native or denatured
conformation), or by any subsequence thereof.
[0017] As used herein the term "bioactive fragment of a
polypeptide" refers to a fragment of a full-length polypeptide,
wherein the fragment specifically agonizes (mimics) or antagonizes
(inhibits) the activity of a wild-type polypeptide. The bioactive
fragment preferably is a fragment capable of interacting with at
least one other molecule, protein or DNA, with which a full length
protein can bind.
[0018] "Cells," "host cells" or "recombinant host cells" are terms
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 cell, but are still included within the
scope of the term as used herein.
[0019] A "chimeric polypeptide" or "fusion polypeptide" is a fusion
of a first amino acid sequence encoding one of the subject
polypeptides with a second amino acid sequence defining a domain
(e.g. polypeptide portion) foreign to and not substantially
homologous with any domain of a polypeptide. A chimeric polypeptide
may present a foreign domain which is found (albeit in a different
polypeptide) in an organism which also expresses the first
polypeptide, or it may be an "interspecies", "intergenic", etc.
fusion of polypeptide structures expressed by different kinds of
organisms.
[0020] The term "nucleotide sequence complementary to the
nucleotide sequence set forth in SEQ ID NO. x" refers to the
nucleotide sequence of the complementary strand of a nucleic acid
strand having SEQ ID NO. x. The term "complementary strand" is used
herein interchangeably with the term "complement". The complement
of a nucleic acid strand can be the complement of a coding strand
or the complement of a non-coding strand.
[0021] A "delivery complex" shall mean a targeting means (e.g. a
molecule that results in higher affinity binding of a gene,
protein, polypeptide or peptide to a target cell surface and/or
increased cellular or nuclear uptake by a target cell). Examples of
targeting means include: sterols (e.g. cholesterol), lipids (e.g. a
cationic lipid, virosome or liposome), viruses (e.g. adenovirus,
adeno-associated virus, and retrovirus) or target cell specific
binding agents (e.g. ligands recognized by target cell specific
receptors). Preferred complexes are sufficiently stable in vivo to
prevent significant uncoupling prior to internalization by the
target cell. However, the complex is cleavable under appropriate
conditions within the cell so that the gene, protein, polypeptide
or peptide is released in a functional form.
[0022] As is well known, genes or a particular polypeptide may
exist in single or multiple copies within the genome of an
individual. Such duplicate genes may be identical or may have
certain modifications, including nucleotide substitutions,
additions or deletions, which all still code for polypeptides
having substantially the same activity. Moreover, certain
differences in nucleotide sequences may exist between individual
organisms, which are called alleles. Such allelic differences may
or may not result in differences in amino acid sequence of the
encoded polypeptide yet still encode a polypeptide with the same
biological activity.
[0023] The terms "epitope" and "epitope tag", as used herein, are
meant to refer to any of various convenient molecular markers known
in the art, such as hemagluttinin or FLAG, so that the level of a
polypeptide can be confirmed in a Western blot using, for example,
a suitable anti-flu or anti-FLAG antibody.
[0024] The term "equivalent" is understood to include nucleotide
sequences encoding functionally equivalent polypeptides of or
functionally equivalent peptides having an activity of an
---protein such as described herein. Equivalent nucleotide
sequences will include sequences that differ by one or more
nucleotide substitutions, additions or deletions, such as allelic
variants; and will, therefore, include sequences that differ from
the nucleotide sequence of the ---gene shown in SEQ ID NOs:, due to
the degeneracy of the genetic code.
[0025] As used herein, the terms "gene", "recombinant gene" and
"gene construct" refer to a nucleic acid comprising an open reading
frame encoding a polypeptide of the present invention, including
both exon and (optionally) intron sequences.
[0026] A "recombinant gene" refers to nucleic acid encoding a
polypeptide and comprising--encoding exon sequences, though it may
optionally include intron sequences which are derived from, for
example, a chromosomal gene or from an unrelated chromosomal gene.
The term "intron" refers to a DNA sequence present in a given gene
which is not translated into protein and is generally found between
exons.
[0027] "Homology" or "identity" or "similarity" refers to sequence
similarity between two peptides or between two nucleic acid
molecules, with identity being a more strict comparison. Homology
and identity can each be determined by comparing a position in each
sequence which may be aligned for purposes of comparison. When a
position in the compared sequence is occupied by the same base or
amino acid, then the molecules are identical at that position. A
degree of homology or similarity or identity between nucleic acid
sequences is a function of the number of identical or matching
nucleotides at positions shared by the nucleic acid sequences. A
degree of identity of amino acid sequences is a function of the
number of identical amino acids at positions shared by the amino
acid sequences. A degree of homology or similarity of amino acid
sequences is a function of the number of amino acids, i.e.
structurally related, at positions shared by the amino acid
sequences. An "unrelated" or "non-homologous" sequence shares less
than 40% identity, though preferably less than 25% identity, with
one of the sequences of the present invention.
[0028] The term "interact" as used herein is meant to include
detectable interactions (e.g. biochemical interactions) between
molecules, such as interaction between protein-protein,
protein-nucleic acid, nucleic acid-nucleic acid, and protein-small
molecule or nucleic acid-small molecule in nature.
[0029] The term "isolated" as used herein with respect to nucleic
acids, such as DNA or RNA, refers to molecules separated from other
DNAs, or RNAs, respectively, that are present in the natural source
of the macromolecule. For example, an isolated nucleic acid
encoding one of the subject polypeptides preferably includes no
more than 10 kilobases (kb) of nucleic acid sequence which
naturally immediately flanks the gene in genomic DNA, more
preferably no more than 5 kb of such naturally occurring flanking
sequences, and most preferably less than 1.5 kb of such naturally
occurring flanking sequence. The term isolated as used herein also
refers to a nucleic acid or peptide that is substantially free of
cellular material, viral material, or culture medium when produced
by recombinant DNA techniques, or chemical precursors or other
chemicals when chemically synthesized. Moreover, an "isolated
nucleic acid" is meant to include nucleic acid fragments which are
not naturally occurring as fragments and would not be found in the
natural state. The term "isolated" is also used herein to refer to
polypeptides which are isolated from other cellular proteins and is
meant to encompass both purified and recombinant polypeptides.
[0030] The term "modulation" as used herein refers to both
upregulation (i.e., activation or stimulation (e.g., by agonizing
or potentiating)) and downregulation (i.e. inhibition or
suppression (e.g., by antagonizing, decreasing or inhibiting)).
[0031] The term "mutated gene" refers to an allelic form of a gene,
which is capable of altering the phenotype of a subject having the
mutated gene relative to a subject which does not have the mutated
gene. If a subject must be homozygous for this mutation to have an
altered phenotype, the mutation is said to be recessive. If one
copy of the mutated gene is sufficient to alter the genotype of the
subject, the mutation is said to be dominant. If a subject has one
copy of the mutated gene and has a phenotype that is intermediate
between that of a homozygous and that of a heterozygous subject
(for that gene), the mutation is said to be co-dominant.
[0032] The "non-human animals" of the invention include mammalians
such as rodents, non-human primates, sheep, dog, cow, chickens,
amphibians, reptiles, etc. Preferred non-human animals are selected
from the rodent family including rat and mouse, most preferably
mouse, though transgenic amphibians, such as members of the Xenopus
genus, and transgenic chickens can also provide important tools for
understanding and identifying agents which can affect, for example,
embryogenesis and tissue formation. The term "chimeric animal" is
used herein to refer to animals in which the recombinant gene is
found, or in which the recombinant gene is expressed in some but
not all cells of the animal. The term "tissue-specific chimeric
animal" indicates that one of the recombinant gene is present
and/or expressed or disrupted in some tissues but not others.
[0033] As used herein, the term "nucleic acid" refers to
polynucleotides such as deoxyribonucleic acid (DNA), and, where
appropriate, ribonucleic acid (RNA). The term should also be
understood to include, as equivalents, analogs of either RNA or DNA
made from nucleotide analogs, and, as applicable to the embodiment
being described, single (sense or antisense) and double-stranded
polynucleotides.
[0034] As used herein, the term "promoter" means a DNA sequence
that regulates expression of a selected DNA sequence operably
linked to the promoter, and which effects expression of the
selected DNA sequence in cells. The term encompasses "tissue
specific" promoters, i.e. promoters, which effect expression of the
selected DNA sequence only in specific cells (e.g. cells of a
specific tissue). The term also covers so-called "leaky" promoters,
which regulate expression of a selected DNA primarily in one
tissue, but cause expression in other tissues as well. The term
also encompasses non-tissue specific promoters and promoters that
constitutively express or that are inducible (i.e. expression
levels can be controlled).
[0035] The terms "protein", "polypeptide" and "peptide" are used
interchangeably herein when referring to a gene product.
[0036] The term "recombinant protein" refers to a polypeptide of
the present invention which is produced by recombinant DNA
techniques, wherein generally, DNA encoding a -- polypeptide is
inserted into a suitable expression vector which is in turn used to
transform a host cell to produce the heterologous protein.
Moreover, the phrase "derived from", with respect to a recombinant
-- gene, is meant to include within the meaning of "recombinant
protein" those proteins having an amino acid sequence of a native
-- polypeptide, or an amino acid sequence similar thereto which is
generated by mutations including substitutions and deletions
(including truncation) of a naturally occurring form of the
polypeptide.
[0037] The term "repression" as used herein is meant to include
"inducible repression" and is used to refer to transcriptional
repression as by a transcriptional repressor such as a DNA binding
transcriptional repressor which binds a target promoter (a
"repressible" promoter) to be repressed.
[0038] The term "degrading" as used herein is meant to include
"inducible degradation" and is used to refer to proteolytic
degradation as may be facilitated by a component of the N-end rule
proteolytic pathway. Such an "inducible degradation," as referred
to herein, is meant to describe the targeted degradation of a
specific "target gene polypeptide."
[0039] "Small molecule" as used herein, is meant to refer to a
composition, which has a molecular weight of less than about 5 kD
and most preferably less than about 4 kD. Small molecules can be
nucleic acids, peptides, polypeptides, peptidomimetics,
carbohydrates, lipids or other organic (carbon containing) or
inorganic molecules. Many pharmaceutical companies have extensive
libraries of chemical and/or biological mixtures, often fungal,
bacterial, or algal extracts, which can be screened with any of the
assays of the invention to identify compounds that modulate a
bioactivity.
[0040] "Transcription" is a generic term used throughout the
specification to refer to DNA sequences, such as initiation
signals, enhancers, and promoters, which induce or control
transcription of protein coding sequences with which they are
operably linked. "Transcriptional repressor," as used herein,
refers to any of various polypeptides of procaryotic, eucaryotic
origin, or which are synthetic artificial chimeric constructs,
capable of repression either alone or in conjunction with other
polypeptides and which repress transcription in either an active or
a passive manner as described elsewhere. It will also be understood
that the recombinant gene can be under the control of
transcriptional regulatory sequences which are the same or which
are different from those sequences which control transcription of
the naturally-occurring forms of polypeptide.
[0041] As used herein, the term "transfection" means the
introduction of a nucleic acid, e.g., via an expression vector,
into a recipient cell by nucleic acid-mediated gene transfer.
"Transformation", as used herein, refers to a process in which a
cell's genotype is changed as a result of the cellular uptake of
exogenous DNA or RNA, and, for example, the transformed cell
expresses a recombinant form of a polypeptide or, in the case of
anti-sense expression from the transferred gene, the expression of
a naturally-occurring form of the polypeptide is disrupted.
[0042] As used herein, the term "target gene" refers to the nucleic
acid which encodes a gene of interest. The target gene can be an
"essential" gene, required for continued cell viability whose
function is to be shut-off by the method of the invention. The term
"target gene" is used to refer to both the original gene to be
targeted for shut-off and the same gene as later modified for
shut-off (such as by the replacement of the native promoter with a
repressible promoter and the addition of a ubiquitin-X encoding
sequence to the amino terminus of the targeted ORF, or open reading
frame). The term "target polypeptide" is used interchangeably with
the term "target gene polypeptide" and refers to the polypeptide
gene product of the target gene as described above.
[0043] As used herein, the term "transgene" means a nucleic acid
sequence (encoding, e.g., one of the polypeptides, or an antisense
transcript thereto) which has been introduced into a cell. A
transgene could be partly or entirely heterologous, i.e., foreign,
to the transgenic animal or cell into which it is introduced, or,
homologous to an endogenous gene of the transgenic animal or cell
into which it is introduced, but which is designed to be inserted,
or is inserted, into the animal's genome in such a way as to alter
the genome of the cell into which it is inserted (e.g., it is
inserted at a location which differs from that of the natural gene
or its insertion results in a knockout). A transgene can also be
present in a cell in the form of an episome. A transgene can
include one or more transcriptional regulatory sequences and any
other nucleic acid, such as introns, that may be necessary for
optimal expression of a selected nucleic acid.
