+

WO1995016694A1 - Genes et proteines de complexe de replication, et procedes associes - Google Patents

Genes et proteines de complexe de replication, et procedes associes Download PDF

Info

Publication number
WO1995016694A1
WO1995016694A1 PCT/US1994/014563 US9414563W WO9516694A1 WO 1995016694 A1 WO1995016694 A1 WO 1995016694A1 US 9414563 W US9414563 W US 9414563W WO 9516694 A1 WO9516694 A1 WO 9516694A1
Authority
WO
WIPO (PCT)
Prior art keywords
leu
lys
ser
glu
asn
Prior art date
Application number
PCT/US1994/014563
Other languages
English (en)
Inventor
Bruce W. Stillman
Stephen P. Bell
Ryuji Kobayashi
Jasper Rine
Margit Foss
Francis J. Mcnally
Patricia Laurenson
Ira Herskowitz
Joachim J. Li
Original Assignee
Cold Spring Harbor Laboratory
The Regents Of The University Of California
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cold Spring Harbor Laboratory, The Regents Of The University Of California filed Critical Cold Spring Harbor Laboratory
Priority to AU13734/95A priority Critical patent/AU700405B2/en
Priority to EP95904929A priority patent/EP0733057A4/fr
Priority to JP7516984A priority patent/JPH09506768A/ja
Publication of WO1995016694A1 publication Critical patent/WO1995016694A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the technical field of this invention concerns Origin of Replication Complex genes which are invovled with DNA transcription and replication.
  • ARS elements derived from yeast chromosomes, a subset of which were subsequently shown to act as chromosomal origins of DNA replication (reviewed in 11). Sequence comparison of a number of ARS elements resulted in the identification of the ARS consensus sequence (ACS, 12). This sequence is essential for the function of yeast origins of DNA replication (7, 12, 13). Three additional elements required for efficient ARSl function have been identified. When mutated individually, these elements, referred to as Bl, B2, and B3, result in a slight reduction of ARSl activity.
  • ARSl function is severely compromised (14).
  • Proteins that recognize two elements of ARSl have been identified.
  • the yeast transcription factor ABF1 binds to and mediates the function of the B3 element (11, 14).
  • ORC binds to more than 10 yeast ARS elements, several of which are known origins of DNA replication (15). Specific DNA binding by ORC requires ATP, suggesting that ORC binds ATP, a property of a number of known initiator proteins (17). ORC also interacts with other sequences outside of the ACS that are known to be important for ARS function (18, 19). Further support for the hypothesis that ORC mediates the function of the ACS is provided by in situ deoxyribonuclease I (DNase I) footprinting experiments that identify a protected region of ARSl remarkably similar to that observed with ORC in vitro (20).
  • DNase I in situ deoxyribonuclease I
  • compositions comprising isolated nucleic acids encoding unique ORC gene portions, especially portions encoding biologically active unique portions of ORC1-ORC6 proteins.
  • Vectors and cells comprising such DNA molecules find use in the production of recombinant ORC peptides.
  • the subject compositions are used to isolate ORC genes from a wide variety of species, including human.
  • the subject ORC peptides also find particular use in screening for ORC selective agents useful in the diagnosis, prognosis or treatment of disease, particulary fungal infections and neoproliferative disease. Particularly useful are agents capable of distinguishing an ORC protein of an infectious organism or transformed cell from the wild-type human homologue.
  • the methods involve transforming an expression library of hybrid proteins into a reporter strain, wherein the library comprises protein-coding sequences fused to a constitutively expressed transcription activation domain and the reporter strain comprises a reporter gene with at least one copy of a selected DNA sequence in its promoter region. Clones expressing the transcription or translation product of the reporter gene are detected and recovered.
  • a preferred method employs an activation domain from GAL4 and a lacZ reporter gene.
  • SEQUENCE ID NO:l DNA Sequence of ORC1.
  • SEQUENCE ID NO: 3. DNA Sequence of ORC2.
  • the recombinant polypeptides of the invention comprise unique portions of the disclosed ORC proteins which retain an binding affinity specific to the subject full-length ORC protein.
  • a "unique portion” has an amino acid sequence unique to subject ORC in that it is not found in previously known protein and has a length at least long enough to define a peptide specific to that ORC.
  • Unique portions are found to vary from about 5 to about 25 residues, usually from 5 to 10 residues in length, depending on the particular amino acid sequence and are readily identified by comparing the subject portion sequences with known peptide/protein sequence data bases.
  • polypeptide as used herein defines an amino acid polymer with as few as five residues.
  • ORCs used in the subject screening assays are frequently smaller deletion mutants of full-length ORC proteins.
  • deletion mutants are readily generated using conventional molecular techniques and screened for an ORC-specific binding affinity using the various assays described below, e.g. footprint analysis, coimmunoprecipitation, etc.
  • ORC-specific retained binding affinities include the ability to selectively bind a nucleic acid of a defined sequence, an ORC protein or an compound such as an antibody which is capable of selectively binding an ORC protein.
  • binding specificity may be provided by an ORC-specific immunological epitope, lectin binding site, etc.
  • Selective binding is conveniently shown by competition with labeled ligand using recombinant ORC peptide either in vitro or in cell based systems as disclosed herein.
