The N-degron is an intracellular degradation signal whose essential determinant is a specific, destabilizing, N-terminal amino acid residue. A set of N-degrons containing different destabilizing residues is manifested as the N-end rule, which relates the in vivo half-life of a protein to the identity of its N-terminal amino acid residue. Disclosed herein is a heat-inducible N-degron module. A heat-inducible N-degron module is a protein or peptide bearing a destabilizing N-terminal amino acid residue which becomes a substrate of the N-end rule pathway only at a temperature high enough to result in at least partial unfolding of the protein. At this elevated (nonpermissive) temperature, the heat-inducible N-degron module (and any protein or peptide attached at its C-terminus) is rapidly degraded in a cell in which the N-end rule pathway is operative. Also disclosed are DNA and protein fusion constructs, methods for screening for additional heat-inducible N-degron modules and methods for using the disclosed heat-inducible N-degron modules.

BACKGROUND OF THE INVENTION 
A conditional mutant retains the function of a gene under one set of 
conditions, called permissive, but lacks that function under a different 
set of conditions, called nonpermissive; the latter must still be 
permissive for the wild-type allele of the gene. Conditional mutants are 
presumed, in most cases, to result from missense mutations in a structural 
gene encoding a protein. In the case of temperature-sensitive (ts) 
mutants, the amino acid replacement resulting from the missense mutation 
partially destabilizes the encoded protein, resulting in the maintenance 
of its three-dimensional integrity only at relatively low temperatures. 
Conditional mutants make possible the analysis of physiological changes 
caused by inactivation of a gene or gene product, and can be used to 
address the function of any gene. This strategy is especially valuable for 
the analysis of essential genes. Several types of conditional mutants and 
methods for producing them have been developed since the original 
demonstration of the utility of ts mutants (Horowitz, Genetics 33, 612 
(1948); Horowitz, Adv. Genetics 3, 33(1950)) but the ts phenotype is still 
the one most frequently used. 
One limitation of the ts approach is the uncertainty as to whether a given 
gene can be mutated to yield a ts product. For example, only six loci were 
identified after repeated searches for ts lethal mutations mapping to the 
S. cerevisiae chromosome I, which contains at least one hundred genes 
(more that six of which are essential) (Kaback et al., Genetics 108: 67 
(1984); Harris and Pringle, Genetics 127: 279 (1991)). Another problem 
with conventional ts mutations is that they are often too "leaky" to be 
useful. That is, the function of a leaky ts protein at nonpermissive 
temperatures is not fully blocked by the mutation. For these and other 
reasons, a method for producing ts mutants which does not require a search 
for a ts mutation in a gene of interest would be extremely useful in a 
variety of applications. 
SUMMARY OF THE INVENTION 
The subject invention relates, in one aspect, to a heat-inducible N-degron 
module and to DNA encoding same. In a preferred embodiment, the DNA 
encoding the heat-inducible N-degron module hybridizes to the DNA 
represented in SEQ ID NO: 1, or its complement, under stringent 
hybridization conditions. 
The DNA encoding the heat-inducible N-degron module can be linked 
covalently at its 3' end to the 5' end of a DNA sequence encoding a 
protein (or peptide) of interest. When expressed in a cell in which the 
N-end rule of protein degradation is operative, the heat-inducible 
N-degron module, and any protein (or peptide) linked to the C-terminus of 
the heat-inducible N-degron module, are rapidly degraded by enzymatic 
components of the N-end rule proteolytic pathway. 
A specific heat-inducible N-degron module is disclosed herein. In addition, 
methods for the identification of additional functional heat-inducible 
N-degron modules are also disclosed. Such methods are useful for the 
isolation of heat-inducible N-degron modules using simple screening 
processes. Finally, it is disclosed that a low molecular weight ligand 
that binds to a heat-inducible N-degron can interfere with its activation 
by heat, thereby allowing modulation of the activity of the N-degron by 
agents other than temperature. 
STATEMENT OF UTILITY 
Prior to the development of ts mutants, the range of genetic analysis was 
severely limited due to the fact that mutants which were defective in an 
essential function could not be studied due to the lethality of the 
genetic lesion. This problem was resolved, to some degree, through the 
development of the ts and other conditional mutants. However, the 
identification of a ts mutant is a laborious, time-consuming procedure 
which includes a first step in which mutations are randomly induced, and a 
second step in which mutants are isolated (e.g., by non-selective 
isolation, enrichment or screening, and by selective isolation procedures 
as well (for a review see Pringle, in Methods in Cell Biology, Academic 
Press, New York, Prescott, ed., 233-271 (1975)). 