[0044] A "transgenic animal" refers to any animal, preferably a
non-human mammal, bird or an amphibian, in which one or more of the
cells of the animal contain heterologous nucleic acid introduced by
way of human intervention, such as by transgenic techniques well
known in the art. The nucleic acid is introduced into the cell,
directly or indirectly by introduction into a precursor of the
cell, by way of deliberate genetic manipulation, such as by
microinjection or by infection with a recombinant virus. The term
genetic manipulation does not include classical cross-breeding, or
in vitro fertilization, but rather is directed to the introduction
of a recombinant DNA molecule. This molecule may be integrated
within a chromosome, or it may be extrachromosomally replicating
DNA. In the typical transgenic animals described herein, the
transgene causes cells to express a recombinant form of one of the
polypeptide, e.g. either agonistic or antagonistic forms. However,
transgenic animals in which the recombinant -- gene is silent are
also contemplated, as for example, the FLP or CRE recombinase
dependent constructs described below. Moreover, "transgenic animal"
also includes those recombinant animals in which gene disruption of
one or more genes is caused by human intervention, including both
recombination and antisense techniques.
[0045] The term "treating" as used herein is intended to encompass
curing as well as ameliorating at least one symptom of the
condition or disease.
[0046] The term "vector" refers to a nucleic acid molecule capable
of transporting another nucleic acid to which it has been linked.
One type of preferred vector is an episome, i.e., a nucleic acid
capable of extra-chromosomal replication. Preferred vectors are
those capable of autonomous replication and/or expression of
nucleic acids to which they are linked. Vectors capable of
directing the expression of genes to which they are operatively
linked are referred to herein as "expression vectors". In general,
expression vectors of utility in recombinant DNA techniques are
often in the form of "plasmids" which refer generally to circular
double stranded DNA loops which, in their vector form are not bound
to the chromosome. In the present specification, "plasmid" and
"vector" are used interchangeably as the plasmid is the most
commonly used form of vector. However, the invention is intended to
include such other forms of expression vectors which serve
equivalent functions and which become known in the art subsequently
hereto.
[0047] The term "wild-type allele" refers to an allele of a gene
which, when present in two copies in a subject results in a
wild-type phenotype. There can be several different wild-type
alleles of a specific gene, since certain nucleotide changes in a
gene may not affect the phenotype of a subject having two copies of
the gene with the nucleotide changes.
[0048] The term "ubiquitin" as used herein refers to an abundant 76
amino acid residue polypeptide that is found in all eukaryotic
cells. The ubiquitin polypeptide is characterized by a
carboxy-terminal glycine residue that is activated by ATP to a
high-energy thiol-ester intermediate in a reaction catalyzed by a
ubiquitin-activating enzyme (E1). The activated ubiquitin is
transferred to a substrate polypeptide via an isopeptide bond
between the activated carboxy-terminus of ubiquitin and the
epsilon-amino group of a lysine residue(s) in the protein
substrate. This transfer requires the action of ubiquitin
conjugating enzymes such as E2 and, in some instances, E3
activities. The ubiquitin modified substrate is thereby altered in
biological function, and, in some instances, becomes a substrate
for components of the ubiquitin-dependent proteolytic machinery
which includes both ubiquitin isopeptidase enzymes as well as
proteolytic proteins which are subunits of the proteasome. As used
herein, the term "ubiquitin" includes within its scope all known as
well as unidentified eukaryotic ubiquitin homologs of vertebrate or
invertebrate origin. Examples of ubiquitin polypeptides as referred
to herein include the human ubiquitin polypeptide which is encoded
by the human ubiquitin encoding nucleic acid sequence (GenBank
Accession Numbers: U49869, X04803) as well as all equivalents.
Equivalent ubiquitin polypeptide encoding nucleotide sequences are
understood to include those sequences that differ by one or more
nucleotide substitutions, additions or deletions, such as allelic
variants; as well as sequences which differ from the nucleotide
sequence encoding the human ubiquitin coding sequence shown in SEQ
ID NO. 2, due to the degeneracy of the genetic code. Another
example of a ubiquitin polypeptide as referred to herein is murine
ubiquitin which is encoded by the murine ubiquitin encoding nucleic
acid sequence (GenBank Accession Number: X51730).
[0049] The term "ubiquitin mutants" as used herein refers to
naturally occurring and synthetically derived altered forms of the
ubiquitin polypeptide molecule described above. Such mutants
include polypeptides encoded by ubiquitin nucleic acid coding
sequences containing missense mutations, which produce altered
amino acid sequences at a specific residue(s), and nonsense
mutations which produce STOP codons resulting in the formation of
truncated polypeptide. These mutations also include insertions and
deletions which produce frame-shifts or amino acid residue
insertions and deletions. These mutants thus produce altered coding
sequences resulting in the synthesis of altered forms of the
ubiquitin polypeptide other than those described by the term
"ubiquitin" as defined above. Examples of ubiquitin mutants
described herein include the Ub-75 polypeptide and Ub(K48R). In the
Ub-75 polypeptide, SEQ ID NO. 3, the carboxy-terminal glycine codon
of ubiquitin (SEQ ID NO. 2) is replaced by a stop codon resulting
in the synthesis of a mutant ubiquitin polypeptide characterized by
a carboxy-terminal glycine residue corresponding to the penultimate
glycine residue of wild type ubiquitin. Ub(K48R) is a mutant in
which the 48.sup.th residue of wild type ubiquitin, which
corresponds to a lysine residue used in a polyubiquitination
cross-linking reaction, is changed to an arginine residue which
cannot accommodate the polyubiquitination cross-linking
reaction.
[0050] The term "ubiquitin-like protein" as used herein refers to a
group of naturally occurring proteins, not otherwise describable as
ubiquitin equivalents, but which nonetheless show strong amino acid
homology to ubiquitin. As used herein this term includes the
polypeptides NEDD8, UBL1, NPVAC, and NPVOC. These "ubiquitin-like
proteins" are at least over 40% identical in sequence to the human
ubiquitin polypeptide and contain a pair of carboxy-terminal
glycine residues which function in the activation and transfer of
ubiquitin to target substrates as described supra.
[0051] As used herein, the term "ubiquitin-related protein" as used
herein refers to a group of naturally occurring proteins, not
otherwise describable as ubiquitin equivalents, but which
nonetheless show some relatively low degree (<40% identity) of
amino acid homology to ubiquitin. These "ubiquitin-related"
proteins include human Ubiquitin Cross-Reactive Protein (UCRP, 36%
identical to huUb, Accession No. P05161), FUBI (36% identical to
huUb, GenBank Accession No. AA449261), and Sentrin/Sumo/Pic1 (20%
identical to huUb, GenBank Accession No. U83117). The term
"ubiquitin-related protein" as used herein further pertains to
polypeptides possessing a carboxy-terminal pair of glycine residues
and which function as protein tags through activation of the
carboxy-terminal glycine residue and subsequent transfer to a
protein substrate.
[0052] The term "ubiquitin-homologous protein" as used herein
refers to a group of naturally occurring proteins, not otherwise
describable as ubiquitin equivalents or ubiquitin-like or
ubiquitin-related proteins, which appear functionally distinct from
ubiquitin in their ability to act as protein tags, but which
nonetheless show some degree of homology to ubiquitin (34-41%
identity). These "ubiquitin-homologous proteins" include RAD23A
(36% identical to huUb, SWISS-PROT. Accession No. P54725), RAD23B
(34% identical to huUb, SWISS-PROT. Accession No. P54727), DSK2
(41% identical to huUb, GenBank Accession No. L40587), and GDX (41%
identical to huUb, GenBank Accession No. J03589). The term
"ubiquitin-homologous protein" as used herein is further meant to
signify a class of ubiquitin homologous polypeptides whose
similarity to ubiquitin does not include glycine residues in the
carboxy-terminal and penultimate residue positions. Said proteins
appear functionally distinct from ubiquitin, as well as
ubiquitin-like and ubiquitin-related polypeptides, in that,
consistent with their lack of a conserved carboxy-terminal glycine
for use in an activation reaction, they have not been demonstrated
to serve as tags to other proteins by covalent linkage.
[0053] The term "ubiquitin conjugation machinery" as used herein
refers to a group of proteins which function in the ATP-dependent
activation and transfer of ubiquitin to substrate proteins. The
term thus encompasses: E1 enzymes, which transform the
carboxy-terminal glycine of ubiquitin into a high energy thiol
intermediate by an ATP-dependent reaction; E2 enzymes (the UBC
genes), which transform the E1-S.about.Ubiquitin activated
conjugate into an E2-S.about.Ubiquitin intermediate which acts as a
ubiquitin donor to a substrate, another ubiquitin moiety (in a
poly-ubiquitination reaction), or an E3; and the E3 enzymes (or
ubiquitin ligases) which facilitate the transfer of an activated
ubiquitin molecule from an E2 to a substrate molecule or to another
ubiquitin moiety as part of a polyubiquitin chain. The term
"ubiquitin conjugation machinery", as used herein, is further meant
to include all known members of these groups as well as those
members which have yet to be discovered or characterized but which
are sufficiently related by homology to known ubiquitin conjugation
enzymes so as to allow an individual skilled in the art to readily
identify it as a member of this group. The term as used herein is
meant to include novel ubiquitin activating enzymes which have yet
to be discovered as well as those which function in the activation
and conjugation of ubiquitin-like or ubiquitin-related polypeptides
to their substrates and to poly-ubiquitin-like or
polyubiquitin-related protein chains.
[0054] The term "ubiquitin-dependent proteolytic machinery" as used
herein refers to proteolytic enzymes which function in the
biochemical pathways of ubiquitin, ubiquitin-like, and
ubiquitin-related proteins. Such proteolytic enzymes include the
ubiquitin C-terminal hydrolases, which hydrolyze the linkage
between the carboxy-terminal glycine residue of ubiquitin and
various adducts; ubiquitin isopeptidases, which hyrolyze the
glycine76-lysine48 linkage between cross-linked ubiquitin moieties
in poly-ubiquitin conjugates; as well as other enzymes which
function in the removal of ubiquitin conjugates from ubiquitinated
substrates (generally termed "deubiquitinating enzymes"). The
aforementioned protease activities function in the removal of
ubiquitin units from a ubiquitinated substrate following or during
ubiquitin-dependent degradation as well as in certain proofreading
functions in which free ubiquitin polypeptides are removed from
incorrectly ubiquitinated proteins. The term "ubiquitin-dependent
proteolytic machinery" as used herein is also meant to encompass
the proteolytic subunits of the proteasome (including human
proteasome subunits C2, C3, C5, C8, and C9). The term
"ubiquitin-dependent proteolytic machinery" as used herein thus
encompasses two classes of proteases: the deubiquitinating enzymes
and the proteasome subunits. The protease functions of the
proteasome subunits are not known to occur outside the context of
the assembled proteasome, however independent functioning of these
polypeptides has not been excluded.
[0055] The term "ubiquitin system" as referred to herein is meant
to describe all of the aforementioned components of the ubiquitin
biochemical pathways including ubiquitin, ubiquitin-like proteins,
ubiquitin-related proteins, ubiquitin-homologous proteins,
ubiquitin conjugation machinery, ubiquitin-dependent proteolytic
machinery, or any of the substrates which these ubiquitin system
components act upon.
[0056] 4.3 Essential Components of the Method
[0057] The following sections describe in detail various
alternative embodiments of each of the elements of the general
methods described above. In particular, section 4.3.1 describes in
detail one necessary component, an inducible promoter, which is
useful for either of the two prongs of the method. Furthermore
section 4.3.5 provides a description of the various target genes
which can be employed in any of the repression methods. Section
4.3.5 also describes methods for modifying the existing native
target gene to make it responsive to either or both prongs of the
repression system. Section 4.3.2 describes in detail the
transcriptional repressors and corresponding repressible promoters
which are essential to the design of the transcriptional repression
prong of the method. Sections 4.3.3 and 4.3.4 describe in detail
the components essential for the polypeptide degradation prong of
the method. In particular, section 4.3.3 describes various
embodiments of the N-end rule gene which is inducibly deployed to
effect proteolysis of the target polypeptide. Section 4.3.4
describes the ubiquitin and ubiquitin equivalent sequences which
are used to produce, through endoproteolytic processing of a
ubiquitin-target polypeptide fusion protein, target polypeptides
possessing unique (generally non-methionine) amino-terminal amino
acids which subject the target polypeptide to N-end rule
proteolytic processes.
[0058] 4.3.1 Inducible Promoters
[0059] In both prongs of the method of the present invention, an
inducible promoter is employed to drive expression of the "effector
of suppression." Thus in the inducible transcriptional repression
prong of the method the inducible promoter is used to drive
expression of the transcriptional repressor, while in the inducible
proteolytic degradation prong of the method the inducible promoter
is used to drive expression of the N-end rule gene (see FIGS. 1A
and 1B). In a preferred mode of the invention, identical or unique
inducible promoters are used to drive the independent or coupled
expression of both a transcriptional repressor and an N-end rule
gene. The inducible promoters of the present invention are capable
of functioning in a eucaryotic host organism. Preferred embodiments
include naturally occurring yeast and mammalian inducible promoters
as well as synthetic promoters designed to function in a eucaryotic
host as described below. The important functional characteristic of
the inducible promoters of the present invention is their ultimate
inducibility by exposure to an environmental inducing agent.