  • selective binding requires a binding affinity of 10 "6 M, preferably 10 "8 M, more preferably 10 "10 M, under in vitro conditions as exemplified below.
  • the subject recombinant polypeptides may be free or covalently coupled to other atoms or molecules. Frequently the polypeptides are present as a portion of a larger polypeptide comprising the subject polypeptide where the remainder of the larger polypeptide need not be ORC-derived.
  • the subject polypeptides are typically "isolated", meaning unaccompanied by at least some of the material with which they are associated in their natural state. Generally, an isolated polypeptide constitutes at least about 1%, preferably at least about 10%, and more preferably at least about 50% by weight of the total poly/peptide in a given sample.
  • pure peptidepolypeptide is intended at least about 60% , preferably at least 80%, and more preferably at least about 90% by weight of total polypeptide. Included in the subject polypeptide weight are any atoms, molecules, groups, etc. covalently coupled to the subject polypeptides, such as detectable labels, glycosylations, phosphorylations, etc.
  • the subject polypeptides may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample and to what, if anything, the polypeptide is covalently linked.
  • Purification methods include electrophoretic, molecular, immunological and chromatographic techniques, especially affinity chromatography and RP-HPLC in the case of peptides.
  • suitable purification techniques see Scopes, R., Protein Purification, Springer- Verlag, NY (1982).
  • polypeptides may be modified or joined to other compounds using physical, chemical, and molecular techniques disclosed or cited herein or otherwise known to those skilled in the relevant art to affect their ORC/receptor binding specificity or other properties such as solubility, membrane transportability, stability, toxicity, bioavailability, localization, detectability, in vivo half-life, etc. as assayed by methods disclosed herein or otherwise known to those of ordinary skill in the art.
  • Other modifications to further modulate binding specificity/affinity include chemical/enzymatic intervention (e.g. fatty acid-acylation, proteolysis, glycosylation) and especially where the poly /peptide is integrated into a larger polypeptide, selection of a particular expression host, etc.
  • Amino and/or carboxyl termini may be functionalized e.g., for the amino group, acylation or alkylation, and for the carboxyl group, esterification or amidification, or the like.
  • glycosylation sites and patterns which may be disrupted or modified, e.g. by enzymes like glycosidases.
  • N or O-linked glycosylation sites of the disclosed poly/peptides may be deleted or substituted for by another basic amino acid such as Lys or His for N- linked glycosylation alterations, or deletions or polar substitutions are introduced at Ser and Thr residues for modulating O-linked glycosylation.
  • Glycosylation variants are also produced by selecting appropriate host cells, e.g. yeast, insect, or various mammalian cells, or by in vitro methods such as neuraminidase digestion.
  • poly/peptides may be introduced by reacting the targeted amino acid residues with an organic derivatizing (e.g. ethyl ⁇ s' [(p-azido-phenyl)dithio] propioimidate) or crosslinking agent (e.g. 1, 1- bis(diazoacetyl)-2-phenylethane) capable of reacting with selected side chains or termini.
  • organic derivatizing e.g. ethyl ⁇ s' [(p-azido-phenyl)dithio] propioimidate
  • crosslinking agent e.g. 1, 1- bis(diazoacetyl)-2-phenylethane
  • the subject poly/peptides thereof may be labeled directly (radioisotopes, fluorescers, etc.) or indirectly with an agent capable of providing a detectable signal, for example, a heart muscle kinase labeling site.
  • ORC poypeptides with ORC binding specificity are identified by a variety of ways including crosslinking, or preferably, by screening such polypeptides for binding to or disruption of ORC-ORC complexes.
  • Additional ORC-specific agents include specific antibodies that can be modified to a monovalent form, such as Fab, Fab', or Fv, specifically binding oligopeptides or oligonucleotides and most
  • ORC peptides are used as immunogens to generate specific polyclonal or monoclonal antibodies. See, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, for general methods.
  • libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily producible.
  • Useful agents are identified with assays employing a compound comprising the subject polypeptides or encoding nucleic acids.
  • assays employing a compound comprising the subject polypeptides or encoding nucleic acids.
  • ORC polypeptide find convenient use.
  • immobilized ORC-ORC or ORC-nucleic acid complexes provide convenient targets for disruption, e.g. as measured by the disassociation of a labelled component of the complex.
  • Such assays are amenable to scale-up, high throughput usage suitable for volume drug screening. While less preferred, cell-based assays may be used to
  • Preferred agents are ORC- and species-specific. Useful agents may be found within numerous chemical classes, though typically they are organic compounds; preferably small organic compounds. Small organic compounds have a molecular weight of more than 150 yet less than about 4,500, preferably less than about 1500, more preferably, less than about 500. Exemplary classes include steroids, heterocyclics, polycyclics, substituted aromatic compounds, and the like. Selected agents may be modified to enhance efficacy, stability, pharmaceutical compatibility, and the like. Structural identification of an agent may be used to identify, generate, or screen additional agents. For example, where peptide agents are identified, they may be modified in a variety of ways as described above, e.g. to enhance their proteolytic stability. Other methods of stabilization may include encapsulation, for example, in liposomes, etc. The subject binding agents are prepared in any convenient way known to those in the art.