The heat-inducible N-degron module of the subject invention is useful, for 
example, for the generation of a ts mutant without the need for the 
time-consuming classical approach to identification of a ts mutant 
(described above). As described in detail below, the heat-inducible 
N-degron module is linked via its C-terminal residue to the N-terminal 
residue of a protein (or peptide) of interest. The protein of interest can 
be either essential or nonessential for cell viability. The resulting 
fusion protein will be rapidly degraded by the N-end rule pathway at a 
nonpermissive temperature, but not at a lower, permissive temperature.

DETAILED DESCRIPTION OF THE INVENTION 
The N-degron is an intracellular degradation signal whose essential 
determinant is a specific ("destabilizing") N-terminal amino acid residue 
of a substrate protein. A set of N-degrons containing different 
destabilizing residues is manifested as the N-end rule, which relates the 
in vivo half-life of a protein to the identity of its N-terminal residue. 
The fundamental principles of the N-end rule, and the proteolytic pathway 
that implements it, are well-established in the literature (see, e.g., 
Bachmair et al., Science 234: 179 (1986); Varshavsky, Cell 69: 725 
(1992)), and are the subject of several issued patents. Specifically, 
aspects of the N-end rule which are relevant to the subject invention are 
patented in U.S. Pat. Nos.: 5,132,213, 5,093,242 and 5,196,321, the 
disclosures of which are incorporated herein by reference. 
In eukaryotes, the N-degron comprises at least two determinants: a 
destabilizing N-terminal residue and a specific internal lysine residue 
(or residues). The latter is the site of attachment of a multiubiquitin 
chain, whose formation is required for the degradation of at least some 
N-end rule substrates. Ubiquitin is a protein whose covalent conjugation 
to other proteins plays a role in a number of cellular processes, 
primarily through routes that involve protein degradation. 
In a stochastic view of the N-degron, each internal lysine of a protein 
bearing a destabilizing N-terminal residue can be assigned a probability 
of being utilized as a multiubiquitination site, depending on 
time-averaged spatial location, orientation and mobility of the lysine. 
For some, and often for all of the Lys residues in a potential N-end rule 
substrate, this probability would be infinitesimal because of the lysine's 
lack of mobility and/or its distance from a destabilizing N-terminal 
residue. 
The present invention is based on the discovery that it is possible to 
construct a thermolabile protein bearing a destabilizing N-terminal 
residue in such a way that the protein becomes a substrate of the N-end 
rule pathway only at a temperature high enough to result in at least 
partial unfolding of the protein. This unfolding activates a previously 
cryptic N-degron in the protein by increasing exposure of its 
(destabilizing) N-terminal residue, by increasing mobilities of its 
internal Lys residues, or because of both effects at once. Since 
proteolysis by the N-end rule pathway is highly processive, any protein of 
interest can be made short-lived at a high (nonpermissive) but not at a 
low (permissive) temperature by expressing it as a fusion to the thus 
engineered thermolabile protein, with the latter serving as a portable, 
heat-inducible N-degron module. 
The heat-inducible N-degron module can be any protein or peptide bearing a 
destabilizing N-terminal residue which becomes a substrate of the N-end 
rule pathway only at a temperature high enough to be useful as a 
nonpermissive temperature. In the Exemplification section which follows, 
an example of such a heat-inducible N-degron module is provided. More 
specifically, the experiments described herein disclose a ts allele of the 
21-kd mouse dihydrofolate reductase protein, in which the wild-type 
N-terminal Val is replaced by Arg. 
The experimental work disclosed herein demonstrates that this ts allele 
functions as a heat-inducible N-degron module. More specifically, when 
this heat-inducible N-degron module is fused at its C-terminus to the 
N-terminus of a protein (or peptide) of interest, the protein (or peptide) 
of interest also becomes short-lived at the nonpermissive temperature due 
to the highly processive nature of the N-end rule pathway. Throughout this 
document, the use of the expression "protein of interest" specifically 
includes a peptide of interest. Processivity, as used in this context, is 
defined as the ability of a pathway to complete the initially started 
degradation of a protein, resulting in protein fragments whose sizes do 
not significantly exceed those of small peptides (e.g., less than about 20 
amino acid residues). This ability is well-established for 
ubiquitin-dependent proteolytic pathways, and in particular for the N-end 
rule pathway. It is indicated in particular by the total disappearance of 
various protein fusions degraded by the N-end rule pathway (see e.g., 
Hershko, J. Biol. Chem. 263: 15237 (1988); Rechsteiner, Cell 66: 615 
(1991); and Varshavsky, Cell 69, 725 (1992)). 