Appropriate environmental inducing agents include exposure to heat,
various steroidal compounds, divalent cations (including Cu.sup.+2
and Zn.sup.+2), galactose, tetracycline, IPTG (isopropyl .beta.-D
thiogalactoside), as well as other naturally occurring and
synthetic inducing agents and gratuitous inducers. It is important
to note that, in certain modes of the invention, the environmental
inducing signal can correspond to the removal of any of the above
listed agents which are otherwise continuously supplied in the
uninduced state (see the tTA based system described below for
example). The inducibility of a eucaryotic promoter can be achieved
by either of two mechanisms included in the method of the present
invention. Suitable inducible promoters can be dependent upon
transcriptional activators which, in turn, are reliant upon an
environmental inducing agent. Alternatively the inducible promoters
can be repressed by a transcriptional repressor which itself is
rendered inactive by an environmental inducing agent. Thus the
inducible promoter can be either one which is induced by an
environmental agent which positively activates a transcriptional
activator, or one which is derepressed by an environmental agent
which negatively regulates a transcriptional repressor. We note
here that the latter class of inducible promoter systems defines
transcriptional repressors and corresponding negative cis
regulatory elements which can also find use as the repressors and
corresponding repressible promoters of the present invention as
described in section 4.3.2.
[0060] The inducible promoters of the present invention include
those controlled by the action of latent transcriptional activators
which are subject to induction by the action of environmental
inducing agents. Preferred examples include the copper inducible
promoters of the yeast genes CUP1, CRS5, and SOD1 which are subject
to copper-dependent activation by the yeast ACE1 transcriptional
activator (see e.g. Strain and Culotta (1996) Mol Gen Genet 251:
139-45; Hottiger et al. (1994) Yeast 10: 283-96; Lapinskas et al.
(1993) Curr Genet 24: 388-93; and Gralla et al. (1991) Proc. Natl.
Acad. Sci. USA 88: 8558-62). Alternatively, the copper inducible
promoter of the yeast gene CTT1 (encoding cytosolic catalase T),
which operates independently of the ACE1 transcriptional activator
(Lapinskas et al. (1993) Curr Genet 24: 388-93), can be utilized.
The copper concentrations required for effective induction of these
genes are suitably low so as to be tolerated by most cell systems,
including yeast and Drosophila cells. Alternatively, other
naturally occurring inducible promoters can be used in the present
invention including: steroid inducible gene promoters (see e.g.
Oligino et al. (1998) Gene Ther. 5: 491-6); galactose inducible
promoters from yeast (see e.g. Johnston (1987) Microbiol Rev 51:
458-76; Ruzzi et al. (1987) Mol Cell Biol 7: 991-7); and various
heat shock gene promoters. Many eucaryotic transcriptional
activators have been shown to function in a broad range of
eucaryotic host cells, and so, for example, many of the inducible
promoters identified in yeast can be adapted for use in a mammalian
host cell as well. For example, a unique synthetic transcriptional
induction system for mammalian cells has been developed based upon
a GAL4-estrogen receptor fusion protein which induces mammalian
promoters containing GAL4 binding sites (Braselmann et al. (1993)
Proc Natl Acad Sci USA 90: 1657-61). These and other inducible
promoters responsive to transcriptional activators which are
dependent upon specific inducing agents are suitable for use with
the present invention.
[0061] The inducible promoters of the present invention also
include those which are repressed by repressors which are subject
to inactivation by the action of environmental inducing agents.
Examples include procaryotic repressors which can transcriptionally
repress eucaryotic promoters which have been engineered to
incorporate appropriate repressor-binding operator sequences.
Preferred repressors for use in the present invention are sensitive
to inactivation by physiologically benign inducing agent. Thus,
where the lac repressor protein is used to control the expression
of a eucaryotic promoter which has been engineered to contain a
lacO operator sequence, treatment of the host cell with IPTG will
cause the dissociation of the lac repressor from the engineered
promoter and allow transcription to occur. Similarly, where the tet
repressor is used to control the expression of a eucaryotic
promoter which has been engineered to contain a tetO operator
sequence, treatment of the host cell with IPTG will cause the
dissociation of the tet repressor from the engineered promoter and
allow transcription to occur.
[0062] In a preferred embodiment of the invention, the repressor of
the inducible promoter is synthesized as a ubiquitin fusion protein
conforming to the formula ubiquitin-X-repressor. This can be
achieved using the ubiquitin fusion vector systems designed to
confer inducible proteolytic sensitivity to the target gene
polypeptide as described below. Thus it will be appreciated by the
skilled artisan that a rapid induction of a repressible promoter
can be achieved by simultaneously delivering an environmental
inducing agent which causes dissociation of the repressor from the
repressed inducible promoter, and simultaneously promoting the
destruction of that repressor by N-end rule directed proteolysis.
Degradation of the repressor prevents rebinding to the operator
which can result in decreased inducibility of the repressible
promoter--a problem which has been recognized in the art (see
Gossen et al. (1993) TIBS 18: 471-5). Furthermore, this aspect of
the invention can be utilized independently of the targeted
shut-off of a gene, to generally increase the inducibility of a
eucaryotic expression system which is subject to repression by a
repressor. Thus the present invention further provides improved
methods for inducible expression of endogenous or heterologous
genes in a eucaryotic cell.
[0063] As suggested above, the inducible promoters of the present
invention include those which are not naturally occurring promoters
but rather synthetically derived inducible promoter systems which
may make use of procaryotic transcriptional repressor proteins. The
advantage of using prokaryotic repressor proteins in the invention
is their specificity to a corresponding bacterial operator binding
site, which can be incorporated into the synthetic inducible
promoter system These procaryotic repressor proteins have no
natural eucaryotic gene targets and affect only the effector of
suppression gene which is put under the transcriptional control of
the inducible synthetic promoter. This system thereby avoids
undesirable side-effects resulting from unintentional alteration of
the expression of nontargeted eucaryotic genes when the inducible
promoter is induced. A preferred example of this type of inducible
promoter system is the tetracycline-regulated inducible promoter
system. Various useful versions of this promoter system have been
described (see Shockett and Schatz (1996) Proc. Natl. Acad. Sci.
USA 93: 5173-76 for review). As suggested above, these
tetracycline-regulated systems generally make use of a strong
eucaryotic promoter, such as human cytomegalovirus (CMV) immediate
early (IE) promoter/enhancer and a tet resistance operator (tetO)
which is bound by the tet repressor protein. In a preferred
embodiment, the system involves a modified version of the tet
repressor protein called a reverse transactivator (rtTA, or
rtTA-nls, which contains a nuclear localization signal) which binds
tetO sequences only in the presence of the tet derivatives
doxycycline or anhydrotetracycline. Using this system, a synthetic
human CMV/IE-tetO-promoter driven construct could be induced by 3
orders of magnitude in 20 hrs by the addition of the tet
derivatives (see Gossen et al. (1995) Science 268: 1766-9). Thus
this system can be used to make the effector of suppression genes
of the present invention inducible in response to the delivery of
tetracycline derivatives to the targeted eucaryotic cell.
Alternatively, a tet repressor fused to a transcriptional
activation domain of VP 16 (tTA) can be used to drive expression of
the inducible promoter of the present invention In this instance,
transcriptional activation of a synthetic human
CMV/IE-tetO-promoter driven construct is achieved by the removal of
tetracycline since the tTA activator only binds to the tetO in the
absence of tet (see Gossen and Bujard (1992) Proc. Natl. Acad. Sci.
USA 89: 5547-51). Other synthetic inducible promoter systems are
also available for use in the present invention. For example, a lac
repressor-VP 16 fusion which exhibits a "reverse" DNA binding
phenotype (i.e., analogous to rtTA described above, it only binds
the lacO operator sequence in the presence of the inducer IPTG)
(see Lambowitz and Belfort (1993) Annu Rev Biochem 62: 587-622).
This particular synthetic inducible promoter is approximately
1000-fold inducible in the presence of IPTG. Since neither the tet
repressor gene nor the lac repressor gene occurs naturally in a
eucaryotic cell, systems involving synthetic inducible promoter
constructs such as these rely on the further delivery of an
expressible copy of the appropriate procaryotic repressor gene.
Suitable expression cassettes for this purpose are readily
available for heterologous expression in many different eucaryotic
cells including various yeast species and mammalian cells.
[0064] The present invention thus allows for considerable
flexibility in choosing a suitable inducible promoter and
corresponding inducing agent. In some embodiments of the invention,
the choice of inducible promoter may be governed by the suitability
of the required inducing agent. Factors such as cytotoxicity or
indirect effects on nontarget genes may be important to consider in
this instance. In other instances the choice may be governed by the
properties of the inducible system as a whole. In particular, the
ease with which the system can be introduced into the appropriate
host cell and the speed and strength with which induction of the
system occurs following exposure to an inducing agent.
[0065] As mentioned above, the inducible promoters of the present
invention are used to drive expression of the effector of
suppression genes utilized in each of the two prongs of the method
of the present invention (see FIGS. 1A & 1B). These effector of
suppression genes include transcriptional repressors (described
below) and N-end rule system genes (described in section
4.3.3).
[0066] 4.3.2 Repressors and Corresponding Repressible Promoters
[0067] Although these two elements--the transcriptional repressor
and the corresponding repressible promoters are understood to be
independently implementable by the method of the invention, the
choice of one of these elements governs the selection of the other
and so they are discussed together here for the sake of
convenience. Nonetheless, in preferred embodiments of the invention
the two components are separately and independently engineered into
the targeted eucaryotic cell. In particular, a host cell engineered
to contain an inducible transcriptional repressor and an inducible
N-end rule system component, can be maintained independently for an
indefinite period of time prior to the introduction of a target
gene construct subject to repression by the transcriptional
repressor. Such a host cell can serve as a "master cell line" which
carries all of the essential components of the two-pronged shutoff
system. In preferred embodiments this "master cell line" comprises
both an inducible repressor of the target gene and an inducible
N-end rule system component for proteolytic destruction of the
target polypeptide. It is understood, however, that in certain
instances a "master cell line" carrying only one or the other
inducible "effector of suppression" of the two-pronged shut-off
system will be desired. For example, a cell-line containing only
the transcriptional shut-off prong may be used to determine the
normal half-life of a target gene. The complete block to target
gene transcription in such a case would allow one to follow the
rate of degradation of the target polypeptide (for instance by
Western analysis) without having to radioactively label the target
as in a "pulse-chase" type experiment. The complete master cell
line carrying both inducible effectors of suppression is, however,
a preferred embodiment of the invention as it allows the creation
of multiple otherwise "isogenic" cell lines which differ only in
the specific gene which has been engineered to be subject to the
inducible transcriptional/proteolytic shut-off system.
[0068] Suitable repressors and repressible promoters for use with
the immediate invention include those utilizing procaryotic
repressors as discussed above in the description of suitable
inducible promoter systems. However, the requirements for
suitability of a repressor for use as a repressor of the target
gene are fewer than are the requirements for a suitable repressor
for use in the inducible promoter system of the invention. In
particular, such target gene repressors need not be inactivatable
by an inducing agent. Thus, the lacI repressor or the tet repressor
could be used in this context without regard to the need to reverse
their repression with IPTG or tetracycline (inducing agents). This
is because reversal of the transcriptional inactivation of the
target gene is not generally desired in the present invention. Thus
virtually any site-specific DNA binding protein, which is not
otherwise a transcriptional activator and for which a high-affinity
binding sequence is known, is suitable for use as a repressor in
the present invention. The only requirement is that the high
affinity binding sequence be incorporated into the repressible
promoter used to express the target gene and that the site-specific
DNA binding protein, when bound to this sequence, is capable of
repressing the transcription of the target gene. Repression can be
achieved by either active or passive processes as is generally
understood in the art. For example, the procaryotic lacI and tet
repressors are generally believed to be capable of repression in
eucaryotes due to a passive ability to block trancription from a
eucaryotic promoter to which they are bound (so-called "steric"
blocking of the transcription apparatus). In contrast, active
repression occurs when a DNA binding protein recruits other
cellular proteins involved in transcriptional repression. For
example, in Saccharomyces cerevisiae the Ssn6-Tup1 corepressor is
recruited by a number of different DNA-binding repressor proteins.
These include ROX1, a preferred repressor of the present invention,
which is described in detail below. Other systems sensitive to
Ssn6-Tup 1 repression in yeast include the DIT1 and DIT2 genes in
yeast which are repressed through a cis sequence called NREDIT
(Friesen et al. (1998) Genetics 150: 59-73). Negative regulation by
the NREDIT sequence responds to mutations in SIN4 and ROX3. Thus,
in the present invention, ROX3 could be used as a repressor and the
DITi or DIT2 promoter (or minimally an heterologous promoter
incorporating the NREDIT element) as the repressible promoter used
to drive expression of the target gene. Alternatively, the MIG1
zinc-finger protein, which recruits the Ssn6-Tup1 repressor complex
to glucose-repressed promoters (Treitel and Carlson (1995) Proc.