  • compositions and agents disclosed herein may be administered by any convenient way.
  • Small organics are preferably administered orally; other compositions and agents are preferably administered parenterally, conveniently in a pharmaceutically or physiologically acceptable carrier, e.g., phosphate buffered saline, or the like.
  • a pharmaceutically or physiologically acceptable carrier e.g., phosphate buffered saline, or the like.
  • the compositions are added to a retained physiological fluid.
  • many of the disclosed therapeutics are amenable to direct injection or infusion, topical, in tratracheal/nasal- administration e.g. through aerosal, intraocularly, or within/on implants e.g. collagen, osmotic pumps, grafts comprising appropriately transformed cells, etc.
  • the ' amount administered will be empirically determined, typically in the range of about 10 to 1000 ⁇ g/kg of the recipient.
  • concentration will generally be in the range of about 50 to 500 ⁇ g/ml in the dose administered.
  • Other additives may be included, such as stabilizers, bactericides, etc. These additives will be present in conventional amounts.
  • the invention provides isolated nucleic acids encoding ORC genes, their transcriptional regulatory regions and the disclosed unique ORC polypeptides which retain ORC-specific function.
  • an "isolated" nucleic acid is present as other than a naturally occurring chromosome or transcript in its natural state and is typically joined in sequence to at least one nucleotide with which it is not normally associated on a natural chromosome; nucleic acids with substantial sequence similarity hybridize under low stringency conditions, for example, at 50°C and SSC (0.9 M saline/0.09 M sodium citrate) and remain bound when subject to washing at 55 °C with SSC, wherein regions of non-identity of substantially similar nucleic acid sequences preferably encode redundant codons; a partially pure nucleotide sequence constitutes at least about 5 % , preferably at least about 30%, and more preferably at least about 90% by weight of total nucleic acid present in a given fraction; unique portions of the disclosed nucleic acids are of length sufficient to distinguish previously known nucle
  • the invention's ORC polypeptide encoding polynucleotides are associated with heterologous sequences.
  • heterologous sequences include regulatory sequences such as promoters, enhancers, response elements, signal sequences, polyadenylation sequences, etc., introns, 5' and 3' noncoding regions, etc.
  • portions of the coding sequence are spliced with heterologous sequences to produce soluble, secreted fusion proteins, using appropriate signal sequences and optionally, a fusion partner such as ⁇ -Gal.
  • especially useful oligonucleotides are between about 10 and 30 nucleotides in length and include sequences surrounding the disclosed ATG start site, especially the oligonucleotides defined by the disclosed sequence beginning about 5 nucleotides before the start site and ending about 10 nucleotides after the disclosed start site.
  • the ORC encoding nucleic acids can be subject to alternative purification, synthesis, modification, sequencing, expression, transfection, administration or other use by methods disclosed in standard manuals such as Current Protocols in Molecular Biology (Eds. Aufubel, Brent, Kingston, More, Feidman, Smith and Guatemala, Greene Publ. Assoc, Wiley-Interscience, NY, NY, 1992) or that are otherwise known in the art.
  • the invention also provides vectors comprising the described ORC nucleic acids.
  • vectors comprising the described ORC nucleic acids.
  • vectors will often include a promotor operably linked to an ORC polypeptide-encoding portion, one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g. antibiotic resistance.
  • the inserted coding sequences may be synthesized, isolated from natural sources, prepared as hybrids, etc.
  • Suitable host cells may be transformed/ transfected/infected by any suitable method including electroporation, CaCl 2 mediated DNA uptake, viral infection, microinjection, microprojectile, or other methods.
  • Appropriate host cells include bacteria, archebacteria, fungi, especially yeast, and plant and animal cells, especially mammalian cells.
  • Prefe ⁇ ed expression systems include COS-7, 293, BHK, CHO, TM4, CV1, VERO-76, HELA, MDCK, BRL 3A, W138, Hep G2, MMT 060562, TRI cells, and baculovirus systems.
  • Preferred replication systems include Ml 3, ColEl, SV40, baculovirus, lambda, adenovirus, AAV, BPV, etc.
  • a large number of transcription initiation and termination regulatory regions have been isolated and shown to be effective in the transcription and translation of heterologous proteins in the various hosts. Examples of these regions, methods of isolation, manner oi " manipulation, etc. are known in the art.
  • the subject nucleic acids may be integrated into a host genome by recombination events.
  • a nucleic acid can be electroporated into a cell, and thereby effect homologous recombination at the site of an endogenous gene, an analog or pseudogene thereof, or a sequence with substantial identity to an ORC- encoding gene.
  • Other recombination-based methods such as nonhomologous recombinations, deletion of endogenous gene by homologous recombination, especially in pluripotent cells, etc. , provide additional applications.
  • compositions and methods disclosed herein may be used to effect gene therapy. See, e.g. Zhu et al. (1993) Science 261, 209-211; Gutierrez et al. (1992) Lancet 339, 715-721.
  • cells are transfected with ORC-encoding sequences operably linked to gene regulatory sequences capable of effecting altered ORC expression or regulation.
  • target cells may be transfected with complementary antisense polynucleotides.
  • administration will depend on a number of variables that are ascertained empirically. For example, the number of cells will vary depending on the stability of the transfered cells.