The DNA sequence of the ts allele of the 21-kd mouse dihydrofolate 
reductase protein is set forth in SEQ ID NO: 1. The amino acid residues 
encoded by this DNA sequence are represented in SEQ ID NOS: 1 and 2. The 
scope of the invention encompasses any heat-inducible N-degron module 
which is encoded by a DNA sequence which hybridizes to the DNA sequence of 
SEQ ID NO: 1, or the complement thereof, under stringent hybridization 
conditions. Stringent hybridization conditions, as used herein, refer to 
hybridization in which the DNA molecule represented in SEQ ID NO: 1 is 
fixed to a solid support and a second DNA molecule to be tested for the 
ability to hybridize to the DNA of SEQ ID NO: 1 is detectably labeled and 
suspended in a hybridization buffer consisting essentially of 50% 
formamide, 5.times.SSPE (1.times.SSPE is 0.15 mM NaCl, 1 mM Na-EDTA, 10 mM 
Na-phosphate (pH 7.0), 5.times.Denhardt's solution (0.1% 
polyvinylpyrrolidone, 0.1% Ficoll)). The hybridization buffer is contacted 
with the solid support at a temperature of about 45.degree. C. for a 
period of several hours. The hybridization solution is then removed, and 
non-specifically bound nucleic acid is removed by repeated washing with 
1.times.SSC at increasing temperatures (up to 65.degree. C.). 
Identification of additional heat-inducible N-degron modules requires 
exclusively straightforward experimental procedures, such as those 
described in the Exemplification section, which follows. More 
specifically, in the experiments described below, a nucleic acid construct 
encoding Ub-Arg-DHFR-ha-Ura3 is described. The fusion protein encoded by 
this construct is carried on a plasmid which also carries a gene for a 
selectable marker, and has several features which facilitate the 
identification of a heat-inducible N-degron module. The N-terminal 
ubiquitin moiety is included as a transient moiety specifying a cleavage 
site in the encoded fusion protein between the Ub and Arg-DHFR moieties. 
Linear fusions of ubiquitin have been demonstrated to be efficiently 
cleaved between the C-terminal glycine and the N-terminal amino acid 
residue of the ubiquitin fusion partner (see, e.g., Bachmair et al., 
Science 234: 179 (1986); Bachmair and Varshavsky, Cell 56: 1019 (1989)). 
This specific cleavage is effected by a ubiquitin-specific protease 
activity which has been identified in all eukaryotes examined. 
Arg-DHFR is a variant of the 21-kd mouse dihydrofolate reductase in which 
the wild-type N-terminal Val is replaced by Arg. Arg is a destabilizing 
residue according to the N-end rule, in that exposure of Arg at the 
N-terminus of a protein should, if other conditions are met as well, 
transform a relatively stable (long-lived) protein (such as DHFR or any 
other protein) to a less stable (more short-lived) protein. The "ha" 
portion is a 14-residue domain containing an ha epitope. The ha epitope 
facilitates immunoprecipitation of the Arg-DHFR-ha-Ura3 fusion with a 
monoclonal anti-ha antibody. The S. cerevisiae Ura3 domain of the fusion 
protein made possible selections for or against the presence of the fusion 
protein in cells, while also serving as a test protein. 
It will be recognized that individual components of the Arg-DHFR-ha-Ura3 
fusion protein can be replaced by functional homologs without compromising 
the value of the fusion protein for use in a method for identifying a 
heat-inducible N-degron module. For example, Ura 3 domain can be replaced 
with another selectable marker domain. Similarly, the ha epitope can be 
replaced by another immunological tag which facilitates 
immunoprecipitation of the fusion protein. 
To identify additional heat-inducible N-degron modules, a modified protein 
or peptide moiety other than DHFR can be substituted for Arg-DHFR in the 
Arg-DHFR-ha-Ura3 fusion protein described above. For example, consider a 
protein designated "Protein X" (pX). A modified pX bearing a destabilizing 
N-terminal amino acid residue (e.g., Arg-pX) can be substituted for 
Arg-DHFR in the fusion construct described in the preceding paragraph. As 
described herein, this is achieved by placing the Ub moiety in front of 
Arg-pX within a fusion; the cotranslational cleavage of the Ub moiety at 
the Ub-Arg junction in vivo will then yield a protein bearing Arg-pX at 
its N-terminus. 
The DNA encoding the Ub-Arg-pX-ha-Ura3 moiety can then be treated with a 
mutagen. In a ts mutant, the gene product is preferably not too 
dramatically altered. Therefore, it is preferable to employ mutagens 
characterized by a tendency to produce missense mutations rather than 
mutagens which tend to induce more extensive genetic lesions such as 
deletions. The experiments disclosed herein demonstrate that hydroxylamine 
is an appropriate mutagen. In addition, a variety of other known mutagens 
including, for example, N-methyl-N'-nitro-N-nitrosoguanidine (NG), nitrous 
acid (NA), ethylmethane sulfonate (EMS), and ultraviolet light are known 
to be useful for the generation of ts mutants (see e.g., Pringle, in 
Methods in Cell Biology, Academic Press, New York, Prescott, ed., 233-271 
(1975)). The listing of appropriate mutagens provided herein is meant to 
provide examples of useful mutagens and is not meant to be comprehensive 
in the listing of useful mutagens. 