Natl. Acad. Sci. USA 92: 3132-6), can be used as the repressor of
the present invention in conjunction with a suitable
glucose-repressible promoter to drive expression of the target
gene. Furthermore, virtually any site specific DNA binding protein
can be adapted for use as a repressor in the present invention by
fusing the DNA binding polypeptide to a protein domain known to
recruit the Ssn6-Tup1 complex. For example, the yeast alpha 2
repressor is known to recruit the Ssn6-Tup1 complex and thus the
appropriate alpha 2 coding sequence could be fused to virtually and
DNA binding polypeptide in order to derive a suitable repressor
protein for use in the present invention.
[0069] Many examples of eucaryotic transcriptional repressors which
"actively" repress transcription through a specific cis element are
known in the art and are of use in the present invention.
Surprisingly, even some eucaryotic transcriptional activators can
be converted into active repressor complexes when bound with an
appropriate corepressor protein. For example, MCM1 functions as an
activator in yeast but the MCM1/alpha 2 complex is an active
repressor complex capable of repressing a cis-linked promoter (see
e.g. Jonson and Herskowitz (1985) Cell 42: 237-47). Similarly, the
Drosophila developmental regulator Dorsal is a transcriptional
activator which behaves as an active repressor when bound to
certain cis regulatory elements such as the zen gene VRE (ventral
repression element, see e.g. Pan and Courey (1992) EMBO 11:
1837-42). In these instances either the activator (MCM1 or Dorsal
for example) or the corepressor with which it acts (alpha 2 or
Groucho (Dubnicoff et al. (1997) Genes Dev 11: 2952-7) is suitable
for use in the present invention where a suitable responsive
promoter element (such as STE6 operator or a zen VRE) is available
to control transcription of the target gene.
[0070] In a preferred embodiment, the transcriptional repressor of
the present invention is ROX1 and the repressible promoter is
selected from the group consisting of: ANB1, HEM13, ERG11 and OLE1.
ROX1 is a well-studied a transcriptional repressor of hypoxic genes
(Lowry, C. V., Cerdan, M. E., and Zitomer, R. (1990) Mol. Cell
Biol. 10: pp. 5921-5926; Balasubramanian, B., Lowry, C. V., and
Zitomer, R. S. (1993) Mol. Cell Biol. 13: pp. 6071-6078). ROX1
binds to specific hypoxic concensus sequences located in the
upstream of the upstream region of these genes and represses
transcription in conjunction with the general repression complex
Tup1-Ssn6 (Deckert et al. (1995) Mol Cell Biol 15: 6109-17). When
placed under the control of an ACE I-dependent and copper inducible
promoter from the yeast genes CUP1, CRS5, or SOD1,
[0071] the inPr-ROX1 construct can be stably integrated into the
yeast genome, for example, by conventional two-step gene
replacement. In the absence of copper, ROX1 is not expressed.
However when copper is added to the strain, ROX1 is expressed to
levels high enough to repress its target genes, but not high enough
to impair cell growth, as occurs with galactose-inducible ROX1
constructs (Deckert, J., Perini, R., Balasubramanian, B., and
Zitomer, R. S. (1995) Genetics 139: pp. 1149-58).
[0072] In preferred embodiments employing the ROX1 repressor, a 5'
fragment of the coding sequence of the target gene of interest can
then be genetically modified so that the ROX1-repressible promoter,
such as the ANB1 promoter, replaces the native promoter (naPr) of
the target gene as shown in FIG. 2A. This genetic modification can
be achieved by either standard recombinant DNA subcloning
manipulations which are known in the art, or by in vivo homologous
cross-over events which occur at a relatively high frequency from
double-stranded DNA ends in Saccharomyces cerevisiae and which can
also be selected for in mammalian systems using a "double
selection" method known in the art. This ANB1 driven allele, when
introduced into the inPr-ROX1 parent strain, renders the resulting
ANB1 driven allele susceptible to repression by the inducing agent
of the inducible promoter. Where the inducible promoter (inPr) is a
copper-inducible promoter such as from CUP1, the resulting host
cell will express the target gene constitutively in the absence of
Cu.sup.+2. Addition of Cu.sup.+2 causes the rapid ROX1-dependent
transcriptional repression of the target gene. In the absence of de
novo synthesis of the target gene mRNA, the existing pool of target
gene mRNA will be degraded through normal mRNA "turnover"
processes. Thus no further de novo target gene polypeptide
synthesis can occur and only the remaining pool of target gene
polypeptide remains to provide function to the host cell. The
second prong of the repression system is therefore specifically
tied to removing this residual target gene polypeptide through a
process of inducible targeted proteolysis.
[0073] 4.3.3 N-End Rule Pathway Components and Corresponding
Amino-Terminal Codons
[0074] The second prong of the two-pronged gene shut-off system is
an inducible proteolytic means for the degradation of the existing
pool of target gene polypeptide. In combination with the
transcriptional shut-off described above, it provides for a
thorough block to continued target gene function.
[0075] The targeted inducible proteolytic prong of the system makes
use of an inducible promoter, as described above, to drive
expression of a components of the so-called "N-end rule" system for
proteolytic degradation (Bachmair et al. (1986) Science 234:
179-86). This system operates to degrade a cellular polypeptide at
a rate dependent upon the amino-terminal amino acid residue of that
polypeptide. Protein translation ordinarily initiates with an ATG
methionine codon and so most polypeptides have an amino-terminal
methionine residue and are typically relatively stable in vivo. For
example, in the yeast S. cerevisiae, a beta-galactosidase
polypeptide with a methionine amino terminus has a half-life of
>20 hours (Varshavsky (1992) Cell 725-35). Under certain
circumstances, however, polypeptides possessing a non-methionine
amino-terminal residue can be created. For example, when an
endoprotease hydrolyzes and thus cleaves a unique polypeptide bond
(Y-X) internal to a polypeptide, it results in the release of two
separate polypeptides--one of which possesses an amino-terminal
amino acid, X, which may not be methionine. For example, the
endoprotease ubiquitin isopeptidase, which is a preferred component
of the present invention, will cleave a polypeptide bond
carboxy-terminal to the final glycine residue (codon 76),
regardless of what the next codon is. In the normal function of the
cell, this isopeptidase serves to cleave a polyubiquitin precursor
into individual ubiquitin units. However it can also be used to
generate a target polypeptide with virtually any amino-terminal
residue by merely fusing the target polypeptide in-frame to a codon
corresponding to the desired amino-terminal amino acid (X), which
codon, in turn, is fused downstream of ubiquitin (typically
contiguous with ubiquitin Gly codon 76). The resulting target gene
chimera construct, has the general structure Ubiquitin-X-Target.
Preferred target constructs further comprise an epitope tag (Ep) so
that the resulting target gene chimera construct has the general
structure Ubiquitin-X-Ep-target, which results in the eventual
production of a polypeptide of the general structure X-Ep-Target.
Constitutively active ubiquitin isopeptidase activities present in
eucaryotic cells will result in the endoproteolytic processing of
the Ubiquitin-X-Target polypeptide into Ubiquitin and X-Target
entities. The X-Target polypeptide is further acted upon by the
components of the N-end rule system as described below.
[0076] It has been determined, with reasonable reliability, the
relative effect of a given amino-terminal residue, X, upon target
polypeptide stability. For example, when all 20 possible
amino-terminal amino acid residues were tested to determine their
effect on the stability of beta-galactosidase (utilizing a
ubiquitin-X-beta-galactosidase chimeric fusion) in Saccharomyces
cerevisiae, drastic differences were discovered (see Varshavsky
(1992) Cell 69: 725-35). For example when X was met, cys, ala, ser,
thr, gly, val, or pro, the resulting polypeptide was very stable
(half-life of >20 hours). When X was tyr, ile, glu, or gln, the
resulting polypeptide possessed moderate protein stability
(half-life of 10-30 minutes). In contrast, the residues arg, lys
phe, leu, trp, his, asp, and asn, all conferred low stability on
the beta-galactosidase polypeptide (half-life of <3 minutes).
The residue arginine (arg), when located at the amino terminus of a
polypeptide, appears to generally confer the lowest stability.
Thus, chimeric constructs and corresponding chimeric polypeptides
employing an arg residue at the position X, described above, are
generally preferred embodiments of the present invention. This is
because a general goal of the invention is to eliminate the
function of the target gene polypeptide in the cell.
[0077] The above described experiments establishing the relative
half-lives conferred by each of the 20 possible amino terminal
residues form the basis of the N-end rule. The N-end rule system
components are those gene products which act to bring about the
rapid proteolysis of polypeptides possessing amino-terminal
residues which confer instability. The N-end rule system for
proteolysis in eucaryotes appears to be a part of the general
ubiquitin-dependent proteolytic system pathways possessed by
apparently all eucaryotic cells. Briefly, this system involves the
covalent tagging of a target polypeptide on one or more lysine
residues by a ubiquitin polypeptide marker (to form a target
(lys)-epsilon amino-gly(76) Ubiquitin covalent bond). Additional
ubiquitin moieties may be subsequently conjugated to the target
polypeptide and the resulting "ubiquitinated" target polypeptide is
then subject to complete proteolytic destruction by a large (26S)
multiprotein complex known as the proteasome. The enzymes which
conjugate the ubiquitin moieties to the targeted protein include E2
and E3 (or ubiquitin ligase) functions. The E2 and E3 enzymes are
thought to possess most of the specificity for ubiquitin dependent
proteolytic processes.
[0078] Indeed a key component of the N-end rule proteolytic pathway
in yeast is UBR1 (Bartel, et al. (1990) EMBO J. 9: 3179-89), a gene
which encodes an E3 like function which appears to recognize
polypeptides possessing susceptible amino terminal residues and
thereby facilitates ubiquitination of such polypeptides (Dohmen et
al. (1991) Proc. Natl. Acad. Sci. USA 88: 7351-55). In preferred
embodiments of the invention, UBR1 is used as the N-end rule
component which is the effector of proteolytic degradation of the
target gene polypeptide. The UBR1 gene has now been cloned from a
mammalian organism (Kwon et al. (1998) Proc. Natl. Acad. Sci. USA
95: 7893-903) as well as from yeast. Thus the construction of a
UBR1 mouse knockout is imminent and so both prongs of the
two-pronged gene function shut-off system can now be set up in both
yeast and mammalian host cells.
[0079] A preferred embodiment of the N-end rule component of the
two-component shut-off is the above described N-end rule ubiquitin
ligase UBR1 gene. This gene is particularly convenient since it can
be used in conjunction with any of the above described "X"
amino-terminal destabilizing residues including: the most
destabilizing--arg; strongly destabilizing residues--such as lys
phe, leu, trp, his, asp, and asn; and moderately destabilizing
residues--such as tyr, ile, glu, or gln. Indeed, it is an object of
the present invention to provide a means, where desired, to not
completely shut-off a target gene's function, but merely to
attenuate it to some set degree. This can be achieved using the
method of the present invention in any of a number of ways. For
example, a moderately destabilizing amino-terminal residue (X=tyr,
ile, glu, or gin) can be deployed on the target
polypeptide--resulting in a less rapid removal of the target
polypeptide pool. Alternatively, only one of the two prongs of the
method could be employed such as only the transcriptional
repression prong or only the targeted proteolysis prong.
[0080] Alternative embodiments of the N-end rule component of the
present invention include S. cerevisiae UBC2 (RAD6), which encodes
an E2 ubiquitin conjugating function which cooperates with the
UBR1-encoded N-end rule E3 to promote multiubiquitination and
subsequent degradation of N-end rule substrates (Dohmen et al.
(1991) Proc. Natl. Acad. Sci. USA 88: 7351-55). Thus N-end rule
directed proteolysis will not occur in the absence of either UBR1
or UBC2. This allows either gene to be used as the inducible
"effector of targeted proteolysis" by the method of the present
invention. Indeed, a target gene polypeptide possessing an N-end
rule destabilizing amino-terminal amino acid (such as arg) will be
stable until expression of either the UBR1 (E3) or the UBC2 (E2) is
induced from the cognate inducible promoter construct.
[0081] Both UBR1 and UBC2 can be used in conjunction with any of
the above described "X" amino-terminal destabilizing residues
including: the most destabilizing--arg; strongly destabilizing
residues--such as lys phe, leu, trp, his, asp, and asn; and
moderately destabilizing residues--such as tyr, ile, glu, or gin.
Still other alternative embodiments of the N-end rule component of
the present invention are components of the N-end rule system which
affect only a subset of the destabilizing residues. For example,
the NTA1 deamidase (Baker and Varshavsky (1995) J Biol Chem 270:
12065-74) functions to deaminate amino-terminal asn or gln residues
(to form polypeptides with asp or glu amino-terminal residues
respectively). Yeast strains harboring nta1 null alleles are unable
to degrade N-end rule substrates that bear amino-terminal asn or
gin residues. Thus, the NTA1 gene is an alternative embodiment of
the N-end rule component of the present invention, but is used
preferably in conjunction with a target gene polypeptide
(X-target), in which X is either asn or gin. Similarly the ATE1
transferase (Balzi et al. (1990) J. Biol Chem 265: 7464-71) is an
enzyme which acts to transfer the arg moiety from a tRNA.about.Arg
activated tRNA to amino-terminal glu or asp bearing polypeptides.