  • Transfer media is typically a buffered saline solution or other pharmacologically acceptable solution.
  • amount of other administered compositions e.g. transfected nucleic acid, protein, etc. , will depend on the manner of administration, purpose of the therapy, and the like.
  • the genes encoding six ORC subunits from S. cerevisiae are used to obtain the functional homologues of the ORC proteins from other species.
  • the ORCI gene is conserved in a related fungi klyuermyces lactis.
  • the ORCI gene in both S. cerevisie and t lactis contain conserved primary protein sequence that are utilized to obtain the ORCI gene from other species including other fungi and from human.
  • PCR is used to identify the ORCI protein in any eukaryotic species.
  • the cloned gene encoding ORCI polypeptide from any fungi or from human cells is used to express the protein in a bacterial expression system to make antibodies against the polypeptide. These antibodies are used to immunoprecipitate the ORC complex from the relevant species.
  • sequence the ORC polypeptides is obtained.
  • other genes or cDNAS encoding the ORC polypeptides from other fungi species and from human cells are obtained. As we demonstrate herein how to reconstitute the ORC complex by expressing each of the S.
  • Inhibitors of ORC protein in fungi provide valuable reagents to selectively inhibit proliferation of fungal cell division by inhibiting the initiation of DNA replication. This offers a powerful, selective target for antifungal agents valuable in controlling fungal infections in human and other species. For example, as disclosed herein, inhibiting the ORC function by mutation in S. cerevisiae can actually cause the death of the mutant cells. In human proliferative disorders such as cancer, cells of the diseased tissue undergo uncontrolled cell proliferation. A key event in this cell proliferation is the initiation of DNA replication. Inhibiting the initiation of DNA replication through inhibition of ORC function provides a valuable target for inhibitors of cell growth.
  • ORC function is reconstituted in vitro. Using this recombinant, expressed protein, inhibitors of ORC function are obtained that block the initiation of DNA replication in cell cycle. As described above, small molecular inhibitors of ORC DNA binding or other activities provide valuable reagents as anti-cancer and anti-proliferation drugs.
  • the following examples are offered by way of illustration and not by way of limitation.
  • ORC binds all four of the mating-type silencers, that ORC can bind sequences other than the ACS and that it plays an important role at HML and HMR.
  • a clear link between ORC function and transcriptional silencing was provided by the finding that a mutation in a gene encoding a subunit of ORC was defective for repression at HMR (below).
  • To clone the genes encoding the various ORC subunits peptides derived from each of the ORC subunits were sequenced (24).
  • a candidate gene, refe ⁇ ed to as ORC2 was isolated by complementation of a temperature sensitive mutation that showed silencing defects at the permissive temperature.
  • ORC2 mediated the silencing function of the ACS at HMR-E, making it a good candidate to encode a subunit of ORC (below).
  • Comparison of the predicted amino acid sequence of ORC2 showed that all of the peptides derived from the 72 kd subunit of ORC were within the open reading frame of the ORC2 gene indicating that it encoded the second largest subunit of ORC.
  • ORC2 mutations alter ORC function in vitro.
  • the resulting protein blot was incubated with polyclonal mouse sera raised against the entire ORC complex. This sera detects all but the 50 kd subunit of ORC. Antibody-antigen complexes were detected with horseradish peroxidase conjugated secondary antibodies followed by incubation with a chemiluminescent substrate.
  • Wild type fractions contained the 120, 72, 62, 56, and 53 kd subunits of ORC in roughly equal quantity.
  • the mutant fractions showed a distinctly different subunit composition. While the amount of the 120 and 56 kd subunits was only slightly reduced relative to the wild type fraction, the amount of the 72, 62, and 53 kd subunits was reduced dramatically.
  • UV cross-linking experiments the same three subunits are specifically cross-linked to DNA in an ACS and ATP dependent manner, suggesting an important role for one or more of these subunits in ORC DNA binding (15).
  • a second gene that represented a strong candidate to encode one of the subunits of ORC was the AAP1 gene.
  • This gene was cloned using a novel screen fop proteins that bound to the ACS in vivo (below).
  • AAP1 As compared to the predicted amino acid sequence of this gene, we found that all of the peptides derived from the 50 kd subunit of ORC were encoded by the open reading frame of the AAP1 gene (28). For this reason we now refer to AAP1 as ORC6 , as it encodes the smallest of the six ORC subunits.
  • the identification of this gene as a subunit of ORC provides direct evidence that ORC is bound to the ACS in vivo.
  • Purified ORC (— 10 ⁇ g per subunit) was separated by SDS-PAGE and stained with 0.1 % Coomassie Brilliant Blue G (Aldrich). After destaining the gel was soaked in water for one hour. The protein bands were excised, transferred to a microcentrifuge tube and treated with 200 ng of Achromobacte ⁇ protease I (Lysylendopeptidase: Wako). The resulting peptides were separated by reverse- phase chromatography and sequenced by automated Edman degradation (Applied Biosystems model 470).
