PAS domain proteins

A novel conserved amino acid motif ("PAS") which provides a binding site for homo and hetero protein interactions has been found in mammalian and insect proteins. Abnormalities in these protein interactions are believed to be responsible for a variety of human diseases or conditions, including behavioral disorders and epithelial tissue cancers. Methods for identifying persons who have a disposition towards these behavioral disorders or epithelial tissue cancers are described. Methods are also described for identifying agonists and antagonists of proteins or related peptides containing PAS domains. Screening such agents involves assessing the ability of candidate compounds to promote or interfere with the binding of certain biologic preparations comprised of PAS-containing proteins. Successful agonists or antagonists should be useful in modifying the effects of human behavioral disorders, as well as certain epithelial cancers.

TECHNICAL FIELD 
This invention relates to methods for identifying and treating certain 
behavioral disorders and cancers, and drugs useful in such treatment. 
BACKGROUND OF THE INVENTION 
Daily fluctuations in physiological and behavioral processes are governed 
by an endogenous circadian (approximately 24 h) pacemaker or clock. While 
the mechanisms which underlie circadian rhythms are not well understood in 
humans, it is believed that certain gene products are required for the 
proper manifestation of circadian rhythms. 
Arguably, the best known "rhythm gene" at present is the product of the 
period locus (per) of Drosophila melanogaster. This gene product contains 
a ca. 270 amino acid motif called PAS. Nambu, J. R., et al., Cell (1991); 
67:1157-1167. A PAS motif is also contained in two proteins critical to 
the aryl hydrocarbon ("AH") receptor system in the liver and lung that 
converts environmental carcinogens (e.g. dioxin, cigarette smoke) into 
carcinogenic compounds. Evidence exists in mice that inheritance of the AH 
receptor is involved in the adverse response to carcinogens and a genetic 
predisposition to certain forms of cancer. Knutson, J. D. and A. Poland, 
Cell (1980); 22:27-36; Knutson, J. C. and A. Poland, Cell (1982); 
30:225-234; Poland, A. et al, Nature (1982); 300:271-273. Evidence also 
exists for this AH receptor system involvement in humans. Kellerman, G. et 
al, Amer. J. Hum. Genet. (1973); 25:327-331; Paigen, B. et al, Amer. J. 
Hum. Genet. (1978); 30:561-571; Borresen A. L. et al, Clinical Genetics 
(1981); 19:281-289. 
SUMMARY OF THE INVENTION 
Applicants are the first to recognize that abnormalities in PAS domain 
protein functions may cause certain conditions or diseases in humans, such 
as human behavioral disorders and epithelial tissue cancers. Thus, the 
present invention concerns identifying, purifying and characterizing 
PAS-containing proteins in humans. The present invention also encompasses 
methods for identifying individuals who may have conditions or diseases 
influenced by abnormalities in PAS-containing protein functions. Further, 
the present invention concerns methods for identifying physiologically 
active materials useful to treat these diseases by assessing the ability 
of these materials to promote or interfere with naturally occurring, 
isolated or cloned PAS-containing protein complexes. 
The best characterized clock gene candidate is the period (per) gene in the 
fruit fly, Drosophila melanogaster. Mutations in the Per gene can shorten, 
lengthen or essentially abolish the circadian rhythms of the fruit fly. 
Konopka, R. J. and S. Benzer, Proc. Natl. Acad. Sci. USA, (1971); 
68:2112-2116; Rosbash, M. and J. C. Hall, Neuron (1989); 3:387-398; Young, 
M. W., et al., Molecular Biology of the Drosophila clock. In: Neuronal and 
Cellular Oscillators, edited by Jacklet, J. W. New York: Marcel Dekker, 
1989, p. 529-542. 
Recent findings by applicants indicate that the per gene product (PER) is 
involved in a feed-back loop that influences the circadian transcription 
of its own gene. The transcription of per is inversely correlated to the 
apparent concentration of PER present in those cells that express the per 
gene. The concentration of PER is also positively correlated to the 
fruitfly circadian clock, reaching maximum concentration at approximately 
two hours before lights on and reaching minimum concentration at 
approximately two hours before lights out when a twelve hour lights on, 
twelve-hour lights off cycle is used. This result is consistent with 
observations in other organisms that the temporal regulation of gene 
expression is an important feature of the circadian oscillator. Takahashi, 
J. S., Science (1992); 258:238-240. Although the biochemical function of 
PER has not been established, a PAS amino acid motif is also present in 
three basic Helix-Loop-Helix transcription factors (BHLH), specifically, 
the D. melanogaster single-minded gene product (SIM) and both subunits of 
the mammalian dioxin receptor complex (AH). Nambu, J. R., et al. Cell 
(1991) 67:1157-1167; Hoffman, E. C., et al., Science (1991); 252:954-958; 
Burbach, K. M., et al., Proc. Natl. Acad. Sci. USA (1992); 89:8185-8189. 
The aryl hydrocarbon or dioxin receptor complex (AH) is actively expressed 
in a number of mammalian tissues and cells. In the liver, where the AH 
complex has been best characterized, the two subunits are known to 
function as a heterodimer and bind DNA to activate transcription of 
detoxification genes, such as members of the cytochrome P-450 family. 
Hoffman, E. C. et al, Science (1991); 252:954-958; Burbach, K. M. et al, 
Proc. Natl. Acad. Sci. USA (1992); 89:8185-8189; Ema, M. et al, Biochem. 