The resulting DNA, carried in an appropriate plasmid, is then used to 
transform cells (e.g., E. coli MC1066 cells) to ampicillin resistance with 
the selection being carried out at 37.degree. C. Transformants are then 
replica-plated, for example, onto M9 plates containing amp, Trp and Leu, 
and lacking uracil. Under this selection scheme, the yeast URA3 gene 
complements the Ura.sup.- phenotype of E. coli pyrF mutants. This E. coli 
screen will eliminate mutant plasmids that do not express a functional 
Ura3 moiety of Ub-Arg-pX-ha-Ura3 at 37.degree. C. 
Appropriate plasmid constructs which express a functional Ura3 moiety of 
Ub-Arg-pX-ha-Ura3 at 37.degree. C. are then screened for the ability to 
confer a ts Ura.sup.+ phenotype whose ts aspect requires the N-end rule 
pathway. This is accomplished, for example, by introducing such plasmids 
into S. cerevisiae YPH500 (ura3), with transformants selected at 
23.degree. C. on SD(-Ura) plates containing 0.1 mM CuSO.sub.4. Resulting 
colonies are replica-plated onto plates appropriate for selection. For 
example, the resulting colonies can be replica-plated onto SD plates 
lacking His and containing 5-fluoroorotic acid (FOA) and uracil. The 
inclusion of FOA serves as a selection against cells expressing Ura3 
(Boeke et al., Mol. Gen. Genet. 197, 345 (1984)). The FOA plates are then 
incubated at 37.degree. C. to select against cells that could yield a 
functional Ura3 at 37.degree. C. After several rounds of the FOA-mediated 
selection against cells that are Ura.sup.+ at 37.degree. C., the ts 
URA.sup.+ phenotype of surviving cell clones can be verified by 
replica-plating them onto SD(-Ura, -His) plates at 37.degree. C. Plasmids 
from cells passing these screens are introduced into cells such as the 
YPH500-derived strain JD15 (described in detail below), with transformants 
selected on SD(-Ura) plates at 37.degree. C. This step narrows the 
selection to plasmids having the ability to confer the ts Ura.sup.+ 
phenotype only in the presence of the N-end rule pathway. Conventional 
sequencing techniques are then used to confirm that the mutation 
responsible for the desired phenotype is present within the pX domain of 
the fusion protein. Upon confirmation of this, the mutant Arg-pX moiety 
has been identified as a heat-inducible N-degron module. 
DNA encoding a heat-inducible N-degron module of the type described above 
can be linked at its 3' end to the 5' end of DNA encoding a protein or 
peptide of interest, yielding a desired gene fusion construct. The gene 
fusion, together with any regulatory sequences required for expression, is 
introduced into cells using conventional techniques. The cells into which 
the gene fusion is introduced can be either cells grown in culture, or 
differentiated tissue cells in a whole organism. It will be recognized 
that these cells should also lack a functioning allele of the gene whose 
ts mutation is being sought. This can be accomplished, for example, 
through the use of targeted mutagenesis techniques which are well known in 
the art. This gene fusion construct is then expressed to produce a protein 
fusion in which the heat-inducible N-degron module is joined covalently at 
its C-terminus to the N-terminus of the protein or peptide of interest. 
Provided that the cell in which the gene fusion construct is expressed is a 
cell in which the N-end rule degradation pathway is operative (e.g., any 
eukaryotic cell), the metabolic fate of the protein or peptide of interest 
in the fusion protein will be determined by the presence of the 
heat-inducible N-degron module. Due to the highly processive nature of the 
N-end rule degradation pathway, the recognition of a destabilizing 
N-terminal amino acid residue by the recognition component of the pathway 
seals the fate of the protein fusion. Specifically, at nonpermissive 
temperatures, the N-terminal residue will be recognized by the recognition 
component of the N-end rule degradation pathway, and the entire fusion 
protein will be rapidly degraded. This typically results in a strong 
decrease of the steady state concentration of the fusion and, 
consequently, in a null phenotype for the protein or peptide of interest. 
EXEMPLIFICATION 
A thermolabile protein was constructed that functions as a substrate of the 
N-end rule pathway only at a temperature high enough to be useful as a 
nonpermissive temperature. This unfolding activates a previously cryptic 
N-degron module in the protein. Since proteolysis by the N-end rule 
pathway is highly processive, any protein of interest can be made 
conditionally short-lived by expressing it as a fusion to the thus 
engineered thermolabile protein, with the latter serving as a portable, 
heat-inducible N-degron module. 