The resulting arg-glu-polypeptide and arg-asp-polypeptide products
are then susceptible to the E2/E3-mediated N-end rule dependent
proteolytic processes described above. Thus, the ATE1 transferase
is an alternative embodiment of the N-end rule component of the
present invention, but its use is preferably tied to target gene
polypeptides (X-target), in which X is asp, glu, asn or gln.
Polypeptides bearing the latter two amino-terminal residues are
first converted to polypeptides bearing one of the former tow
amino-terminal residues by NTA1 deamidase function described
above.
[0082] It is important to note here that, as is the case for the
repressor of the present invention which is made subject to
induction by an inducible promoter of the present invention, the
N-end rule component must be available as a clone so that it can be
put under the control of an inducible promoter (using standard
subcloning methods known in the art). This can be achieved by first
introducing a genetically engineered copies of the inducible
repressor and the inducible N-end rule component constructs, and
subsequently deleting the normal chromosomal copies of these genes
from the host by "knockout" methods. Such methods, we note here are
well developed in the art--particularly in the case of both the
yeast Saccharomyces cerevisiae and the mammal mouse. More
convenient, however, is the availability of "knock-in" technology
which allows the existing chromosomal copy of the gene to be
modified to so that its native promoter is deleted and an inducible
promoter is inserted in a single step. FIG. 2A diagrams this
process for the replacement of the native promoter of the target
gene with a repressible promoter, but this principle is also
applicable to the replacement of the native promoter of the
effector of suppression (i.e. the transcriptional repressor and/or
the N-end rule component) with a suitable inducible promoter.
[0083] 4.3.4 Ubiquitin Polypeptide Sequences
[0084] As shown in FIG. 1B, the target gene must be fused
downstream of a codon which encodes an N-end rule susceptible
residue (X, as described above) and this residue, in term, must be
fused in-frame to the carboxy-terminus of a ubiquitin coding
sequence (generally gly76 of ubiquitin). The reason for
constructing this extensive chimeric gene construct is to take
advantage of the ability of constitutive ubiquitin proteases to
cleave any peptide bond which is carboxy-terminal to gly76 of a
ubiquitin moiety. This isopeptidase normally functions to process
poly-ubiquitin chains (the translational product of the tandem
ubiquitin encoding sequences of eucaryotic genomes) into discrete
(normally 76 aa) ubiquitin moieties which are used in
ubiquitin-system pathways. In the method of the present invention,
the ubiquitin isopeptidases serve as a convenient means to generate
target gene polypeptides bearing specific amino-terminal residues
(X). Nonetheless, it is understood that other alternatives to
mammalian or yeast ubiquitin exist which can function in the method
of the present invention. Such ubiquitin equivalents include, for
example, ubiquitin mutants, ubiquitin-like proteins,
ubiquitin-related proteins, and ubiquitin-homologous proteins. For
example, ubiquitin-like proteins such as NEDD8, UBL1, FUBI, and
UCRP, as well as analogous ubiquitin-related proteins such as
SUMO/Sentrin/Pic1 may be used as ubiquitin equivalents in the
method of the invention. Other proteins related to ubiqutin, but
which are somewhat less homologous to it, include
ubiquitin-homologous proteins such as Rad23 and Dsk2 whose
similarity to ubiquitin does not include the presence of a
carboxyl-terminal pair of glycines. These ubiquitin-like proteins
share the common features of being related to ubiquitin by amino
acid sequence homology and, with the apparent exception of the
ubiquitin homologous proteins, of being covalently transferred to
cellular protein targets post-translationally.
[0085] Indeed, the intended scope of the immediate invention
encompasses any means known in the art by which a target
polypeptide bearing an N-end rule susceptible residue (X=arg, lys,
his, leu, phe, try, ile, trp, asn, gln, asp, or glu) can be
generated.
[0086] 4.3.5 Target Genes
[0087] As discussed above, the method of the present invention is
ideally suited to the analysis and exploitation of genes whose
function is essential for viability. Moreover, the methods
developed here allow virtually any gene to be made subject to
either or both of the gene function shut-off prongs of the present
invention. FIG. 2A diagrams the manner in which virtually any
desired target gene comprising a native promoter (napr) which
drives expression of the target ORF (open reading frame) can be put
under the control of a repressible promoter (repr) which is subject
to the transcriptional repression prong of the present invention.
Furthermore, FIG. 2B diagrams the manner in which virtually any
desired target gene target ORF can be fused in-frame with a
Ubiquitin-X-Ep (in which Ep is an optional epitope tag which can be
used to facilitate the measurement of target gene polypeptide
levels) to create a gene sequence which encodes a target gene
polypeptide which is subject to the targeted proteolytic
destruction prong of the present invention. As noted earlier these
construct can be created through either standard subcloning
techniques known in the art, or by means of a "knock-in" construct
which has the advantage of yielding the desired altered target gene
in a single step. A detail description of the suitable mouse cell
technology is provided below. FIG. 3 further diagrams how a single
target gene can be made subject to control by both the
transcriptional repression and the targeted proteolytic destruction
prong of the invention.
[0088] Specific examples of preferred target genes include various
components of the RNApoIII transcriptional machinery, as described
in the Example sections below. These include TAF60, TAF19, TAF90,
TAF130, TFIIB, and TBP (see Moqtader et al. (1996) Nature 383:
188-91).
[0089] 4.3.6 Other Methods
[0090] Methods for obtaining transgenic and knockout non-human
animals are well known in the art. Knock out mice are generated by
homologous integration of a "knock out" construct into a mouse
embryonic stem cell chromosome which encodes the gene to be knocked
out. In one embodiment, gene targeting, which is a method of using
homologous recombination to modify an animal's genome, can be used
to introduce changes into cultured embryonic stem cells. By
targeting a Target gene of interest in ES cells, these changes can
be introduced into the germlines of animals to generate chimeras.
The gene targeting procedure is accomplished by introducing into
tissue culture cells a DNA targeting construct that includes a
segment homologous to a target Target gene locus, and which also
includes an intended sequence modification to the Target genomic
sequence (e.g., insertion, deletion, point mutation). The treated
cells are then screened for accurate targeting to identify and
isolate those which have been properly targeted.
[0091] Gene targeting in embryonic stem cells is in fact a scheme
contemplated by the present invention as a means for disrupting a
Target gene function through the use of a targeting transgene
construct designed to undergo homologous recombination with one or
more Target genomic sequences. The targeting construct can be
arranged so that, upon recombination with an element of a Target
gene, a positive selection marker is inserted into (or replaces)
coding sequences of the gene. The inserted sequence functionally
disrupts the Target gene, while also providing a positive selection
trait. Exemplary Target gene targeting constructs are described in
more detail below.
[0092] Generally, the embryonic stem cells (ES cells) used to
produce the knockout animals will be of the same species as the
knockout animal to be generated. Thus for example, mouse embryonic
stem cells will usually be used for generation of knockout
mice.
[0093] Embryonic stem cells are generated and maintained using
methods well known to the skilled artisan such as those described
by Doetschman et al. (1985) J. Embryol. Exp. MoMFGFhol. 87:27-45).
Any line of ES cells can be used, however, the line chosen is
typically selected for the ability of the cells to integrate into
and become part of the germ line of a developing embryo so as to
create germ line transmission of the knockout construct. Thus, any
ES cell line that is believed to have this capability is suitable
for use herein. One mouse strain that is typically used for
production of ES cells, is the 129J strain. Another ES cell line is
murine cell line D3 (American Type Culture Collection, catalog no.
CKL 1934) Still another preferred ES cell line is the WW6 cell line
(loffe et al. (1995) PNAS 92:7357-7361). The cells are cultured and
prepared for knockout construct insertion using methods well known
to the skilled artisan, such as those set forth by Robertson in:
Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.
J. Robertson, ed. IRL Press, Washington, D.C. [1987]); by Bradley
et al. (1986) Current Topics in Devel. Biol. 20:357-371); and by
Hogan et al. (Manipulating the Mouse Embryo: A Laboratory Manual,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
[1986]).
[0094] A knock out construct refers to a uniquely configured
fragment of nucleic acid which is introduced into a stem cell line
and allowed to recombine with the genome at the chromosomal locus
of the gene of interest to be mutated. Thus a given knock out
construct is specific for a given gene to be targeted for
disruption. Nonetheless, many common elements exist among these
constructs and these elements are well known in the art. A typical
knock out construct contains nucleic acid fragments of not less
than about 0.5 kb nor more than about 10.0 kb from both the 5' and
the 3' ends of the genomic locus which encodes the gene to be
mutated. These two fragments are separated by an intervening
fragment of nucleic acid which encodes a positive selectable
marker, such as the neomycin resistance gene (neo.sup.R). The
resulting nucleic acid fragment, consisting of a nucleic acid from
the extreme 5' end of the genomic locus linked to a nucleic acid
encoding a positive selectable marker which is in turn linked to a
nucleic acid from the extreme 3' end of the genomic locus of
interest, omits most of the coding sequence for Target gene or
other gene of interest to be knocked out. When the resulting
construct recombines homologously with the chromosome at this
locus, it results in the loss of the omitted coding sequence,
otherwise known as the structural gene, from the genomic locus. A
stem cell in which such a rare homologous recombination event has
taken place can be selected for by virtue of the stable integration
into the genome of the nucleic acid of the gene encoding the
positive selectable marker and subsequent selection for cells
expressing this marker gene in the presence of an appropriate drug
(neomycin in this example).
[0095] Variations on this basic technique also exist and are well
known in the art. For example, a "knock-in" construct refers to the
same basic arrangement of a nucleic acid encoding a 5' genomic
locus fragment linked to nucleic acid encoding a positive
selectable marker which in turn is linked to a nucleic acid
encoding a 3' genomic locus fragment, but which differs in that
none of the coding sequence is omitted and thus the 5' and the 3'
genomic fragments used were initially contiguous before being
disrupted by the introduction of the nucleic acid encoding the
positive selectable marker gene. This "knock-in" type of construct
is thus very useful for the construction of mutant transgenic
animals when only a limited region of the genomic locus of the gene
to be mutated, such as a single exon, is available for cloning and
genetic manipulation. Alternatively, the "knock-in" construct can
be used to specifically eliminate a single functional domain of the
targetted gene, resulting in a transgenic animal which expresses a
polypeptide of the targetted gene which is defective in one
function, while retaining the function of other domains of the
encoded polypeptide. This type of "knock-in" mutant frequently has
the characteristic of a so-called "dominant negative" mutant
because, especially in the case of proteins which homomultimerize,
it can specifically block the action of (or "poison") the
polypeptide product of the wild-type gene from which it was
derived. In a variation of the knock-in technique, a marker gene is
integrated at the genomic locus of interest such that expression of
the marker gene comes under the control of the transcriptional
regulatory elements of the targeted gene. A marker gene is one that
encodes an enzyme whose activity can be detected (e.g.,
b-galactosidase), the enzyme substrate can be added to the cells
under suitable conditions, and the enzymatic activity can be
analyzed. One skilled in the art will be familiar with other useful
markers and the means for detecting their presence in a given cell.
All such markers are contemplated as being included within the
scope of the teaching of this invention.
[0096] As mentioned above, the homologous recombination of the
above described "knock out" and "knock in" constructs is very rare
and frequently such a construct inserts nonhomologously into a
random region of the genome where it has no effect on the gene
which has been targeted for deletion, and where it can potentially
recombine so as to disrupt another gene which was otherwise not
intended to be altered. Such nonhomologous recombination events can
be selected against by modifying the abovementioned knock out and
knock in constructs so that they are flanked by negative selectable
markers at either end (particularly through the use of two allelic
variants of the thymidine kinase gene, the polypeptide product of
which can be selected against in expressing cell lines in an
appropriate tissue culture medium well known in the art--i.e. one
containing a drug such as 5-bromodeoxyuridine). Thus a preferred
embodiment of such a knock out or knock in construct of the
invention consist of a nucleic acid encoding a negative selectable
marker linked to a nucleic acid encoding a 5' end of a genomic
locus linked to a nucleic acid of a positive selectable marker
which in turn is linked to a nucleic acid encoding a 3' end of the
same genomic locus which in turn is linked to a second nucleic acid
encoding a negative selectable marker Nonhomologous recombination
between the resulting knock out construct and the genome will
usually result in the stable integration of one or both of these
negative selectable marker genes and hence cells which have
undergone nonhomologous recombination can be selected against by
growth in the appropriate selective media (e.g. media containing a
drug such as 5-bromodeoxyuridine for example). Simultaneous
selection for the positive selectable marker and against the
negative selectable marker will result in a vast enrichment for
clones in which the knock out construct has recombined homologously
at the locus of the gene intended to be mutated. The presence of
the predicted chromosomal alteration at the targeted gene locus in
the resulting knock out stem cell line can be confirmed by means of
Southern blot analytical techniques which are well known to those
familiar in the art. Alternatively, PCR can be used.
[0097] Each knockout construct to be inserted into the cell must
first be in the linear form. Therefore, if the knockout construct
has been inserted into a vector (described infra), linearization is
accomplished by digesting the DNA with a suitable restriction
endonuclease selected to cut only within the vector sequence and
not within the knockout construct sequence.