Biophys. Res. Commun. (1992); 184:246-253. In the presence of a toxin or 
ligand, such as aryl hydrocarbons or dioxin, the two subunits dimerize and 
contribute to initiating biological functions that make the toxins water 
soluble, so that the toxins can be eliminated from the body. But the 
heterodimer also activates expression of genes whose products convert 
toxins into carcinogens. In other words, the PAS-containing proteins are 
required intermediaries in a necessary detoxification process, but as a 
consequence also participate in a coupled toxification process. 
Applicants have shown in D. melanogaster that the PAS domain of the PER 
protein functions in vitro as a novel protein dimerization motif, and can 
mediate associations between different members of the PAS protein family. 
One implication of this finding is that the PAS motif also functions as a 
dimerization domain in the three BHLH proteins of known functions. These 
findings also establish a link between PER and proteins of known 
biochemical function and indicate that PER itself might affect circadian 
rhythms by modulating transcription. 
The dimerization efficiency of the PER proteins can be decreased by several 
missense mutations in the PAS domain. In particular, mutating the 
hydrophobic valine at position 243, which lies in a conserved hydrophobic 
region just N-terminal to the first PAS repeat, to a hydrophilic aspartic 
acid results in vitro in a 7 fold decrease in PER self-association 
efficiency and in vivo in lengthening the fruitfly circadian period from 
24 hours to 29 hours. These results indicate a biological mechanism 
whereby PER may regulate circadian gene transcription by interacting with 
the PAS domain of BHLH-PAS-containing transcription factors. 
Therefore, the invention features methods for identifying and 
characterizing new proteins in humans that contain the PAS motif and 
purifying the nucleic acid that encodes these protein(s). The purified 
nucleic acid may then be introduced into host cells and the PAS-containing 
proteins expressed in quantities sufficient to characterize the PAS-PAS 
interactions of the newly identified proteins. Accordingly, once the PAS 
sequences have been identified and expressed, the PAS sequences can be 
screened to determine the binding affinity of the PAS-PAS interactions in 
the presence or absence of various molecules. Such screens can be used to 
identify therapeutic molecules for treatment of human behavioral 
disorders, such as seasonal affective disorder, sleep disorders and jet 
lag, as well as treatment or prevention of toxic molecule build-up to 
prevent epithelial tissue cancers. 
Thus, in the first aspect, the present invention features a method for 
identifying, isolating and characterizing the activity of PAS-domain 
containing proteins in humans or other animals which contain PAS domains. 
The invention also includes methods for identifying, isolating and 
characterizing the genes that encode PAS-containing proteins. Once a gene 
encoding a PAS-containing protein is located, it can be cloned, amplified 
and purified into a substantially pure form. Genes isolated from various 
individuals can be obtained and sequenced according to these methods. The 
sequences then can be compared to determine the correlation of DNA 
sequence abnormalities to behavioral disorders and cancers. 
The gene can also be cloned into a plasmid, the plasmid expressed and 
recombinant protein isolated in substantially pure form, such that large 
quantities of the protein are available for further investigation. For 
example, one could also determine the amino acid sequence of 
PAS-containing proteins from various individuals to find correlations 
between the amino acid sequence of the PAS-containing protein to 
behavioral disorders and cancers. 
In the second aspect, the invention features an in vitro assay method for 
identifying, screening and characterizing compounds potentially useful for 
treatment of diseases or disorders arising from abnormal PAS-PAS binding 
affinities. This method includes bringing together a test sample and a 
PAS-containing protein preparation. The test sample contains one or more 
test compounds, and the PAS-containing protein preparation contains one or 
more human peptides comprising at least the PAS region being investigated. 
The test sample is incubated with the PAS-containing protein preparation 
under conditions that would allow the PAS domain containing proteins to 
interact in the absence of the test sample. Those test samples containing 
one or more test compounds that affect PAS-containing protein binding 
functions can then be identified. 
In the third aspect, the invention features an in vivo assay method for 
identifying, screening and characterizing compounds potentially useful for 
treatment of diseases or disorders arising from abnormal PAS-PAS binding 
affinities. The method includes transfecting purified nucleic acid 
encoding a PAS-containing protein into a host cell which contains, or is 
manipulated to contain nucleic acid of a reporter gene whose transcription 
is regulated by the presence or absence of dimerized PAS-containing 
proteins. Test sample compounds can then be introduced and the effect on 
reporter gene transcription assayed. 
In the fourth aspect, the invention features a method for diagnosis of a 
disease or condition which is characterized by abnormal PAS-PAS binding 
affinities. The method includes isolating nucleic acid from a patient, and 
locating the nucleic acid encoding one or more PAS-containing proteins 
sought to be investigated. In one preferred embodiment, this nucleic acid 
can be sequenced and the portion encoding the PAS domain region compared 
to the nucleic acid sequence of patients with normal PAS-PAS binding 
affinities. In another preferred embodiment, abnormal nucleic acid can be 
transfected into a host cell or used in the in vitro assay as described in 
the preceding paragraphs and the effect on reporter gene transcription or 
PAS protein interactions can be assayed. 
These and other aspects of the invention will be apparent upon review of 
the detail description of the preferred embodiments.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The present invention relates to the discovery of a novel conserved amino 
acid motif, PAS, found in mammalian and fruitfly proteins. Thus, the 
invention includes identifying and purifying mammalian proteins that 
contain PAS domains and can dimerize either with an identical protein or 
with a different protein that also contains a PAS motif. 