Arg-DHFR, a variant of the 21-kd mouse dihydrofolate reductase in which the 
wild-type N-terminal Val is replaced by Arg, is long-lived in the yeast S. 
cerevisiae (t.sub.1/2 &gt;6 hr at 30.degree. C.), even though Arg (unlike 
Val) is a destabilizing residue in the N-end rule. A search was conducted 
for a ts allele of Arg-DHFR whose cryptic N-degron would be activated at 
37.degree. C. but not at 23.degree. C. A plasmid (pPW17R) was constructed 
that expressed Ub-Arg-DHFR-ha-Ura3 in S. cerevisiae. 
Briefly, referring to FIG. 1, a fusion protein on the left contains an 
N-terminal ubiquitin (Ub) moiety, a ts dihydrofolate reductase 
(DHFR.sup.ts) moiety, with a destabilizing residue such as Arg (R) at the 
Ub-DHFR junction, and a test protein moiety at the C-terminus of the 
fusion. In the experiments described herein, the test proteins were Ura3 
and Cdc28 of S. cerevisiae. Some of the Lys (K) residues of DHFR.sup.ts 
are indicated in FIG. 1 as well. Expression of this fusion in a eukaryote 
such as the yeast S. cerevisiae results in rapid cleavage at the Ub-DHFR 
junction and the exposure of a destabilizing Arg (R) residue at the 
N-terminus of a deubiquitinated fusion. At permissive temperature 
(23.degree. C.), the N-degron module of the Arg-DHFR.sup.ts moiety is 
inactive. However, at nonpermissive temperature (37.degree. C.), a 
conformational destabilization of Arg-DHFR.sup.ts results in at least some 
of its lysines becoming available as ubiquitination sites of the 
previously cryptic N-degron module. The processive degradation of the 
fusion by the N-end rule pathway then ensues, greatly reducing its level 
in the cell. In the examples shown, the yeast Ura3 (orotidine-5'-phosphate 
decarboxylase) as the C-terminal moiety of the fusion resulted in 
Ura.sup.+ cells at 23.degree. C. but in Ura.sup.- cells at 37.degree. C. 
Similarly, when the essential kinase Cdc28 was expressed as an 
Arg-DHFR.sup.ts -Cdc28 fusion, cells grew at 23.degree. C., but not at 
37.degree. C. With either Arg-DHFR.sup.ts -Ura3 or Arg-DHFR.sup.ts -Cdc28, 
the absence of the N-end rule pathway (in ubr1.DELTA. cells) precluded 
these conditional phenotypes at 37.degree. C. Thus, Arg-DHFR.sup.ts can be 
used as a portable, heat-inducible N-degron that yields ts mutants of a 
new class, called td (temperature-inducible degron). 
More specifically, the CEN6, HIS3-based plasmid pPW17R, which expressed 
Ub-Arg-DHFR-ha-Ura3 from the S. cerevisiae P.sub.CUP1 promoter, was 
constructed in the background of pRS313 (R. S. Sikorski and P. Hieter, 
Genetics 122, 19 (1989)). Briefly, a .about.0.4 kb fragment from pJDC22-2 
(K. Madura, R. J. Dohmen, A. Varshavsky, J. Biol. Chem. 268, 12046 (1993)) 
that contained the P.sub.CUP1 promoter was ligated to a separately 
constructed fragment encoding Ub-Arg-DHFR-ha-Ura3. The DHFR moiety was 
followed by a 14-residue, ha epitope-containing sequence. The Ura3 moiety 
of Ub-Arg-DHFR-ha-Ura3 was actually a fusion of the last 91 residues of S. 
cerevisiae His4 to residue 6 of the Ura3 protein (E. Alani and N. 
Kleckner, Genetics 117, 5 (1987)). 
The ubiquitin (Ub) moiety of this fusion protein was required for 
production of the desired residue, such as Arg, at the N-terminus of the 
DHFR moiety. Ubiquitin fusions are rapidly cleaved in vivo after the last 
residue of ubiquitin, making possible the production of otherwise 
identical proteins bearing different N-terminal residues (FIG. 1) (see 
e.g., Varshavsky, Cell 69, 725 (1992)). The "ha" epitope allowed 
immunoprecipitation of the Arg-DHFR-ha-Ura3 fusion with a monoclonal 
anti-ha antibody. The S. cerevisiae Ura3 moiety made possible selections 
for or against the fusion's presence in cells, while also serving as a 
test protein (FIG. 1). 
Purified pPW17R was mutagenized with hydroxylamine (S. Busby, M. Irani, B. 
deCrombrugghe, J. Mol. Biol. 154, 197 (1982)). The resulting DNA was used 
to transform the Ura.sup.- (pyrF) E. coli MC1066 (M. J. Casadaban, A. 