  • Antibodies were raised against the entire ORC complex using a single mouse. The resulting sera was able to recognize all but the 50 kd subunit of ORC. Proteins were transfe ⁇ ed to nitrocellulose and antigen-antibody complexes were detected with horse radish peroxidase conjugated secondary anitbodies and a chemiluminescent substrate. 23. Yeast cells were grown to a density of 1-4 x 10 7 cells per ml at 24°C then diluted to a density of 2-4 x 10 6 cells per ml into YPD containing 6 ⁇ M alpha- factor and incubated for 2-2.5 hours at 24°C (> 90% unbudded cells).
  • hydroxyurea a ⁇ est experiments alpha factor was washed away and the cells were resuspended in YPD containing 100 mM hydroxyurea and incubated an additional 2.5 hours ( > 90% large budded cells). After incubation with the growth inhibitor, cells were briefly sonicated and plated on YPD plates pre-incubated at either 24°C or 37°C and observed at 0, 3, and 6 hours after plating.
  • Yeast cells were grown to a density of 1-4 x 10 7 cells per ml at 24°C and diluted into fresh YPD at either 37°C or 24°C and a density of 2-4 x 10 6 cells per ml. At times after dilution, 3 x 10 6 cells were processed as described (42).
  • ORC2 a gene required for viability and silencing
  • a temperature-sensitive mutation called orc2-l was isolated that, at the permissive temperature, resulted in derepression of HMRa. flanked by the synthetic silencer and did not cause derepression of HMRa. flanked by the wild-type silencer (20). Because the orc2-l mutant was temperature- sensitive and silencing defective, it merited further analysis.
  • the temperature resistance of a heterozygus orc2-l/ORC2 diploid (JRY2640) established that the mutation was recessive. The diploid was transformed with a plasmid containing HMRa flanked by a mutant silencer (pJR1212), to provide MATal function required for sporulation.
  • the temperature-sensitive growth phenotype segregated 2 ts 2 wild type in each of 23 tetrads, indicating that it was caused by a single nuclear mutation.
  • An HMLOL m ⁇ tal HMR orc2-l segregant JRY3683 was obtained from the diploid following sporulation.
  • MAT ⁇ / MAT ⁇ diploid formed by a mating event between these two strains. This diploid was able to sporulate due to the low level of expression of HMRa in the diploid caused by the RAPl -site mutation in the HMR-E silencer (21). One of these strains had the orc2-l mutation (JRY4133) and the other did not.
  • a second screen was performed to identify additional mutations in essential genes with a role in silencer function.
  • This second screen produced 50 mutants that were temperature sensitive for growth, and in which HMRa (flanked by a mutation in the RAPl -binding site) was derepressed at a semi-permissive temperature. Complementation tests for both growth at 37 °C and for mating phenotype were performed between orc2-l and the collection of temperature-sensitive mutants from the second screen. The collection of temperature sensitive mutants had the matal stel4 genotype, but were able to mate as ⁇ ;'s due to the derepression of HMR ⁇ .
  • the new mutations (in strains JRY4136, 4137 and 4138) were designated orc2-2, orc2-3, and orc2-4. To investigate the possibility that the new mutations were in a gene other than ORC2 yet still failed to complement orc2-l, the allelism between orc2-l and orc2-3 was tested.
  • the original m ⁇ tal orc2-3 stel4 mutant was cured of its HMR ⁇ plasmid, creating JRY 4137, and mated with a MAT ⁇ HMRa-e-r ⁇ pl-10 orc2-l strain (JRY3685). In 24 tetrads from this diploid, all segregants were temperature sensitive for growth, indicating strong linkage between orc2-l and orc2-3 ( ⁇ 2 centimorgans). All further studies were performed using the orc2-l allele, which provided the stronger mutant phenotypes. Map position of ORC2
  • JRY4130 x JRY4134 tested the linkage between seel 8, which is centromere proximal to LYS2, and ORC2. Because both orc2-l and secl8 are temperature sensitive, an ORC2 allele marked by URA3 (from pJR1423) was used to determine that SEC18 and ORC2 were separated by 6.6 centimorgans (Table 1).
  • the ORC2 mutants a ⁇ ested with a cell cycle terminal phenotype.
  • mutant orc2-l strains were grown in liquid medium at 23 °C, the permissive temperature, and then shifted to 37 °C to test whether the cells a ⁇ ested with a single terminal morphology.
  • orc2-l cells JRY3683
  • JRY3683 were grown to log phase at the permissive temperature (23°C) and the culture was split.
  • Half of the culture was grown an additional five hours at the permissive temperature and the other half was shifted to the nonpermissive temperature (37°C) and grown for an additional five hours. At that time, both cultures were fixed and stained with DAPI to allow visualization of the nucleus.
  • ORC2 cells harvested either after continuous growth at the permissive temperature or after a shift to the nonpermissive temperature were fixed and stained with DAPI allowing visualization of DNA with fluorescence microscopy.
  • the cells shifted to the nonpermissive temperature looked very different: the majority a ⁇ ested as large budded cells, and for the most part, each mother-daughter pair contained only a single brightly-staining region, often at or near the neck.
  • the ORC2 gene was cloned by complementation of the orc2-l temperature sensitivity (22).