The present invention also generally relates to methods for identifying 
patients with a disease or condition characterized by abnormal PAS-PAS 
binding affinities. This method is characterized by comparing the sequence 
of PAS-containing proteins observed in normal individuals to an individual 
suspected of having the disease or condition. The invention also describes 
methods for identifying compounds that may promote or interfere with these 
PAS-PAS binding affinities. The invention further includes identifying and 
purifying the nucleic acid which encodes the proteins containing a PAS 
motif. This purified nucleic acid can be expressed in a host cell and 
various test compounds may be assayed either in vitro or in vivo for the 
ability to alter the PAS-PAS interactions. 
FIG. 2A depicts a representation of the PAS domain from proteins found in 
mammals and fruit flies. The PAS domain is indicated at the top and the 
PAS repeats are shown as dark stippling. Two stretches of amino acids that 
are highly conserved are shown below the asterisks. These conserved 
hydrophobic regions within the PAS domain are believed by applicants to be 
important for association or binding of proteins containing these PAS 
motifs. AHR, SIM, ARNT and PER are designations for the four proteins in 
which the PAS motif has been located, namely the mammalian aryl 
hydrocarbon receptor, the Drosophila single-minded gene product, the human 
aryl hydrocarbon receptor nuclear translocator protein and the Drosophila 
period protein. The one letter symbols following the protein designations 
are abbreviations for the amino acid found at each location. The 
cross-references for these symbols and amino acids can be found in Table 
1. 
A PAS-containing protein is therefore any protein or peptide having a motif 
which shares a primary sequence homology to the PAS motif of the four 
proteins described above. Thus, any protein that contains an amino acid 
sequence identical to, or substantially similar to one of the four 
PAS-sequences shown in FIG. 2A is a PAS-containing protein. The motif 
should also be capable of associating, binding or dimerizing either to an 
identical protein or to another protein containing a PAS motif. Those 
individuals versed in the art will understand that changes can be made to 
the amino acid sequence of the PAS motif which do not affect the ability 
of the PAS motif to associate, bind or dimerize with another protein 
containing the PAS domain. Thus, any changes which do not affect the 
ability of the PAS motif to associate, bind or dimerize with another 
PAS-containing protein do not affect the ability to practice the present 
invention. 
TABLE 1 
______________________________________ 
Abbreviations for amino acids 
Three-letter 
One-letter 
Amino Acid abbreviation 
symbol 
______________________________________ 
Alanine Ala A 
Arginine Arg R 
Asparagine Asn N 
Aspartic acid Asp D 
Asparagine or aspartic acid 
Asx B 
Cysteine Cys C 
Glutamine Gln Q 
Glutamic acid Glu E 
Glutamine or glutamic acid 
Glx Z 
Glycine Gly G 
Histidine His H 
Isoleucine Ile I 
Leucine Leu L 
Lysine Lys K 
Methionine Met M 
Phenylalanine Phe F 
Proline Pro P 
Serine Ser S 
Threonine Thr T 
Tryptophan Trp W 
Tyrosine Tyr Y 
Valine Val V 
______________________________________ 
Stryer, Biochemistry, W. H. Freeman and Company, New York (3rd. Ed. 1988) 
p. 21. 
Thus, in the first aspect of the present invention, methods for identifying 
additional mammalian proteins that contain the PAS motif are described. 
The present methods capitalize on the discovery that PAS is a protein 
dimerization motif. Novel PAS-containing proteins can be identified in 
screens by contacting samples believed to contain novel PAS proteins with 
known proteins that contain the PAS motif under conditions permitting 
interaction. The known PAS proteins can be derived from any organism, 
including for example, flies and mammals. Any novel protein that 
associates with the known PAS proteins can then be sequenced to determine 
whether, in fact, it contains the PAS motif. 
Preferably, applicants employ a two-hybrid yeast system to identify in vivo 
protein-protein interactions. This method is a modification of known 
methods reported in Fields, S. and O. Song, Nature (1989); 340:245-248 and 
Zervos, A. S., et al., Cell (1993); 72:223-232 which are incorporated by 
reference. The two-hybrid system reported in these papers takes advantage 
of the properties of the transcriptional activator protein GAL4 from the 
yeast Saccharomyces cerevisiae. This protein is required for the 
expression of genes encoding enzymes for galactose utilization and 
consists of two separable and functionally essential domains: a N-terminal 
domain that binds to specific DNA sequences; and a C-terminal domain that 
is necessary to activate transcription. 
As described by Fields and Song, the two-hybrid system requires that the 
GAL4 protein be cleaved into its two functional domains. The N-terminal 
DNA binding region then is fused to one unit of the subject dimerization 
protein pair and the C-terminal transcription activating region is fused 
to the other protein dimer. If the proteins dimerize after introduction 
into the yeast, the DNA binding and transcription activity regions of GAL4 
are brought into close proximity and transcription of the reporter gene is 
activated. If the proteins do not dimerize, the reporter gene is not 
transcribed. Thus, a simple system for investigating protein-protein 
interactions is possible. 