[0098] For insertion, the knockout construct is added to the ES
cells under appropriate conditions for the insertion method chosen,
as is known to the skilled artisan. For example, if the ES cells
are to be electroporated, the ES cells and knockout construct DNA
are exposed to an electric pulse using an electroporation machine
and following the manufacturer's guidelines for use. After
electroporation, the ES cells are typically allowed to recover
under suitable incubation conditions. The cells are then screened
for the presence of the knock out construct as explained above.
Where more than one construct is to be introduced into the ES cell,
each knockout construct can be introduced simultaneously or one at
a time.
[0099] After suitable ES cells containing the knockout construct in
the proper location have been identified by the selection
techniques outlined above, the cells can be inserted into an
embryo. Insertion may be accomplished in a variety of ways known to
the skilled artisan, however a preferred method is by
microinjection. For microinjection, about 10-30 cells are collected
into a micropipet and injected into embryos that are at the proper
stage of development to permit integration of the foreign ES cell
containing the knockout construct into the developing embryo. For
instance, the transformed ES cells can be microinjected into
blastocytes. The suitable stage of development for the embryo used
for insertion of ES cells is very species dependent, however for
mice it is about 3.5 days. The embryos are obtained by perfusing
the uterus of pregnant females. Suitable methods for accomplishing
this are known to the skilled artisan, and are set forth by, e.g.,
Bradley et al. (supra).
[0100] While any embryo of the right stage of development is
suitable for use, preferred embryos are male. In mice, the
preferred embryos also have genes coding for a coat color that is
different from the coat color encoded by the ES cell genes. In this
way, the offspring can be screened easily for the presence of the
knockout construct by looking for mosaic coat color (indicating
that the ES cell was incorporated into the developing embryo).
Thus, for example, if the ES cell line carries the genes for white
fur, the embryo selected will carry genes for black or brown
fur.
[0101] After the ES cell has been introduced into the embryo, the
embryo may be implanted into the uterus of a pseudopregnant foster
mother for gestation. While any foster mother may be used, the
foster mother is typically selected for her ability to breed and
reproduce well, and for her ability to care for the young. Such
foster mothers are typically prepared by mating with vasectomized
males of the same species. The stage of the pseudopregnant foster
mother is important for successful implantation, and it is species
dependent. For mice, this stage is about 2-3 days
pseudopregnant.
[0102] Offspring that are born to the foster mother may be screened
initially for mosaic coat color where the coat color selection
strategy (as described above, and in the appended examples) has
been employed. In addition, or as an alternative, DNA from tail
tissue of the offspring may be screened for the presence of the
knockout construct using Southern blots and/or PCR as described
above. Offspring that appear to be mosaics may then be crossed to
each other, if they are believed to carry the knockout construct in
their germ line, in order to generate homozygous knockout animals.
Homozygotes may be identified by Southern blotting of equivalent
amounts of genomic DNA from mice that are the product of this
cross, as well as mice that are known heterozygotes and wild type
mice.
[0103] Other means of identifying and characterizing the knockout
offspring are available. For example, Northern blots can be used to
probe the mRNA for the presence or absence of transcripts encoding
either the gene knocked out, the marker gene, or both. In addition,
Western blots can be used to assess the level of expression of the
MFGF gene knocked out in various tissues of the offspring by
probing the Western blot with an antibody against the particular
MFGF protein, or an antibody against the marker gene product, where
this gene is expressed. Finally, in situ analysis (such as fixing
the cells and labeling with antibody) and/or FACS (fluorescence
activated cell sorting) analysis of various cells from the
offspring can be conducted using suitable antibodies to look for
the presence or absence of the knockout construct gene product.
[0104] Yet other methods of making knock-out or disruption
transgenic animals are also generally known. See, for example,
Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1986). Recombinase dependent
knockouts can also be generated, e.g. by homologous recombination
to insert target sequences, such that tissue specific and/or
temporal control of inactivation of a Target-gene can be controlled
by recombinase sequences (described infra).
[0105] Animals containing more than one knockout construct and/or
more than one transgene expression construct are prepared in any of
several ways. The preferred manner of preparation is to generate a
series of mammals, each containing one of the desired transgenic
phenotypes. Such animals are bred together through a series of
crosses, backcrosses and selections, to ultimately generate a
single animal containing all desired knockout constructs and/or
expression constructs, where the animal is otherwise congenic
(genetically identical) to the wild type except for the presence of
the knockout construct(s) and/or transgene(s).
[0106] A Target transgene can encode the wild-type form of the
protein, or can encode homologs thereof, including both agonists
and antagonists, as well as antisense constructs. In preferred
embodiments, the expression of the transgene is restricted to
specific subsets of cells, tissues or developmental stages
utilizing, for example, cis-acting sequences that control
expression in the desired pattern. In the present invention, such
mosaic expression of a Target gene protein can be essential for
many forms of lineage analysis and can additionally provide a means
to assess the effects of, for example, lack of Target gene
expression which might grossly alter development in small patches
of tissue within an otherwise normal embryo. Toward this and,
tissue-specific regulatory sequences and conditional regulatory
sequences can be used to control expression of the transgene in
certain spatial patterns. Moreover, temporal patterns of expression
can be provided by, for example, conditional recombination systems
or prokaryotic transcriptional regulatory sequences.
[0107] Genetic techniques, which allow for the expression of
transgenes can be regulated via site-specific genetic manipulation
in vivo, are known to those skilled in the art. For instance,
genetic systems are available which allow for the regulated
expression of a recombinase that catalyzes the genetic
recombination of a target sequence. As used herein, the phrase
"target sequence" refers to a nucleotide sequence that is
genetically recombined by a recombinase. The target sequence is
flanked by recombinase recognition sequences and is generally
either excised or inverted in cells expressing recombinase
activity. Recombinase catalyzed recombination events can be
designed such that recombination of the target sequence results in
either the activation or repression of expression of one of the
subject Target gene proteins. For example, excision of a target
sequence which interferes with the expression of a recombinant
Target gene, such as one which encodes an antagonistic homolog or
an antisense transcript, can be designed to activate expression of
that gene. This interference with expression of the protein can
result from a variety of mechanisms, such as spatial separation of
the Target gene from the promoter element or an internal stop
codon. Moreover, the transgene can be made wherein the coding
sequence of the gene is flanked by recombinase recognition
sequences and is initially transfected into cells in a 3' to 5'
orientation with respect to the promoter element. In such an
instance, inversion of the target sequence will reorient the
subject gene by placing the 5' end of the coding sequence in an
orientation with respect to the promoter element which allow for
promoter driven transcriptional activation.
[0108] The transgenic animals of the present invention all include
within a plurality of their cells a transgene of the present
invention, which transgene alters the phenotype of the "host cell"
with respect to regulation of cell growth, death and/or
differentiation. Since it is possible to produce transgenic
organisms of the invention utilizing one or more of the transgene
constructs described herein, a general description will be given of
the production of transgenic organisms by referring generally to
exogenous genetic material. This general description can be adapted
by those skilled in the art in order to incorporate specific
transgene sequences into organisms utilizing the methods and
materials described below.
[0109] In an illustrative embodiment, either the cre/loxP
recombinase system of bacteriophage P1 (Lakso et al. (1992) PNAS
89:6232-6236; Orban et al. (1992) PNAS 89:6861-6865) or the FLP
recombinase system of Saccharomyces cerevisiae (O'Gorman et al.
(1991) Science 251:1351-1355; PCT publication WO 92/15694) can be
used to generate in vivo site-specific genetic recombination
systems. Cre recombinase catalyzes the site-specific recombination
of an intervening target sequence located between loxP sequences.
loxP sequences are 34 base pair nucleotide repeat sequences to
which the Cre recombinase binds and are required for Cre
recombinase mediated genetic recombination. The orientation of loxP
sequences determines whether the intervening target sequence is
excised or inverted when Cre recombinase is present (Abremski et
al. (1984) J. Biol. Chem. 259:1509-1514); catalyzing the excision
of the target sequence when the loxP sequences are oriented as
direct repeats and catalyzes inversion of the target sequence when
loxP sequences are oriented as inverted repeats.
[0110] Accordingly, genetic recombination of the target sequence is
dependent on expression of the Cre recombinase. Expression of the
recombinase can be regulated by promoter elements which are subject
to regulatory control, e.g., tissue-specific, developmental
stage-specific, inducible or repressible by externally added
agents. This regulated control will result in genetic recombination
of the target sequence only in cells where recombinase expression
is mediated by the promoter element. Thus, the activation
expression of a recombinant Target gene protein can be regulated
via control of recombinase expression.
[0111] Use of the cre/loxP recombinase system to regulate
expression of a recombinant Target gene protein requires the
construction of a transgenic animal containing transgenes encoding
both the Cre recombinase and the subject protein. Animals
containing both the Cre recombinase and a recombinant Target gene
can be provided through the construction of "double" transgenic
animals. A convenient method for providing such animals is to mate
two transgenic animals each containing a transgene, e.g., a Target
gene and recombinase gene.
[0112] One advantage derived from initially constructing transgenic
animals containing a Target transgene in a recombinase-mediated
expressible format derives from the likelihood that the subject
protein, whether agonistic or antagonistic, can be deleterious upon
expression in the transgenic animal. In such an instance, a founder
population, in which the subject transgene is silent in all
tissues, can be propagated and maintained. Individuals of this
founder population can be crossed with animals expressing the
recombinase in, for example, one or more tissues and/or a desired
temporal pattern. Thus, the creation of a founder population in
which, for example, an antagonistic Target transgene is silent will
allow the study of progeny from that founder in which disruption of
Target gene mediated induction in a particular tissue or at certain
developmental stages would result in, for example, a lethal
phenotype.
[0113] Similar conditional transgenes can be provided using
prokaryotic promoter sequences which require prokaryotic proteins
to be simultaneous expressed in order to facilitate expression of
the Target transgene. Exemplary promoters and the corresponding
trans-activating prokaryotic proteins are given in U.S. Pat. No.
4,833,080.
[0114] Moreover, expression of the conditional transgenes can be
induced by gene therapy-like methods wherein a gene encoding the
trans-activating protein, e.g. a recombinase or a prokaryotic
protein, is delivered to the tissue and caused to be expressed,
such as in a cell-type specific manner. By this method, a Target A
transgene could remain silent into adulthood until "turned on" by
the introduction of the trans-activator.
[0115] In an exemplary embodiment, the "transgenic non-human
animals" of the invention are produced by introducing transgenes
into the germline of the non-human animal. Embryonal target cells
at various developmental stages can be used to introduce
transgenes. Different methods are used depending on the stage of
development of the embryonal target cell. The specific line(s) of
any animal used to practice this invention are selected for general
good health, good embryo yields, good pronuclear visibility in the
embryo, and good reproductive fitness. In addition, the haplotype
is a significant factor. For example, when transgenic mice are to
be produced, strains such as C57BL/6 or FVB lines are often used
(Jackson Laboratory, Bar Harbor, Me.). Preferred strains are those
with H-2.sup.b, H-2.sup.d or H-2.sup.q haplotypes such as C57BL/6
or DBA/1. The line(s) used to practice this invention may
themselves be transgenics, and/or may be knockouts (i.e., obtained
from animals which have one or more genes partially or completely
suppressed).
[0116] In one embodiment, the transgene construct is introduced
into a single stage embryo. The zygote is the best target for
micro-injection. In the mouse, the male pronucleus reaches the size
of approximately 20 micrometers in diameter which allows
reproducible injection of 1-2 pl of DNA solution. The use of
zygotes as a target for gene transfer has a major advantage in that
in most cases the injected DNA will be incorporated into the host
gene before the first cleavage (Brinster et al. (1985) PNAS
82:4438-4442). As a consequence, all cells of the transgenic animal
will carry the incorporated transgene. This will in general also be
reflected in the efficient transmission of the transgene to
offspring of the founder since 50% of the germ cells will harbor
the transgene.
[0117] Normally, fertilized embryos are incubated in suitable media
until the pronuclei appear. At about this time, the nucleotide
sequence comprising the transgene is introduced into the female or
male pronucleus as described below. In some species such as mice,
the male pronucleus is preferred. It is most preferred that the
exogenous genetic material be added to the male DNA complement of
the zygote prior to its being processed by the ovum nucleus or the
zygote female pronucleus. It is thought that the ovum nucleus or
female pronucleus release molecules which affect the male DNA
complement, perhaps by replacing the protamines of the male DNA
with histones, thereby facilitating the combination of the female
and male DNA complements to form the diploid zygote.
[0118] Thus, it is preferred that the exogenous genetic material be
added to the male complement of DNA or any other complement of DNA
prior to its being affected by the female pronucleus. For example,
the exogenous genetic material is added to the early male
pronucleus, as soon as possible after the formation of the male
pronucleus, which is when the male and female pronuclei are well
separated and both are located close to the cell membrane.
Alternatively, the exogenous genetic material could be added to the
nucleus of the sperm after it has been induced to undergo
decondensation. Sperm containing the exogenous genetic material can
then be added to the ovum or the decondensed sperm could be added
to the ovum with the transgene constructs being added as soon as
possible thereafter.
[0119] Introduction of the transgene nucleotide sequence into the
embryo may be accomplished by any means known in the art such as,
for example, microinjection, electroporation, or lipofection.