  • One complementing clone (pJR1416) was chosen for further analysis. Subclones missing various fragments from the insert were retransformed into an orc2 strain to assay whether the deletion affected the clone's ability to complement orc2-F temperature sensitivity. The key observations were that the deletion of a 2.8-kb Sstl-SstI fragment destroyed complementation activity, whereas the deletions of flanking sequences (Xbal, and the larger_>_-tl fragment) had no effect. The 2.8-kb fragment was subcloned (pJR1263), and shown to possess complementing activity.
  • ORC2 was disrupted by URA3, (23), and integrated into a diploid homozygous for ura3 and ORC2, (JRY3444). Of the 41 tetrads dissected, 40 tetrads had two live and two dead segregants, and one tetrad had only one live segregant. The colonies that grew were, without exception, Ura-. By inference, the dead segregants contained the URA3 gene, and thus the ORC2 disruption, indicating that ORC2 function was essential for cell viability at all temperatures. The dead segregants were examined under a microscope to gain some insight into the true null phenotype. Most of the spores germinated into cells that were elongated or otherwise deformed and had not divided. In no case did the cell divide more than two times. Thus in many spores, the absence of ORC2 blocked cell division but not growth.
  • ARSl a chromosomal origin of replication (YRP14/CEN4/ARS1/ARS1; (24, 25), selecting for uracil prototrophy.
  • Transformants were grown on selective medium at 23°C, the permissive temperature for orc2-l. The colonies were picked from the selective plate, serially diluted, plated onto solid rich medium and grown to colonies at 23°C. The wild-type transformants grew into colonies most of which were white with a few exhibiting red sectors. The small fraction of red colonies were from cells in the selectively grown colony that had lost the plasmid. In contrast, the majority of colonies from the orc2-l mutant were red, reflecting a high degree of plasmid loss among the cells in the selectively grown colony. Moreover, in the orc2-l strain, red sectors were present in the majority of white colonies with some white colonies displaying multiple red sectors.
  • the sequence of the 2.8-kb Sstl-Sstl ⁇ rc2-complementing fragment was determined and deposited in Genbank (Accession # L23924). The only open reading frame of significant length was deduced to be ORC2, and predicted a 620 residue protein of approximately 68 kD.
  • the Sstl fragment included 806-bp of upstream sequence and 140-bp of downstream sequence.
  • Orc2p protein was 15% basic residues and 16% serine/threonines. Fully 50% of the N-terminal residues (residues 15-280) were lysine, arginine, proline, serine, or threonine.
  • the KeyBank motif program revealed several matches to peptide motifs within Orc2p.
  • Orc2p contained many potential phosphorylation sites: 3 for cAMP- and cGMP-dependent protein kinase (starting at residues -57, 433 and 546), 12 for protein kinase C (24, 41, 42, 89, 101, 102, 176, 321, 335, 431, 521, and 549) and 14 for caseine kinase II (60, 148, 149, 182, 238, 270, 389, 481, 486, 491, 505, 552, 595, and 605), and match to the nuclear targeting sequence (residues 103-107).
  • Another homolgy is with the region near the catalytic domain of human topoisomerase I proteins which has diverged among topoisomerase I proteins from other species except for the region su ⁇ ounding the invariant active-site tyrosine.
  • This region includes a consensus sequence consisting of a serine and lysine residue near the tyrosine (25).
  • the Orc2p protein also contained such a consensus sequence near its C-terminus.
  • mutation of this putative active-site tyrosine to phenylalanine had no detectable effect on the ability of ORC2 to complement the temperature-sensitivity or mating defect of an orc2-l strain.
  • HMRa-e- r ⁇ pl-10 refers to the allele of HMR-E, originally described as PAS1-1, that contains a mutation in the RAPl binding site (21).
  • Cells bearing mutations causing derepression of the plasmid-borne a cassette could be distinguished from the other classes of mutations by exploiting a feature of yeast plasmids. Approximately 10% of the cells in these colonies lacked the plasmid and thus could, in principle, mate with the tester strain and form Ura " diploids capable of growth on the plates supplemented with uracil. If a colony had a mutation in the mating response pathway, the cells would be unable to mate even in the absence of the plasmid, and thus would be unable to form diploids capable of growth on medium supplemented with uracil.
  • An orc2-l strain (JRY3683) was transformed with a CEN EE/2-based Saccharomyces cerevisiae genomic library (32) Approximately 1000 to 1500 transformants formed colonies at 23 °C. Replica prints of these colonies were incubated at 37 °C to screen for the ability to grow at elevated temperatures. Plasmids were isolated from temperature-resistant strains and retested. Those plasmids that complemented the defect a second time were analyzed by restriction digestion. One plasmid from the CEN-LEU2 library (pJR1416) was chosen for further analysis. 22.
  • ORC2 was disrupted with the TnlO LUK transposon (33), which inserted within the ORC2 coding sequence on the plasmid (pJR1146) carrying the Sstl orc2- 1 complementing fragment. Plasmid pJRl 147 had the Tnl QLUK insertion within the ORC2 coding region.
  • a mutation was introduced into the RAPl binding site at HMR-E adjacent to the HMRa locus by oligonucleotide-directed mutagenesis (35), and the change confirmed by sequencing.
  • the RAPl site mutation was identical to the PAS1-1 mutation of HMR-E characterized previously that blocks RAPl protein binding in vitro (21), and is described here as HMRa-e-rapl-10.