The two-hybrid method can be modified to identify new proteins by detecting 
protein-protein interactions of known proteins with the new protein. In 
this system cDNA libraries are constructed using methods known in the art 
from various tissues that are believed to express the proteins of 
interest. See, e.g., Chien, C. T., et al., Proc. Nat'l. Acad. Sci. USA 
(1991); 88:9578-9582; Dalton and Treisman, Cell (1992); 68:597-612. A 
library that conditionally expresses cDNA-encoded proteins fused to an 
epitope tag, a nuclear localization sequence and an acidic transcription 
activation region of GAL4 is introduced into a special yeast strain. That 
special yeast strain contains a plasmid that directs the synthesis of a 
construct that includes the DNA binding region of GAL4 and the protein 
interacting domain of the known protein. The strain also contains a 
reporter gene, the transcription of which is stimulated if the library 
encoded protein complexes or dimerizes with the known protein construct. 
Again, by monitoring the transcription of the reporter gene, new proteins 
that interact with the known protein may be identified. 
In applicant's preferred method, DNA libraries are constructed using 
methods known in the art from various tissues of interest (e.g., brain 
tissue will be used to locate potential PAS-containing proteins relevant 
to behavioral disorders, liver tissue for epithelial cancers, etc.). The 
library is designed such that cDNA-encoded proteins are fused to an 
epitope tag, a nuclear localization sequence and the acidic transcription 
domain from GAL4 and are conditionally expressed. 
This library DNA is then introduced into a yeast strain containing a 
plasmid that expresses a known PAS-containing protein, or only the PAS 
motif of that protein, fused to the DNA binding region of GAL4. The strain 
also contains two different reporter genes whose expression is influenced 
by the GAL4 transcription activating protein. One reporter gene will allow 
growth of the yeast strain in the absence of leucine. The other reporter 
gene directs the synthesis of beta-galactosidase and turns the yeast 
colony blue in the presence of galactose. 
By plating a pool of these transformed yeast cells onto galactose-Leu.sup.- 
selection plates, yeast cells containing new proteins that interact with 
the known PAS protein can be identified. Since these individual yeast 
colonies only contain a single cDNA-containing plasmid, the relevant cDNA 
encoding a mammalian protein fragment can be cloned into E. coli, 
amplified, identified and sequenced using methods known in the art to 
verify that it contains the PAS motif. 
The DNA encoding the PAS-containing protein can be separated from other 
cellular components and purified to a homogenous form using methods known 
in the art. See, e.g., Chien, C. T., et al., Proc. Nat'l. Acad. Sci. USA 
(1991); 88:9578-9582; Dalton and Treisman, Cell (1992); 68:597-612. By 
isolating and purifying the DNA sequence for the whole gene encoding the 
PAS-containing protein, the sequence of the bases also may be determined 
by known methods, such as Maxam and Gilbert, Proc. Natl. Acad. Sci. USA 
(1977); 74:560. The sequence can be used for determination of the amino 
acid sequence of the protein expressed by the gene. By identifying codons 
for methionine followed by a sequence which does not have stop codons 
which prevent expression, one can usually find a single sequence in frame 
with a methionine codon for defining the gene. 
Once the DNA sequence of the mammalian PAS-containing protein is obtained, 
individuals can be screened to determine the significance of PAS domain 
functions from changes in the DNA sequence. For example, a library of DNA 
can be obtained using methods known in the art from individuals who 
exhibit symptoms of human behavioral disorders, as well as from control 
(normal) individuals. The PAS domain segments of these individuals can be 
located and sequenced. Using standard statistical correlation procedures, 
methods for diagnosing whether a subject individual has a behavioral 
disorder then readily can be created by comparing the subject individual's 
PAS domain DNA sequence to a control sequence. See, e.g., Barker, D., et 
al., Science (1987); 236:1100-1102; Wallace, M. R., et al., Science 
(1990); 249:181-186; Li, et al., Cell (1992); 69:275-281. This type of 
analysis also is currently possible to characterize the dioxin receptor 
and to determine the role of sequence abnormalities of that gene in cancer 
development. The literature indicates that a particular allele may be 
inherited at this locus that affects tumor incidence. Knutson, J. D. and 
A. Poland, Cell (1980); 22:27-36; Knutson, J. C. and A. Poland, Cell 
(1982); 30:225-234; Poland, A. et al, Nature (1982); 300:271-273; 
Kellerman, G. et al, Amer. J. Hum. Genet. (1973); 25:327-331; Paigen, B. 
et al, Amer. J. Hum. Genet. (1978); 30:561-571; Borresen A. L. et al, 
Clinical Genetics (1981); 19:281-289. 
Hybrid DNA technology known in the art also may be employed to obtain 
expression of purified DNA encoding a PAS-containing protein. The DNA 
sequence may be restriction mapped and appropriate sites for cleavage 
defined. In this way, the sequence encoding the PAS-containing protein may 
be excised and introduced into a vector having appropriate regulatory 
signals. After introducing the vector into a host cell, the DNA sequence 
is expressed and purified recombinant human protein containing a PAS 
region may be obtained using methods known in the art. See, e.g., Zervos, 
et al., Cell (1993); 72:223-232. 
Similarly, once the mammalian PAS-containing protein sequences are known, 
individuals can be screened to determine the significance of changes in 
the amino acid sequence among individuals. Again, PAS-containing proteins 
isolated from individuals who exhibit symptoms of behavioral disorders and 
control individuals can be sequenced and compared. Using the information 
from this analysis, methods for diagnosing behavioral disorders in subject 
individuals can easily be devised. 