Martinez-Ariaz, S. K. Shapira, J. Chow, Methods Enzymol. 100, 293 (1983)) 
to ampicillin (amp) resistance, with selection on Luria Broth/amp plates 
at 37.degree. C. Transformants were replica-plated onto M9 plates 
containing amp, Trp and Leu, and lacking uracil. The yeast URA3 gene 
complements the Ura.sup.- phenotype of E. coli pyrF mutants (M. Rose, P. 
Grisafi, D. Botstein, Gene 29, 113 (1984)). This E. coli screen eliminated 
mutant plasmids that did not express a functional Ura3 moiety of 
Ub-Arg-DHFR-ha-Ura3 at 37.degree. C. However, those (potentially relevant) 
plasmids that expressed a mutant DHFR moiety were expected to pass this 
test since E. coli lacks the ubiquitin system. The N-terminal ubiquitin 
moiety of Ub-Arg-DHFR-ha-Ura3 was therefore retained in E. coli, 
precluding the formation of an N-degron. 
A screen was carried out for derivatives of pPW17R that could confer onto 
Ura.sup.- cells a ts Ura.sup.+ phenotype whose ts aspect required the 
N-end rule pathway. More specifically, plasmids that passed the E. coli 
screen described above were introduced in S. cerevisiae YPH500 (ura3), (R. 
S. Sikorski and P. Hieter, Genetics 122, 19 (1989)), with transformants 
selected at 23.degree. C. on SD(-Ura) plates containing 0.1 mM CuSO.sub.4. 
The colonies were replica-plated onto SD plates lacking His and containing 
5-fluoroorotic acid (FOA) and uracil (J. D. Boeke, F. Lacroute, G. R. 
Fink, Mol. Gen. Genet. 197, 345 (1984)). The FOA plates were incubated at 
37.degree. C. to select against cells (carrying pPW17R plasmids) that 
could yield a functional Ura3 at 37.degree. C. After several rounds of the 
FOA-mediated selection against cells that were Ura.sup.+ at 37.degree. C., 
the ts URA.sup.+ phenotype of surviving cell clones was verified by 
replica-plating them onto SD(-Ura, -His) plates at 37.degree. C. Plasmids 
from cells that passed these screens were introduced into the 
YPH500-derived strain JD15 (ubr1-.DELTA.1::LEU2 ura3, produced identically 
to ubr1.DELTA. strains described previously (B. Bartel, I. Wunning, A. 
Varshavsky, EMBO J. 9, 3179 (1990); K. Madura, R. J. Dohmen, A. 
Varshavsky, J. Biol. Chem. 268, 12046 (1993)), with transformants selected 
on SD(-Ura) plates at 37.degree. C. This step narrowed the selection to 
plasmids whose ability to confer the ts Ura.sup.+ phenotype required the 
presence of the N-end rule pathway. 
This screen yielded two mutant plasmids with the desired properties: at 
23.degree. C., these plasmids conferred a Ura.sup.+ phenotype, whereas at 
37.degree. C. they conferred a Ura.sup.- phenotype in [UBR1 ura3] cells 
but a Ura.sup.+ phenotype in congenic [ubr1.DELTA. ura3] cells. The 
[ubr1.DELTA. ura3] strain lacked the N-end rule pathway because it lacked 
N-recognin (encoded by UBR1), the recognition component of the degradation 
pathway. The relevant change in both plasmids was a single missense 
mutation that replaced Pro with Leu at position 66 in the DHFR moiety of 
Ub-Arg-DHFR-ha-Ura3, yielding Ub-Arg-DHFR.sup.ts -ha-Ura3. The Pro.sup.66 
region of DHFR connects its .alpha.II helix to the .beta.C strand (C. 
Oefner, A. D'Arcy, F. K. Winkler, Eur. J. Biochem. 174, 377 (1988); K. W. 
Volz et al., J. Biol. Chem. 257, 2528 (1982)). The final construct, termed 
pPW43R, was produced from the unmutagenized pPW17R by replacing its EcoRI 
fragment encoding Ub-Arg-DHFR-ha-Ura3 with the otherwise identical 
fragment from one of the above plasmids encoding Ub-Arg-DHFR.sup.ts 
-ha-Ura3. 
Arg-DHFR.sup.ts was then used to produce a ts version of the S. cerevisiae 
Cdc28 protein kinase--an essential component of the cell cycle oscillator. 
The chromosomal CDC28 gene was replaced with a gene that expressed 
Ub-Arg-DHFR.sup.ts -ha-Cdc28. More specifically, the plasmid pPW66R was 
constructed in the background of the integration vector pRS306, (R. S. 