  • the plasmid consisting of the HMRa-e-rapl-10 Hin ⁇ lll fragment in pRS316 was named pJR1425.
  • the wild- type HMRa version of the same plasmid was named pJR1426.
  • the ORC2 gene was defined by the orc2-l mutation.
  • An orc2- complementing plasmid (pJR1416) was obtained by complementation of the temperature sensitivity of orc2-l.
  • six derivatives of pJR1416 were made and tested for complementation.
  • the S ⁇ R-S ⁇ U. fragment was removed from the insert to yield pJR1418.
  • Three adjacent Xb ⁇ l-Xb ⁇ l fragments were removed to yield pJR1422.
  • Sphl cleaved once in the insert and once just inside the vector. Deleting this Sphl-Sphl fragment produced pJR1417.
  • the first, pJR1423, contained an Xhol/Kpnl insert (from pJR1416) which extended from a few kb upstream of the ORC2 start codon to about 60-bp upstream of the stop codon inserted into Xh ⁇ l- Kpnl-cnt pRS306 (36), a yeast integrating vector marked by URA3.
  • the second plasmid, pJR1424 contained the Sstl ⁇ rc2-complementing fragment inserted into the Sstl site of pRS306. 34.
  • the one-hybrid system In order to identify potential yeast initiators, we developed a genetic strategy, the one-hybrid system, to find proteins that recognize a target sequence of interest.
  • the one-hybrid system has two basic components: (i) a hybrid expression library, constructed by fusing a transcriptional activation domain to random protein segments, and (ii) a reporter gene containing a binding site of interest in its promoter region. Hybrid proteins that recognize this site are expected to induce expression of the reporter gene, because of their dual ability to bind the promoter region and activate transcription (8). This association may be indirect, since hybrids that interact with endogenous proteins already occupying the binding site will also activate transcription (7).
  • the protein inco ⁇ orated in the hybrid should be functionally relevant.
  • the protein component of this screen was provided by a set of three complementary yeast hybrid expression libraries, YL1-3, containing random yeast protein segments fused to the GAL4 transcriptional activation domain (GAL ⁇ * 0 ) (9).
  • the reporter gene for our screen contained four direct repeats of the ACS in its promoter region and was integrated into the yeast strain GGY1 to form JLY363(ACS W ) (10).
  • JLY365(ACS MU ⁇ ANT ) To determine the dependence of lacZ induction on the ACS, we constructed in parallel JLY365(ACS MU ⁇ ANT ), which harbors a reporter gene carrying four copies of a nonfunctional multiply-mutated ACS (Fig. 4) (10).
  • reporter constructs containing direct repeats of four ACS point mutants were each integrated into GGY1 to generate the set of reporter strains(l ⁇ ).
  • the five AAP clones were individually examined in these strains for the ability to induce lacZ expression.
  • AAPl displayed a co ⁇ espondence between the induction of this set of reporter genes and the ARS function (12) of their ACS.
  • the AAP5 hybrid exhibited a slightly weaker co ⁇ elation, and the remaining clones showed poor co ⁇ elation.
  • AAP2-4 encoded hybrid proteins with only short peptide extensions (10, 22, and 38 amino acids respectively) fused to the GAL4 AD , suggesting that these hybrids were not responsible for the transcriptional induction attributed to these clones. Because of this finding and the lack of proper sequence specificity for the ACS element, AAP2-4 were not studied further.
  • AAPl The full-length gene for AAPl was cloned from a yeast genomic library and sequenced (15) (Genbank accession no. L23323).
  • AAPl contains an open reading frame for a protein 435 amino acids long with a predicted molecular weight of 50,302 daltons.
  • the hybrid GAL4 AD -AAP1 protein obtained from the screen was a fusion of the GAL4 AD to the C-terminal two-thirds of the predicted full-length protein (residues 135-435) , indicating that this portion of the molecule is sufficient for association with the ACS.
  • Orc6p contains four phosphorylation sites, (S/T)PXK, for cyclin- dependent protein kinases (20) clustered in the first half of the molecule. Using the more relaxed consensus site (S/T)P adds two more sites to this cluster.
  • S/T more relaxed consensus site
  • Orc ⁇ p contains a potential nuclear localization signal (NLS) within the (S/T)PXK cluster and one in the C- terminal domain (amino acid residues 117-122 and 263-279). Orc6p can be seen in the nucleus by immunofluoresence.
  • NLS nuclear localization signal
  • ORC6 deletion experiments were complicated by the presence of a second open reading frame (ORF2) of 250 amino acids on the antisense strand of the ORC6 gene.
  • ORF2 spans nucleotides 1617 to 868 of the Genbank sequence and overlaps the C-terminal two-thirds of the ORC6 coding sequence.
  • a marked deletion that removed the N-terminal third of the ORC6 coding sequence without affecting ORF2 (pJL733) was introduced into diploids (21). Tetrad analysis again showed the ORC6 deletion cosegregating with cell death.
  • an ORC6 gene was constructed that contains a silent codon change for the ORC6 ORF but introduces a UGA stop codon in ORF2 (22). This gene was able to rescue a haploid strain containing a full deletion of the ORC6 ORF.