Once novel PAS-containing proteins are identified and isolated, one can 
then use the protein or subunit peptides as an antigen for the production 
of antibodies. Antibodies can be prepared using a variety of methods well 
known in the art. Either monoclonal or polyclonal antibodies may be 
desired. For polyclonal antibodies, a vertebrate, normally a domestic 
animal, is hyperimmunized with the antigen and blood collected shortly 
after repeat immunizations and gamma globulin isolated. For monoclonal 
antibodies, a small animal is hyperimmunized, the spleen removed and the 
lymphocytes fused with an appropriate fusing partner. The resulting 
hybridomas are then grown under limiting dilution and clones providing the 
desired antibodies selected. The tissues that express novel PAS-containing 
proteins can then be identified by known procedures of antibody staining 
or in situ hybridization. See, e.g., Harlow and Lane, Antibodies A 
Laboratory Manual, Cold Spring Harbor Laboratory, 1988 which is 
incorporated herein by reference. 
Various compounds also can be tested to determine the ability of the 
compound to interfere with PAS-PAS interactions. An in vivo yeast system 
is utilized and a plasmid is constructed that encodes the PAS-containing 
proteins of interest. The first protein is comprised of a PAS-containing 
protein, or a subunit containing the PAS-motif, fused to the DNA binding 
domain of the GAL4 protein. The second protein is comprised of a second 
PAS-containing protein whose interaction with the first PAS-containing 
protein is sought to be investigated, fused to the transcription 
activating domain of the GAL4 protein. The third protein is a reporter 
protein whose transcription is influenced by GAL4 as described above. 
Once this yeast strain is prepared, it can be grown in large quantities. 
Drug candidates can then be screened by contacting the host cell with the 
test compound and assessing the ability of the test compound to interfere 
with the PAS-PAS interaction. 
An in vitro assay may also be employed to screen drug candidates. To study 
the stoichiometry of the PER protein oligomers in vitro, applicants have 
designed a chemical cross-linking and immunoprecipitation procedure. This 
in vitro procedure is described in detail in Example 3 below. 
This in vitro procedure can be modified to screen drug candidates by 
introducing the test compounds before the PAS-containing proteins are 
cross-linked by glutaraldehyde. Any compounds which inhibit or promote 
this cross-linking reaction are then candidates for further research. 
The following examples are provided for illustration of the ability of the 
PAS motif to mediate protein-protein interactions and are not intended to 
limit the scope of the invention. 
EXAMPLE 1 
Co-Immuno Precipitation Assay 
To test whether the PAS domain can mediate protein-protein interactions, we 
designed a co-immunoprecipitation assay: per cDNA encoding PER 233-685/H 
was generated by the polymerase chain reaction (PCR) from the full length 
per cDNA (pSP65ATper). Citri, Y., et al., Nature (1987); 326:42-47. The 5' 
primer contains, from 5' to 3', an XbaI site, an ATG codon and nucleotides 
697 to 717 of per cDNA; the 3' primer contains, from 5' to 3', an XbaI 
site, a TAG stop codon, the sequence corresponding to the HA peptide 
(YPYDVPDVASL) (Kolodziej, P. A., et al., Meth. Enzymol. (1991), SEQ. ID. 
NO. 9; 194:508-519), and nucleotides 2052 to 2035 of per cDNA. The PCR 
products were inserted into the pBluescript KS(-) vector (Stratagene) at 
the XbaI site such that the per cDNA was under the control of the T7 
promoter (pBSC2H), and the construct was verified by DNA sequencing. 
cDNAs coding for PER 295-685/H and PER 391-685/H were obtained by PCR using 
pBSC2H as a template. The 5' primers contain a T7 promoter, the 5' leader 
and ATG from the human .beta.-globin gene (Schindler, U., et al., EMBO J. 
(1992); 11:1261-1273), and nucleotides 883 to 902 (PER 295-685/H) , or 
nucleotides 1174 to 1193 (PER 391-685/H) of the per cDNA; the 3' primer 
for both cDNAs contains nucleotides 806 to 787 of the (+) strand of the 
pBluescript (KS-) vector. More than two independent PCR products for each 
cDNA were used for analysis and gave identical results in the experiments 
described below. The cDNA for PER 233-568 was generated by linearizing 
pBSC2H with SmaI at nucleotide 1704 of per cDNA. 
Approximately 1 .mu.g of DNA was used for in vitro transcription. RNAs were 
translated using a rabbit reticulocyte lysate in the presence of .sup.35 
S-labeled methionine according to the manufacturer's instructions 
(Promega). 1 .mu.l of translation reaction was used to measure 
TCA-precipitable cpm. Translation products of approximately equal cpm were 
mixed and incubated at 37.degree. C. for 30 min. Typically ca. 
5.times.10.sup.4 cpm of a translation or of a mixing reaction were 
analyzed by SDS-PAGE. For immunoprecipitation approximately 5-fold more 
material was diluted in 250 .mu.l of ice cold HND buffer (20 mM HEPES, 100 
mM KC1, 10% glycerol, 0.4% NP-40, 5 mM EGTA, 5 mM EDTA, 100 .mu.g/ml BSA, 
1 mM DTT, pH 7.4). This and subsequent procedures were done at 4.degree. 
C. The incubation mixture was first pre-cleared by adding 7 .mu.l of 
Gamma-Bind Plus Sepharose (Pharmacia), followed by 10 min. rocking. After 
centrifugation, the supernatant was transferred and incubated with 2 .mu.l 
of monoclonal antibody 12CA5 for 2 h. 10 .mu.l of Gamma-Bind Plus were 
then added with further incubation for 1 h. Samples were washed 3 times 
with HND buffer. They were then heated to 95.degree. C. for 5 min. in SDS 
sample buffer, centrifuged for 2 min. and electrophoresed by SDS-PAGE on a 
12% gel. The gels were fixed, amplified and fluorographed. 