Following introduction of the transgene nucleotide sequence into
the embryo, the embryo may be incubated in vitro for varying
amounts of time, or reimplanted into the surrogate host, or both.
In vitro incubation to maturity is within the scope of this
invention. One common method in to incubate the embryos in vitro
for about 1-7 days, depending on the species, and then reimplant
them into the surrogate host.
[0120] For the purposes of this invention a zygote is essentially
the formation of a diploid cell which is capable of developing into
a complete organism. Generally, the zygote will be comprised of an
egg containing a nucleus formed, either naturally or artificially,
by the fusion of two haploid nuclei from a gamete or gametes. Thus,
the gamete nuclei must be ones which are naturally compatible,
i.e., ones which result in a viable zygote capable of undergoing
differentiation and developing into a functioning organism.
Generally, a euploid zygote is preferred. If an aneuploid zygote is
obtained, then the number of chromosomes should not vary by more
than one with respect to the euploid number of the organism from
which either gamete originated.
[0121] In addition to similar biological considerations, physical
ones also govern the amount (e.g., volume) of exogenous genetic
material which can be added to the nucleus of the zygote or to the
genetic material which forms a part of the zygote nucleus. If no
genetic material is removed, then the amount of exogenous genetic
material which can be added is limited by the amount which will be
absorbed without being physically disruptive. Generally, the volume
of exogenous genetic material inserted will not exceed about 10
picoliters. The physical effects of addition must not be so great
as to physically destroy the viability of the zygote. The
biological limit of the number and variety of DNA sequences will
vary depending upon the particular zygote and functions of the
exogenous genetic material and will be readily apparent to one
skilled in the art, because the genetic material, including the
exogenous genetic material, of the resulting zygote must be
biologically capable of initiating and maintaining the
differentiation and development of the zygote into a functional
organism.
[0122] The number of copies of the transgene constructs which are
added to the zygote is dependent upon the total amount of exogenous
genetic material added and will be the amount which enables the
genetic transformation to occur. Theoretically only one copy is
required; however, generally, numerous copies are utilized, for
example, 1,000-20,000 copies of the transgene construct, in order
to insure that one copy is functional. As regards the present
invention, there will often be an advantage to having more than one
functioning copy of each of the inserted exogenous DNA sequences to
enhance the phenotypic expression of the exogenous DNA
sequences.
[0123] Any technique which allows for the addition of the exogenous
genetic material into nucleic genetic material can be utilized so
long as it is not destructive to the cell, nuclear membrane or
other existing cellular or genetic structures. The exogenous
genetic material is preferentially inserted into the nucleic
genetic material by microinjection. Microinjection of cells and
cellular structures is known and is used in the art.
[0124] Reimplantation is accomplished using standard methods.
Usually, the surrogate host is anesthetized, and the embryos are
inserted into the oviduct. The number of embryos implanted into a
particular host will vary by species, but will usually be
comparable to the number of off spring the species naturally
produces.
[0125] Transgenic offspring of the surrogate host may be screened
for the presence and/or expression of the transgene by any suitable
method. Screening is often accomplished by Southern blot or
Northern blot analysis, using a probe that is complementary to at
least a portion of the transgene. Western blot analysis using an
antibody against the protein encoded by the transgene may be
employed as an alternative or additional method for screening for
the presence of the transgene product. Typically, DNA is prepared
from tail tissue and analyzed by Southern analysis or PCR for the
transgene. Alternatively, the tissues or cells believed to express
the transgene at the highest levels are tested for the presence and
expression of the transgene using Southern analysis or PCR,
although any tissues or cell types may be used for this
analysis.
[0126] Alternative or additional methods for evaluating the
presence of the transgene include, without limitation, suitable
biochemical assays such as enzyme and/or immunological assays,
histological stains for particular marker or enzyme activities,
flow cytometric analysis, and the like. Analysis of the blood may
also be useful to detect the presence of the transgene product in
the blood, as well as to evaluate the effect of the transgene on
the levels of various types of blood cells and other blood
constituents.
[0127] Progeny of the transgenic animals may be obtained by mating
the transgenic animal with a suitable partner, or by in vitro
fertilization of eggs and/or sperm obtained from the transgenic
animal. Where mating with a partner is to be performed, the partner
may or may not be transgenic and/or a knockout; where it is
transgenic, it may contain the same or a different transgene, or
both. Alternatively, the partner may be a parental line. Where in
vitro fertilization is used, the fertilized embryo may be implanted
into a surrogate host or incubated in vitro, or both. Using either
method, the progeny may be evaluated for the presence of the
transgene using methods described above, or other appropriate
methods.
[0128] The transgenic animals produced in accordance with the
present invention will include exogenous genetic material. As set
out above, the exogenous genetic material will, in certain
embodiments, be a DNA sequence which results in the production of a
Target protein (either agonistic or antagonistic), and antisense
transcript, or a Target mutant. Further, in such embodiments the
sequence will be attached to a transcriptional control element,
e.g., a promoter, which preferably allows the expression of the
transgene product in a specific type of cell.
[0129] Retroviral infection can also be used to introduce transgene
into a non-human animal. The developing non-human embryo can be
cultured in vitro to the blastocyst stage. During this time, the
blastomeres can be targets for retroviral infection (Jaenich, R.
(1976) PNAS 73:1260-1264). Efficient infection of the blastomeres
is obtained by enzymatic treatment to remove the zona pellucida
(Manipulating the Mouse Embryo, Hogan eds. (Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, 1986). The viral vector
system used to introduce the transgene is typically a
replication-defective retrovirus carrying the transgene (Jahner et
al. (1985) PNAS 82:6927-6931; Van der Putten et al. (1985) PNAS
82:6148-6152). Transfection is easily and efficiently obtained by
culturing the blastomeres on a monolayer of virus-producing cells
(Van der Putten, supra; Stewart et al. (1987) EMBO J. 6:383-388).
Alternatively, infection can be performed at a later stage. Virus
or virus-producing cells can be injected into the blastocoele
(Jahner et al. (1982) Nature 298:623-628). Most of the founders
will be mosaic for the transgene since incorporation occurs only in
a subset of the cells which formed the transgenic non-human animal.
Further, the founder may contain various retroviral insertions of
the transgene at different positions in the genome which generally
will segregate in the offspring. In addition, it is also possible
to introduce transgenes into the germ line by intrauterine
retroviral infection of the midgestation embryo (Jahner et al.
(1982) supra).
[0130] A third type of target cell for transgene introduction is
the embryonal stem cell (ES). ES cells are obtained from
pre-implantation embryos cultured in vitro and fused with embryos
(Evans et al. (1981) Nature 292:154-156; Bradley et al. (1984)
Nature 309:255-258; Gossler et al. (1986) PNAS 83: 9065-9069; and
Robertson et al. (1986) Nature 322:445-448). Transgenes can be
efficiently introduced into the ES cells by DNA transfection or by
retrovirus-mediated transduction. Such transformed ES cells can
thereafter be combined with blastocysts from a non-human animal.
The ES cells thereafter colonize the embryo and contribute to the
germ line of the resulting chimeric animal. For review see
Jaenisch, R. (1988) Science 240:1468-1474.
5. EXAMPLES
[0131] The present invention is further illustrated by the
following examples which should not be construed as limiting in any
way. The contents of all cited references (including literature
references, issued patents, published patent applications as cited
throughout this application are hereby expressly incorporated by
reference.
[0132] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of cell biology, cell
culture, molecular biology, microbiology and recombinant DNA, which
are within the skill of the art. Such techniques are explained
fully in the literature. See, for example, Molecular Cloning A
Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis
(Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I
and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J.
Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic
Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984);
Transcription And Translation (B. D. Hames & S. J. Higgins eds.
1984); B. Perbal, A Practical Guide To Molecular Cloning (1984);
the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.);
Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.),
Immunochemical Methods In Cell And Molecular Biology (Mayer and
Walker, eds., Academic Press, London, 1987).
5.1 Example 1
[0133] Use of Double Shutoff Yeast Strain Containing
Copper-Inducible Yeast Alleles of Both ROX1 and UBR1 to Determine
that TBP-Associated Factors are not Generally Required for
Transcriptional Activation in Yeast
[0134] Materials and Methods
[0135] The parent strain ZMY60, containing copper-inducible alleles
of UBR1 and ROX1, was created as follows. A cassette containing the
copper-inducible derivative of the H1S3 promoter (Klein, C and K.
Struhl (1994) Science 266: pp. 280-282) and 2 kb of upstream
flanking sequence was inserted at the initial ATG of a
plasmid-borne genomic fragment of ROX1 to create the URA3
integrating plasmid ZM195. The same cassette was inserted at the
initial ATG of UBR1 to create ZM197. Both of these copper-driven
alleles were introduced into yeast strain KY114 (Iyer, V and K.
Struhl (1995) Mol. Cell. Biol. 116: pp. 7069-7086) in successive
two-step gene replacements. To create TAF disruption molecules,
another cassette comprising an inframe fusion of ubiquitin,
arginine, LacI and the HA epitope-driven by the ANB1 promoter, was
fused in-frame to a short 5' fragment of TAF coding sequence
beginning at the initial TAF ATG. To create a given conditional
knockout strain, the relevant TAF knockout molecule on a URA3
integrating plasmid was linearized within the TAF coding sequence
fragment and transformed into ZMY60. This integration results in
homologous recombination at the TAF locus to yield a short,
non-functional 5' TAF piece under its normal promoter, followed
immediately downstream by a full-length copy of the tagged TAF
under the ANB1 promoter. Selection for uracil prototrophy was
maintained in all experiments to avoid loss of the integrated
plasmid.
[0136] RNA levels were determined by the quantitative S1 analysis
as described (Cormack, B. P. et. al., (1994) Genes Dev. 8: pp.
1335-1343; Iyer, V., and K. Struhl (1996) Proc. Natl. Acad. Sci.
USA 98: pp. 5208-5212). All hybridization reactions contained at
least two probes, such that the relative levels of all transcripts
were internally controlled. The error for any particular RNA
determination is .+-.30%. TAF levels were determined by western
blotting, with bands being detected by chemiluminescence (ECL).
Relative levels of TAFs at various times were determined by
comparing band intensities to serially diluted samples from
wild-type cells.
[0137] Results
[0138] As described above, a two-pronged approach was used to
create strains with conditional TAF alleles in which the addition
of copper ion leads to the simultaneous cessation of TAF messenger
RNA synthesis and destruction of any TAF protein present in the
cell. Strains with conditional alleles of TAF130 (TAF145), TAF90,
TAF60 and TAF19 (Fun81), which are homologous to human TAF250,
Drosophila TAF80, Drosophila TAF60 and human TAF18, respectively,
were generated. Of these, TAF130 is particularly interesting, as it
appears to be the scaffold on which the remaining TAFs assemble
into the TFIID complex (Chen, J.-L., et. al., (1994) Cell 79: pp.
93-105) As controls, strains containing conditional alleles of
TFIIB and TBP were generated. In all cases, the conditional
knockout strains fail to grow on copper-containing medium, and the
addition of copper ion caused cells to stop growing within about
six hours. TAF90-depleted cells arrest frequently as large, budding
cells, whereas cells depleted of other TAFs display variable and
abnormal morphologies.
[0139] In general, transcription was analysed 8 hours after copper
ion addition, when more than 95% of the cells were dead (they were
unable to grow when returned to medium lacking copper). At this
time, western blotting reveals that levels of TAF130, TAF90 and TBP
are less than 5% of wild type. Although TAFs may not be completely
eliminated by this procedure, they are reduced to less than 100-200
molecules per cell (Walker, S. S., et. al., (1996) Nature 385 pp.
185-188), which is considerably less than the number of Pol II
promoters per cell (-6,000).
[0140] When cells are grown under standard conditions, TAF
depletion affects transcription of selected Pol II promoters.
Depletion of TAF130, TAF60, TAF90 and TAF 19 does not significantly
affect transcription of ded1 or his3+13, promoters with canonical
TATA elements (Chen, W. and K. Struhl (1985) EMBO J, 4: pp.
3272-3280; Iyer, V. and K. Struhl (1995) Mol. Cell. Biol., 15: pp.
7059-7066). However, depletion of TAF130 significantly reduces the
level of the trp3 and his3+1 transcripts, which arise from
promoters with suboptimal, nonconsensus TATA elements (Iyer, V. and
K. Struhl (1995) Mol. Cell. Biol. 15: pp. 7059-7066; Martens, J. A.
and Brandt, C. J. (1994) J. Biol. Chem., 269: pp. 15661-15667).
This preferential effect on transcription from promoters containing
weak TATA elements is also observed when TAF 19 is depleted, albeit
to a lesser extent and with slower kinetics, but it does not occur
upon depletion of TAF90 or TAF60. Interestingly, the
transcriptional pattern resulting from TAF130 or TAF19 depletion is
similar to that mediated in yeast by human TBP, which has been
suggested to interact inefficiently with yeast TAFs (Cormack, B.