Sikorski and P. Hieter, Genetics 122, 19 (1989)). Briefly, the previously 
described DNA fragment encoding Ub-Arg-DHFR.sup.ts -ha was ligated to a 
fragment (produced using PCR and S. cerevisiae genomic DNA) that 
encompassed the first 284 nucleotides of the CDC28 ORF (S. I. Reed, Annu. 
Rev. Cell Biol. 8, 529 (1992); A. Murray, Nature 359,599 (1992); A. B. 
Futcher, Semi. Cell Biol. 2, 205 (1991); K. Nasmyth, L. Dirick, U. Surana, 
A. Amon, F. Cvrckova, Cold Spring Harbor Symp. Quant. Biol. 56, 9 (1991); 
P. Nurse, Nature 344, 503 (1990)). The resulting fragment, encoding 
Ub-Arg-DHFR.sup.ts -ha-Cdc28.sub.1-95, was positioned downstream from the 
P.sub.CUP1 promoter in pRS306, yielding pPW66R. This plasmid was 
linearized at the Msc I site (nucleotide 92 in the CDC28 ORF) and 
transformed into S. cerevisiae YPH500. In the resulting Ura.sup.+ 
integrants, homologous recombinations (R. Rothstein, Methods Enzymol. 194, 
281 (1991)) between the proximal regions of CDC28 in pPW66R and in 
Chromosome II resulted in the integration of pPW66R and formation of an 
ORF encoding Ub-Arg-DHFR.sup.ts -ha-Cdc28 (which contained the full-length 
CDC28.sub.1-299 moiety), in addition to a nearby sequence encoding 
Cdc28.sub.1-95. This truncated allele of CDC28 was neither functional nor 
dominant negative. 
The resulting S. cerevisiae strains were compared to the wild-type (CDC28) 
strain YPH500. Whereas the wild-type strain grew at both 23.degree. C. and 
37.degree. C., a representative strain expressing Ub-Arg-DHFR.sup.ts 
-ha-Cdc28 (instead of the wild-type Cdc28) grew at 23.degree. C. but was 
inviable at 37.degree. C. The morphology of these cells was examined 
following the temperature upshift in liquid culture. After 2 hr at 
37.degree. C., cells that expressed Ub-Arg-DHFR.sup.ts -ha-Cdc28 became 
larger but lacked buds (G1 phase morphology); however, by 4 hr at 
37.degree. C., many of these cells developed abnormal (elongated) buds and 
arrested in this configuration, which is similar to the arrest phenotype 
observed with some of the conventional ts alleles of CDC28. This 
Cdc28-mediated ts lethal phenotype required the presence of the N-end rule 
pathway, inasmuch as ubr1.DELTA. cells that expressed Ub-Arg-DHFR.sup.ts 
-ha-Cdc28 grew at both 23.degree. C. and 37.degree. C., and remained 
morphologically normal at 37.degree. C. 
Pulse-chase experiments confirmed that Arg-DHFR.sup.ts -ha-Cdc28 was 
long-lived at 23.degree. C. but short-lived at 27.degree. C. (t.sub.1/2 
&lt;10 min). More specifically, exponential cultures of either UBR1 or 
ubr1.DELTA. S. cerevisiae that expressed Arg-DHFR.sup.ts -ha-Cdc28.sup.td, 
were labeled with .sup.35 S-methionine for 5 min at 23.degree. C., 
followed by a chase at 23.degree. C. or 37.degree. C. for zero, 10 and 30 
min, extraction, immunoprecipitation with anti-ha antibody, and SDS-PAGE 
analysis. The onset of metabolic instability of Arg-DHFR.sup.ts -ha-Cdc28 
upon the temperature upshift was extremely rapid. As could be expected 
from the results of phenotypic analysis, Arg-DHFR.sup.ts -ha-Cdc28 was 
long-lived at both temperatures in ubr1.DELTA. cells which lacked the 
N-end rule pathway. 
In addition, it was found that the addition of a specific DHFR ligand, 
methotrexate (MTX), to cells whose essential Cdc28 protein is expressed as 
the ts degron-bearing fusion (Ub-Arg-DHFR.sup.ts -ha-Cdc28) resulted in 
the inhibition of heat induction of the ts N-degron upon transfer of cells 
to nonpermissive (N-degron-inducing) temperature. Specifically, cells 
expressing Ub-Arg-DHFR.sup.ts -ha-Cdc28 remained viable at 37.degree. C. 
in the presence of MTX at a sufficiently high concentration in the medium, 
whereas in the absence of MTX at 37.degree. C. these cells ceased division 
and died, as described above. Pulse-chase experiments confirmed that this 
viability-rescuing effect of MTX (which is known to bind DHFR tightly and 
specifically) was due to the inhibition of degradation of 
Ub-Arg-DHFR.sup.ts -ha-Cdc28 at 37.degree. C. as a result of binding of 
MTX to DHFR. 