Thus, PER fragments containing complete or truncated PAS regions were 
synthesized in vitro, either with or without a hemagglutinin epitope (HA 
tag) at the C-terminus (FIG. 1A). Without an HA tag, PER 233-568 did not 
react with the anti-HA antibody 12CA5 (FIG. 1B, compare lanes 10-12 with 
lane 13). It was, however, immunoprecitated after mixing with PER 
233-685/H (FIG. 1B, lane 14), indicating that the two polypeptides 
associate in vitro. 10-15% of PER 233-568 co-immunoprecipitated with PER 
233-685/H when they were mixed in an approximately 1:1 molar ratio (based 
on four experiments) This is an underestimate of PER self-association 
since PER 233-568/PER 233-568 associations are not detectable in the assay 
and the association conditions may not be optimal. Full length PER protein 
also associated with PER 233-685/H (data not shown). Significantly, the 
immunoprecipitation of PER 233-568 with PAS deletion fragments was greatly 
reduced (FIG. 1, lanes 15 and 16), indicating that the PAS domain is 
necessary for PER self-association in vitro. 
EXAMPLE 2 
Site-Directed Mutagenesis of the PAS Motif Decreases PER Self-Association 
To support the above observations, conserved amino acids in the PAS domain 
were mutated, and the association of the mutant polypeptides with 
wild-type PER fragments was assayed (FIG. 2). To generate amino acid 
substitutions, site-directed mutagenesis (Amersham Kit) was carried out 
with pBSC2H as template and verified by DNA sequencing. These mutant 
pBSC2H templates were linearized with SmaI to obtain cDNAs for various 
mutant PER 233-568 fragments. In vitro translations and 
immunoprecipitations were performed as described in Example 1. 
Of particular interest was the valine at position 243 (V243), which lies in 
a conserved hydrophobic region just N-terminal to the first PAS repeat 
(FIG. 2A). In the classical per mutation, which gives rise to 29 h 
circadian rhythms, V243 is mutated to aspartic acid (D243). Konopka, R. 
J., et al., Proc. Natl. Acad. Sci. USA (1971); 68:2112-2116; Yu, Q., et 
al., Proc. Natl. Acad. Sci. USA (1987); 84:784-788; Jackson, R. F., et 
al., Nature (1986); 320:185-188. The sequence of PER 233-685 was modified 
by site-directed mutagenesis to change V243 to D243 as well as to leucine 
and arginine (FIG. 2A). Another highly conserved stretch of amino acids in 
the second PAS repeat was also mutated (FIG. 2A). With one exception, all 
of the mutations resulted in a significant reduction in 
co-immunoprecipitation with wild-type PER 233-685/H (FIG. 2B, lanes 9-14). 
The original per.sup.L (V/D) mutation showed the most severe phenotype 
(FIG. 2B, compare lanes 9 and 10), a ca. 7-fold reduction in association 
with PER 233-685/H (based on three experiments). It also resulted in a 
slower migration on SDS-polyacrylamide gel, suggestive of a substantial 
structural change in the protein fragment (FIG. 2B, compare lanes 2 and 
1). A further decrease in PER self-association was observed when both PER 
fragments carried the per.sup.L mutation (data not shown; also see FIG. 
3). Only the conservative change of V243 to leucine 243 (V/L) resulted in 
a mild reduction in the co-immunoprecipitation assay (FIG. 2B, lane 11). 
These observations strengthen the conclusion that the PAS domain engages 
in PAS-PAS associations. 
EXAMPLE 3 
Chemical Cross-Linking and Immunoprecipitation Procedure 
To study the stoichiometry of the PER protein oligomers in vitro, we 
designed a chemical cross-linking and immunoprecipitation procedure. 
Approximately 1.times.10.sup.5 cpm of .sup.35 S-labeled in vitro 
translated PER 233-685/H were incubated in a final volume of 40 .mu.l in 
the presence of 0.005% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) 
at 25.degree. C. for 30 min. The cross-linking reactions were terminated 
by adding 2 M Tris (pH 7.5) to a final concentration of 100 mM. Samples 
were subsequently diluted into 250 .mu.l of HND buffer and 
immunoprecipitated as described in Example 1. To produce cold PER 
233-685/H, in vitro translations were carried out with complete amino acid 
mixture without radio-labeled methionine. Unlabeled (cold) PER 233-685/H 
was then mixed with .sup.35 S-labeled PER 233-568 at 37.degree. C. for 30 
min. Further cross-linking and immunoprecipitation procedures were 
performed as described in Example 1. 
In vitro translation of wild-type (FIG. 3, lanes 1 to 3) or per.sup.L 
mutant (FIG. 3, lanes 4 to 6) versions of PER 233-685/H were incubated 
with glutaraldehyde to produce covalent cross-links, immunoprecipitated 
with anti-HA antibody and analyzed by SDS-PAGE. After incubation, only the 
wild-type fragment gave rise to dimerized cross-linked products that 
migrated as a doublet at ca. 140-160 kD (FIG. 3, compare lanes 2 and 3 to 
lanes 5 and 6; doublet indicated by arrowhead on the left; monomers are 
ca. 75 kD polypeptides, lanes 1 and 4). This doublet most likely 
represents two differentially cross-linked forms of the dimers. To ensure 
that two PAS-containing proteins can dimerize, an additional 
cross-linking-immunoprecipitation experiment was carried out in which 
non-radioactive PER 233-685/H (cold) was mixed with the smaller, 45 kD 
radiolabeled PER 233-568 (either the wild-type or the PERL version) . As 
the assay only detects radioactive oligomers that also contain an HA 
epitope, the formation of radioactive dimers must result from an associate 
between the cold and radioactive polypeptides. An appropriately sized 
doublet (ca. 110-130 kD; indicated by the arrowhead on the right) was 
detected only with the wild-type PER fragment (FIG. 3, compare lanes 8 and 
9 to 11 and 12), indicating that the PAS domain dimerizes in vitro and 
that the PER.sup.L fragment is at least 10 times less efficient than its 
wild-type counter-part in this dimerization assay. 