P., et. al. (1994) Genes. Dev. 8: pp. 1335-1343). As expected,
depletion of TBP or TFIIB results in a rapid and large reduction of
all mRNA species tested. At a late time point (11 hours), depletion
of TAF90 confers a moderate decrease of all transcripts. It is
unclear whether this effect reflects a specific function of TAF90
or arises indirectly from cell death.
[0141] Surprisingly, when the conditional knockout strains are
grown under conditions that support activation by Gcn4 or Ace1, TAF
depletion does not significantly affect the level of activated
transcription (Ace1-dependent activation appears slightly reduced
upon TAF90 depletion). In contrast, depletion of TFIIB causes the
loss of activated transcription in both situations. The observed
Gcn4- and Ace1-activated transcription reflects initiation events
that occur under conditions of TAF depletion, because mRNA
half-lives are very short in comparison to the timescale of the
experiment.
[0142] One explanation for the maintenance of Gcn4 and Ace1
activation after TAF depletion is that TAFs present in active
transcription complexes might be preferentially sequestered from
Ubr 1-dependent degradation. To examine whether active
transcription complexes could be assembled after TAF depletion, the
conditional knockout strains were grown in non-inducing conditions,
treated with copper for 8 hours, and then tested for the ability to
mediate activator-dependent transcription de novo. All the
TAF-depleted strains show significant activation by Gal4 and
heat-shock factor upon exposure to the relevant inducer, whereas
activation is not observed in the TFIIB-depleted strain. Similarly,
efficient Ace1-dependent activation was observed after TAFs were
depleted either by placing the TAF genes under the control of the
GAL1, 10 promoter and shifting cells to glucose or by shifting a
tsm1 (dTAF150 homologue) strain to the restrictive temperature. The
heat-shock and Ace1 activation responses in the TAF-depleted
strains are comparable to the parental strain; Gal4 activation is
reduced 3-4 fold. However, as very small fluctuations in Gal4
levels or changes in growth potential can have pronounced effects
on Gal4-dependent transcription (Griggs, D. W. and M. Johnston
(1991) Proc. Natl. Acad. Sci. USA, 88 pp. 8597-8601), it is unclear
whether the decrease reflects a mild activation defect or whether
Gal4 expression is slightly perturbed for other reasons. Previously
described activation-defective yeast strains are considerably more
impaired for Gal4-dependent activation, and they are defective in
the response to other acidic activators (Arndt, K. M. et. al.,
(1995) EMBO J, 14: pp. 1490-1497; Lee, M. and K. Struhl (1995) Mol.
Cell. Biol. 15: 5461-5469; Stargell, L. A. and Struhl, K. (1995)
Science, 269: pp. 75-78).
[0143] Since TAF depletion does not significantly affect activation
by Gcn4, Ace1, Gal4, Hsf, and unidentified activators involved in
ded1 and his3+13 transcription, TAFs do not appear to be required
for transcriptional activation in yeast cells. This conclusion was
reached independently in experiments where TAF depletion was
obtained using temperature-sensitive mutants or a glucose shutoff
procedure (Hernandez, N. (1993) Genes Dev., 7: pp. 1291-1308). It
is particularly striking that this is true of TAF130, which
provides the scaffold for TAF assembly and without which TFIID is
likely to be disrupted. Although TAFs are not generally required
for transcriptional activation, they are essential for cell growth.
One possibility is that TAFs are required for the response to a
subset of activators that affect one or more essential genes.
Alternatively, TAFs could subtly affect activation of many genes,
such that the cumulative effects lead to cell inviability. Finally,
as suggested by the effects on trp3 and his3+1 transcription, TAFs
may be important for transcription from promoters with weak TATA
elements.
[0144] This conclusion is in apparent contrast to numerous
experiments in vitro, which indicate that TAFs are crucial in all
activated transcription. This probably does not indicate that yeast
TAFs are less important than their mammalian and Drosophila
counterparts because: (a) TAFs are strongly conserved among
eukaryotes; (2) TAF-dependent activation in vitro can be achieved
with yeast components (Reese, J. et. al. (1994) Nature, 371:
523-527; Poon, D. et. al. (1995) Proc. Natl. Acad. Sci USA, 92:
8224-8228); and (3) activation can occur in a hamster cell line in
which TAF250 (yeast TAF130 homologue) has been thermally
inactivated (Fos transcription occurs normally, and it is unclear
whether the reduction of cyclin A transcription is an indirect
effect of cell-cycle arrest or a direct effect of TAF250) (Wang, E.
H. and Tijian, R., (1994) Science, 263: pp. 811-814). A more likely
explanation is that TAFs are functionally redundant with other
factors that are absent in typical in vitro reactions. Indeed,
activated transcription in the apparent absence of TAFs can occur
in vitro when reactions either contain Pol II holoenzyme (Koleske,
A. J. and Young, R. A. (1994) Nature, 368: pp. 446-469; Kim, Y.-J.
et. al., (1994), Cell, 77: 599-608) or are performed on chromatin
templates (Balasubramanian, B., et. al., (1993) Mol. Cell. Biol.
13: pp. 6071-6078). Moreover, most in vitro transcription reactions
are reconstituted with core Pol II, and hence may lack components
of the Pol II holoenzyme (for example, Srb proteins, Gal 11) that
are functionally important in vivo (Koleske, A. J. and Young, R. A.
(1995) Trends Biochem. Sci., 20: pp. 113-116).
[0145] A common view of the transcriptional activation process is
that activator proteins stabilize the Pol II machinery at the
promoter, thereby permitting increased transcriptional initiation
(Struhl, K. (1996) Cell, 84: pp. 179-182). In principle, activator
proteins can interact with individual components of the Pol II
machinery, and indeed, artificial connection of enhancer-bound
proteins to TBP (Chatterjee, S. and Struhl, K., (1995) Nature, 374:
pp. 820-822; Klages, N. and Strubin, M., (1995), Nature, 374:
822-823), TAFs and components of the Pol II holoenzyne (Barberis,
A. et. al. (1995) Cell, 81: 359-368) can bypass the need for an
activation domain. If natural activators interact with multiple
components, individual components such as TAFs are likely to be
non-essential for activation, even if they are potential targets.
Thus, although it is possible to generate conditions in which TAFs
are required for activation in vitro, they do not appear to be
generally required in vivo. However, at promoters lacking
conventional TATA elements, which are inherently weak targets for
TFIID, interactions of TAFs with basic transcription factors or
with promoter DNA may be important for stabilizing the Pol II
machinery.
5.2 Example 2
[0146] The following example demonstrates how any given target gene
can be configured for the double shutoff system in saccharomyces
cervisiae. The system requires two basic components: first, a
parent strain containing copper-inducible alleles of both ROX1 and
UBR1; and second, a short 5' fragment of the target gene of
interest fused in frame to a ubiquitin-arginine-lacI-HA ("URLF")
cassette and driven by the ANB1 promoter.
[0147] The strain ZMY60 has the genotype: MAT ay, ACE-UBR1,
Ace-ROX1, trp1-D1, ura3-52, LEU2, HIS3, ade2-101 in a KY114
background. To generate a parent strain in a different background,
one utilizes the URA3 integrating plasmids ZM195 and AM197, which
must be used in successive two-step gene replacements to generate a
parent strain containing copper-inducible ROX1 and UBR1. To create
double-shutoff parent strain, one integrates ZM195 into a desired
strain with AflII. The URA3 marker is the loopout on FOA
(5-fluorotica acid). One would then check for correct loopouts by
Southern blotting analysis. Next, one transforms a correctly
ROX1-replaced strain with AatII-digested AM197 and one loopout on
FOA. Check by Southern.
[0148] {Southern details: With the ZM195 integration, digest with
PvuII, and probe with a 5' piece of the ROX1 ORF (for example, the
550 bp cla1-Pst1 fragment of ZM195). Correct loopouts will pick up
a band at about 3 kb, wildtype ROX1 at about 1 kb.
[0149] With ZM197, digest with Stu1 and Bgl2, and probe with a 5'
piece of the UBR1 ORF (e.g., the 550 bp Hind3 fragment of ZM197).
Correct loopouts will again be 2 kb larger than incorrect
ones.}
[0150] To create the ANB-URLF gene fusion, digest or PCR out a
short, nonfunctional (usually about 300 bp) fragment the gene
beginning at the initial ATG. The fragment should contain a
convenient unique restriction site (not too close to either end,
and preferably closer to the 3' end of your fragment than to the
fusion junction) to allow for efficient integration of the final
plasmid at the normal gene locus. Using the reading frame
information provided, clone the fragment in frame into ZM168, and
then transfer the resulting ANB-URLF-gene fragment into a desired
yeast integrating vector. Alternatively, both steps can be done at
once with a 3-piece ligation.
[0151] To use the system:
[0152] 1. Digest the ANB-URLF-gene construct with the internal
restriction enzyme, and transform the resulting product into ZMY60
or the parent strain. Plate the transformants onto synthetic
complete plates lacking the plasmid marker.
[0153] 2. Test the transformants on plates lacking or containing
500 .mu.M copper sulfate. Be sure to maintain selection for the
marker so as not to allow looping out of the integrated plasmid. If
the gene is essential, the cells should fail to grow in the
presence of copper. More or less copper may be used depending on
the level of shutoff seen with the particular gene, but in general,
500 .mu.M is effective.
5.3 Example 3
[0154] DKO strains and constructs of TAF 19 (strain ZMY67), TAF60
(ZMY66), TAF90 (ZMY68), TAF130 (ZMY69, TBP, AND TFIIB (ZMY71).
[0155] Strain ZMY60 (the DKO parent strain)
[0156] Useful saccharomyces cervisiae strains:
[0157] Parent strains:
[0158] ZMY59 (Ace-UBR1 only strain)
[0159] ZMY61 (Ace-ROX1 only strain)
[0160] ZMY103 (ZMY60 his3.DELTA.200)
[0161] ZMY117 (ZMY60 leu2::PET56)
[0162] ZMY118 (ZMY60 his3.DELTA.200, leu2::PET56)
[0163] shutoff strains:
[0164] ZMY65 (TSM1 DKO)
[0165] ZMY70 (HIS3 DKO))
[0166] ZMY75 (triple-HA-tagged TAF 130 DKO)
[0167] ZMY76 (has complete gene replacement of TAF 130 for
ANB-URLF-TAF 130)
[0168] ZMY119 (TOA1 DKO strain)
[0169] ZMY95 (triple-HA-tagged TAF23 DKO)
[0170] ZMY96 (triple-HA-tagged TAF40 DKO)
[0171] ZMY97 (triple-HA-tagged TAF67 KO)
[0172] ZMY131 (TAF17 DKO)
[0173] ZMY133 (triple-HA-tagged TAF60 KO)
[0174] ZMY134 (TAF60 DKO-complete gene replacement)
[0175] Strains with single-prong shutoffs:
[0176] ZMY62 (TSM1 Ub only)
[0177] ZMY63 (TAF90 ub only)
[0178] ZMY64 (AF130 ub only)
[0179] HIS3 rox-only KO strain
[0180] The TAFs were shut-off by expressing each under direct
control of the GAL 1,10 UAS+switching to glucose and by using the
Ace-HIS3 (Klein and Struhl) promoter, growing in the presence of
copper and then shifting by removing the copper (this, however, is
less efficient than with galactose--cell growth never actually
stopped, only slowed.)
[0181] The ub-only prong was tested by using a reporter plasmid
(obtained from Dan Finley) called pUB23L (incorporating a Leu as
the N-end residue in front of a ub-b-gal reporter. In the presence
of 200 .mu.M copper and X-gal, the ub-only strain ZMY59 with this
reporter is white. Without copper, it is blue.
[0182] A HIS3 DKO and a HIS3 Rox-only strain was tested and found
that in the presence of 5001M CuSO.sub.4, both failed to grow on
plates lacking histidine. Without copper, growth was entirely
normal on plates lacking histidine.
[0183] The urlf allele is usually introduced by means of a one-step
integration that simultaneously truncates the endogenous copy.
(This could also be done by shuffling in a cen plasmid in a null
strain). The strain ZMY76) can stably integrate the urlf allele
with a 2-step gene replacement. This means that there is no
truncated piece left upstream, and no remaining repeated sequence
fragments requiring maintenance of selection for the integrating
plasmid marker.
1 Testing the system: Growth of the indicated strains (+ = strong
growth; - = no growth; +/- = intermediate growth; ND = not
determined). Allele 100 .mu.M Cu 250 .mu.M Cu 500 .mu.M Cu 1 mM
ub-only TAF90 + ND - ND DKO TAF90 +/- - - - ub-only TAF130 + + + ND
DKO TAF130 +/- - - - DKO TFIIB ND - - - ub-only TSM1 + + + + DKO
TSM1 + + +/- -
[0184] Lee and Lis (1998) have used the system to shut off SRB4 and
KIN28 (Nature 393:389-92)
[0185] I have used the system to shut off TOA1 (loss of much PolII
tx-manuscript in prep), TAFs17, 40 and 67 (submitted 17 causes a
general loss of tx, and 40 and 67 cause gene-specific defects), and
TAF23.
[0186] Equivalents
[0187] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, numerous
equivalents to the specific procedures described herein. Such
equivalents are considered to be within the scope of this invention
and are covered by the following claims.
* * * * *