This discovery indicated that the activity of an N-degron can also be 
modulated by agents other than temperature, making possible new classes of 
conditional mutants. Specifically, these results identified MTX as an 
agent that inhibits the activity of the N-degron based on the MTX ligand 
DHFR. They also pointed out the way to identify other such agents for 
N-degrons other than those based on DHFR. Specifically, these results 
indicated that the binding of a low molecular weight ligand to a protein 
component of an N-degron can interfere with the unfolding of this protein 
module at nonpermissive temperatures, and thereby can interfere with the 
targeting of the said degron by the corresponding proteolytic pathway such 
as the N-end rule pathway. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 2 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 579 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..579 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
AGGCACGGATCCGGCATCATGGTTCGACCATTGAACTGCATCGTCGCC48 
ArgHisGlySerGlyIleMetValArgProLeuAsnCysIleValAla 
151015 
GTGTCCCAAAATATGGGGATTGGCAAGAACGGAGACCTACCCTGGCCT96 
ValSerGlnAsnMetGlyIleGlyLysAsnGlyAspLeuProTrpPro 
202530 
CCGCTCAGGAACGAGTTCAAGTACTTCCAAAGAATGACCACAACCTCT144 
ProLeuArgAsnGluPheLysTyrPheGlnArgMetThrThrThrSer 
354045 
TCAGAGGAAGGTAAACAGAATCTGGTGATTATGGGTAGGAAAACCTGG192 
SerGluGluGlyLysGlnAsnLeuValIleMetGlyArgLysThrTrp 
505560 
TTCTCCATTCCTGAGAAGAATCGACTTTTAAAGGACAGAATTAATATA240 
PheSerIleProGluLysAsnArgLeuLeuLysAspArgIleAsnIle 
65707580 
GTTCTCAGTAGAGAACTCAAAGAACCACCACGAGGAGCTCATTTTCTT288 
ValLeuSerArgGluLeuLysGluProProArgGlyAlaHisPheLeu 
859095 
GCCAAAAGTTTGGATGATGCCTTAAGACTTATTGAACAACCGGAATTG336 
AlaLysSerLeuAspAspAlaLeuArgLeuIleGluGlnProGluLeu 
100105110 
GCAAGTAAAGTAGACATGGTTTGGATAGTCGGAGGCAGTTCTGTTTAC384 
AlaSerLysValAspMetValTrpIleValGlyGlySerSerValTyr 
115120125 
CAGGAAGCCATGAATCAACCAGGCCACCTCAGACTCTTTGTGACAAGG432 
GlnGluAlaMetAsnGlnProGlyHisLeuArgLeuPheValThrArg 
130135140 
ATCATGCAGGAATTTGAAAGTGACACGTTTTTCCCAGAAATTGATTTG480 
IleMetGlnGluPheGluSerAspThrPhePheProGluIleAspLeu 
145150155160 
GGGAAATATAAACTTCTCCCAGAATACCCAGGCGTCCTCTCTGAGGTC528 
GlyLysTyrLysLeuLeuProGluTyrProGlyValLeuSerGluVal 
165170175 
CAGGAGGAAAAAGGCATCAAGTATAAGTTTGAAGTCTACGAGAAGAAA576 
GlnGluGluLysGlyIleLysTyrLysPheGluValTyrGluLysLys 
180185190 
GAC579 
Asp 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 193 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
ArgHisGlySerGlyIleMetValArgProLeuAsnCysIleValAla 
151015 
ValSerGlnAsnMetGlyIleGlyLysAsnGlyAspLeuProTrpPro 
202530 
ProLeuArgAsnGluPheLysTyrPheGlnArgMetThrThrThrSer 
354045 
SerGluGluGlyLysGlnAsnLeuValIleMetGlyArgLysThrTrp 
505560 
PheSerIleProGluLysAsnArgLeuLeuLysAspArgIleAsnIle 
65707580 
ValLeuSerArgGluLeuLysGluProProArgGlyAlaHisPheLeu 
859095 
AlaLysSerLeuAspAspAlaLeuArgLeuIleGluGlnProGluLeu 
100105110 
AlaSerLysValAspMetValTrpIleValGlyGlySerSerValTyr 
115120125 
GlnGluAlaMetAsnGlnProGlyHisLeuArgLeuPheValThrArg 
130135140 
IleMetGlnGluPheGluSerAspThrPhePheProGluIleAspLeu 
145150155160 
GlyLysTyrLysLeuLeuProGluTyrProGlyValLeuSerGluVal 
165170175 
GlnGluGluLysGlyIleLysTyrLysPheGluValTyrGluLysLys 
180185190 
Asp 
__________________________________________________________________________