EXAMPLE 4 
PER Associates with other PAS-Containing DNA Proteins 
The apparent contradiction between PER's possible role in gene regulation 
and the absence of an obvious DNA binding domain and DNA binding activity 
(data not shown) can be reconciled by proposing that it regulates 
transcription through interactions with other PAS-containing DNA binding 
proteins. To address this possibility, we tested whether PER can associate 
with other PAS family members that contain DNA binding regions. A SIM 
fragment containing the BHLH and PAS domain with a Myc epitope at its 
N-terminus (SIM M/1-355) was translated in vitro (FIG. 4A). This fragment 
was immunoprecipitated by the anti-Myc antibody 9E10, but not by the 
anti-HA antibody 12CA5 (FIG. 4, lanes 2 and 3). When mixed with PER 
233-685/H, it was co-precipitated by the anti-HA reagent (FIG. 4B, lanes 1 
and 5), indicating that the two fragments can associate in vitro. To show 
that the SIM PAS domain was sufficient for the association, a SIM fragment 
containing only the PAS domain and an HA tag (SIM 89-355/H) was used (FIG. 
4A). The anti-HA antibody precipitated PER 233-568 along with SIM 89-355/H 
only when the two fragments were mixed (FIG. 4, lanes 6-9). The PER PAS 
domain can also mediate a similar association between protein fragments of 
PER and ARNT, a subunit of the mammalian dioxin receptor complex. Taken 
together, the observations demonstrate that the PAS domain is a novel 
protein-protein interaction domain. 
In the known BHLH-PAS proteins from both mammals and flies, it is 
intriguing that PAS domains are located just C-terminal to the BHLH 
motifs, similar to the leucine zipper (LZ) domain in the BHLH-LZ protein 
family (e.g., MYC and MAX). Blackwood, E. M., Science (1991); 
251:1211-1217. The BHLH-PAS proteins thus may represent a new subfamily of 
BHLH proteins. As the dioxin binding region of AHR is apparently localized 
to the PAS domain. (Burbach, K. M., et al., Proc. Natl. Acad. Sci. USA 
(1992); 89:8185-8189), there may be some relationship between 
ligand-binding and the PAS mediated protein-protein interaction (i.e., 
heterodimer formation between AHR and ARNT), not unlike what has been 
observed for the steroid hormone receptors. Forman, B. M., et al., Mol. 
Endocrinol. (1990); 4:1293-1301; Fawell, S. E., et al., Cell (1990); 
60:953-962. In addition, it is possible that unknown ligands participate 
in the regulation of PER or SIM function in Drosophila. 
The foregoing is intended to be illustrative of the present invention, but 
not limiting. Numerous variations and modifications may be effected 
without departing from the true spirit and scope of the novel concepts of 
the invention. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 9 
(2) INFORMATION FOR SEQ ID NO: 1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Protein 
(v) FRAGMENT TYPE: Internal Fragment 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Human 
(G) CELL TYPE: Hepatoma 
(H) CELL LINE: HepG2 
(x) PUBLICATION INFORMATION: 
(A) AUTHORS: Hoffman, E.C., H. Reyes, 
F.-F. Chu, F. Sander, 
L.H. Conley, B.A. Brooks, 
and O. Hankinson 
(B) TITLE: CLONING OF A FACTOR REQUIRED 
FOR ACTIVITY OF THE Ah (DIOXIN) 
RECEPTOR 
(C) JOURNAL: Science 
(E) ISSUE: 252 
(F) PAGES: 954-958 
(G) DATE: May 17, 1991 
(K) RELEVANT RESIDUES IN SEQ ID NO: from 
240- 245 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: 
PheValLeuValValThr 
15 
(2) INFORMATION FOR SEQ ID NO: 2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Protein 
(v) FRAGMENT TYPE: Internal Fragment 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Human 
(G) CELL TYPE: Hepatoma 
(H) CELL LINE: HepG2 
(x) PUBLICATION INFORMATION: 
(A) AUTHORS: Hoffman, E.C., H. Reyes, 
F.-F. Chu, F. Sander, 
L.H. Conley, B.A. Brooks, 
and O. Hankinson 
(B) TITLE: CLONING OF A FACTOR REQUIRED 
FOR ACTIVITY OF THE Ah (DIOXIN) 
RECEPTOR 
(C) JOURNAL: Science 
(E) ISSUE: 252 
(F) PAGES: 954-958 
(G) DATE: May 17, 1991 
(K) RELEVANT RESIDUES IN SEQ ID NO: from 
412- 420 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: 
LeuGlyTyrThrGluValGluLeuCys 
15 
(2) INFORMATION FOR SEQ ID NO: 3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Protein 
(v) FRAGMENT TYPE: Internal Fragment 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Drosophila 
(x) PUBLICATION INFORMATION: 
(A) AUTHORS: Crews, S.T., J.B. Thomas, 
and C.S. Goodman 
(B) TITLE: THE DROSOPHILA SINGLE- MINDED 
GENE ENCODES A NUCLEAR PROTEIN 
WITH SEQUENCE SIMILARITY TO THE 
PER GENE PRODUCT 
(C) JOURNAL: Cell 
(E) ISSUE: 52 
(F) PAGES: 143-152 
(G) DATE: January 15, 1988 
(K) RELEVANT RESIDUES IN SEQ ID NO: from 
240- 245 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: 
PheIlePheValValAla 
15 
(2) INFORMATION FOR SEQ ID NO: 4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Protein 
(v) FRAGMENT TYPE: Internal Fragment 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Drosophila 
(x) PUBLICATION INFORMATION: 
(A) AUTHORS: Crews, S.T., J.B. Thomas, 
and C.S. Goodman 
(B) TITLE: THE DROSOPHILA SINGLE- MINDED 
GENE ENCODES A NUCLEAR PROTEIN 
WITH SEQUENCE SIMILARITY TO THE 
PER GENE PRODUCT 
(C) JOURNAL: Cell 
(E) ISSUE: 52 
(F) PAGES: 143-152 
(G) DATE: January 15, 1988 
(K) RELEVANT RESIDUES IN SEQ ID NO: from 
412- 420 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: 
ThrGlyTyrGluProGlnAspLeuIle 
15 
(2) INFORMATION FOR SEQ ID NO: 5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Protein 
(v) FRAGMENT TYPE: Internal Fragment 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Mouse 
(G) CELL TYPE: Hepatoma 
(H) CELL LINE: Hepa 1c1c7 
(x) PUBLICATION INFORMATION: 
(A) AUTHORS: Burbach, K.M., A. Poland, 
and C.A. Bradfield 
(B) TITLE: CLONING OF THE Ah- RECEPTOR cDNA 
REVEALS A NOVEL LIGAND ACTIVATED 
TRANSCRIPTION FACTOR 
(C) JOURNAL: Proc. Natl. Acad. Sci. USA 
(E) ISSUE: 89 
(F) PAGES: 8185-8189 
(G) DATE: September, 1992 
(K) RELEVANT RESIDUES IN SEQ ID NO: from 
240- 245 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: 
PheLeuPheIleValSer 
15 
(2) INFORMATION FOR SEQ ID NO: 6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Protein 
(v) FRAGMENT TYPE: Internal Fragment 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Mouse 
(G) CELL TYPE: Hepatoma 
(H) CELL LINE: Hepa 1c1c7 
(x) PUBLICATION INFORMATION: 
(A) AUTHORS: Burbach, K.M., A. Poland, 
and C.A. Bradfield 
(B) TITLE: CLONING OF THE Ah- RECEPTOR cDNA 
REVEALS A NOVEL LIGAND ACTIVATED 
TRANSCRIPTION FACTOR 
(C) JOURNAL: Proc. Natl. Acad. Sci. USA 
(E) ISSUE: 89 
(F) PAGES: 8185-8189 
(G) DATE: September, 1992 
(K) RELEVANT RESIDUES IN SEQ ID NO: from 
412- 420 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: 
ValGlyTyrGlnProGlnGluLeuIle 
15 
(2) INFORMATION FOR SEQ ID NO: 7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Protein 
(v) FRAGMENT TYPE: Internal Fragment 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Drosophila 
(x) PUBLICATION INFORMATION: 
(A) AUTHORS: Reddy, P., A.C. Jacquier, 
N. Abovich, G. Petersen, and 
M. Rosbash 
(B) TITLE: THE PERIOD CLOCK LOCUS OF D. 
MELANOGASTER CODES FOR A 
PROTEOGLYCAN 
(C) JOURNAL: Cell 
(E) ISSUE: 46 
(F) PAGES: 53-61 
(G) DATE: 1986 
(K) RELEVANT RESIDUES IN SEQ ID NO: from 
240- 245 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: 
PheCysCysValIleSer 
15 
(2) INFORMATION FOR SEQ ID NO: 8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Protein 
(v) FRAGMENT TYPE: Internal Fragment 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Drosophila 
(x) PUBLICATION INFORMATION: 
(A) AUTHORS: Reddy, P., A.C. Jacquier, 
N. Abovich, G. Petersen, and 
M. Rosbash 
(B) TITLE: THE PERIOD CLOCK LOCUS OF D. 
MELANOGASTER CODES FOR A 
PROTEOGLYCAN 
(C) JOURNAL: Cell 
(E) ISSUE: 46 
(F) PAGES: 53-61 
(G) DATE: 1986 
(K) RELEVANT RESIDUES IN SEQ ID NO: from 
412- 420 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: 
LeuGlyTyrLeuProGlnAspLeuIle 
15 
(2) INFORMATION FOR SEQ ID NO: 9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Protein 
(v) FRAGMENT TYPE: Internal Fragment 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Influenza 
(x) PUBLICATION INFORMATION: 
(A) AUTHORS: P.A. Kolodziej and R.A. Young 
(B) TITLE: EPITOPE TAGGING AND PROTEIN 
SURVEILLANCE 
(C) JOURNAL: Methods In Enzymology 
(E) ISSUE: 194 
(F) PAGES: 508-519 
(G) DATE: 1991 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: 
TyrProTyrAspValProAspValAlaSerLeu 
1510 
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