  • ORC6 is essential for cell viability.
  • pJL749 (28), a plasmid that overexpresses Orc ⁇ p several hundred-fold, was introduced into a virtually isogenic set of temperature-sensitive cdc mutants a ⁇ esting at various points in the cell cycle (29).
  • the C-terminal portions were derived from random yeast protein segments which have been fused to the end of the GAL4 AD . These segments are encoded by short (l-3kb) fragments from a Sau3a partial digest of yeast genomic DNA. Together, YL1-3 ensure that all three reading frames of these fragments can be expressed. 10.
  • pLRlDl is described in R.W. West Jr., R.R. Rogers, M.
  • ARSl domain A and several of its mutant derivatives were inserted into the Bgl II site of pBgl-lacZ to generate all the reporter genes used in this work.
  • the inserted repeat elements derived from complementary oligonucleotides, were oriented with the TATA box to their right.
  • Each reporter gene construct was integrated into the URA3 locus of GGY1 ⁇ MATa Dg ⁇ l4 Dg ⁇ l80 ur ⁇ 3 leu2 his3 ⁇ de2 tyr) [G. Gill and M. Ptashne, Cell 51, 121 (1987)] to create a reporter strain. Integration of pBgl- lacZ into GGY1 generated JLY387.
  • the ARS function of the mutant sequences was analyzed in the context of ARSl domain B (Bglll-Hinfl fragment, nt 853-734) in the following CEN-based URA3-containing plasmids: pJL347 (wt), pJL243 (multiple), pJL326 (A863T), pJL338 (T869A), pJL330 (T862C), and pJL316 (T867G). These plasmids were transformed into JLY106 ⁇ MATa ur ⁇ 3 leu2 his3 trpl lys2 ⁇ de2) and its homozygous diploid counterpart JLY162.
  • pJL243, pJL326, and pJL338 did not yield a high frequency of transformation and could not be assayed quantitatively for ARS function.
  • pJL347, pJL330, and pJL316 transformed cells with high efficiency and were assayed for mitotic stability [Stinchcomb, et al. Nature 282, 39 (1979)].
  • pGAD3R (11) the parent vector for the YL3 library, contains no ORC6 sequence.
  • ORC6 deletion analysis was performed in JLY461 ⁇ MATa/MATa ur ⁇ 3/ur ⁇ 3 Ieu2/leu2 his3/his3 trpl/trpl ⁇ de2/ ⁇ de2 [cir°J), JLY462 ⁇ MAT ⁇ /MATa ur ⁇ 3/ur ⁇ 3 Ieu2/leu2 trpl/trpl his4/his4 c ⁇ nl/c ⁇ nl), and JLY463 ⁇ MAT ⁇ /MATa ur ⁇ 3/ur ⁇ 3 Ieu2/leu2 trpl/trpl his3/HIS3); their respective genetic backgrounds are S288c, EG123, and A364a.
  • JLY461, JLY462, and JLY463 by pJL731 full deletion
  • JLY481, JLY475, and JLY469 respectively.
  • Disruption of JLY461, JLY462, and JLY463 by pJL733 N-terminal deletion
  • pJL749 contains the GAL1 promoter (nt 146-816) driving the expression of ORC6 (nt 443-2298) in the high-copy yeast shuttle vector RS425 [T. W. Christianson, et al., Gene 110, 119 (1992)].
  • 26. The cdc mutant strains have been backcrossed 4-5 times against two congenic strains derived from A364a , RDY487 ⁇ MATa leu2 ur ⁇ 3 trpl) and RDY488 ⁇ MAT ⁇ leu2 ur ⁇ 3 trpl). All are ur ⁇ S ' leu2 trpl.
  • RDY510, RDY664, JLY310, and JLY179 are MATa; the rest are MAT ⁇ . Additional markers can be found in JLY310(a_fe2), RDY543( ⁇ w3), and RDY619 (pep4D::TRPl his3 ⁇ de2).
  • pJL749, pJL772, and RS425 (28) were transformed into these strains and plated on SD-LEU at 22° C.
  • Four colony-purified isolates from each transformation were patched onto SD-LEU plates and replica-plated to SGAL-LEU plates, all at 22° C.
  • the patches on SGAL-LEU were replica-plated to a series of pre-warmed SGAL- LEU plates at 22°, 25°, 27°, 30°, 32.5°, 35°, 37°, and 38° C.
  • the viability of cdc mutants containing pJL749 was compared to those containing pJL772 and pRS425.
  • Acetate, and 20% glycerol was added to the cells and after thawing the cells were broken using a bead beater (Biospec Products) until greater than 90% cell breakage was achieved (twenty 30 second pulses separated by 90 second pauses). After breakage is complete, the volume of the broken cells was measured and one twelfth volume of a saturated (at 4°C) solution of ammonium sulfate was added and sti ⁇ ed for 30 minutes. This solution was then spun at 13,000 x g for 20 minutes. The resulting supernatant was transfe ⁇ ed to 45Ti bottle assemblies (Beckman) and spun in a 45Ti rotor at 44,000 RPM for 1.5 hrs.
  • the volume of the resulting supernatant was measured and 0.27g/ml of ammonium sulfate was added. After stirring for 30 minutes, the precipitate was collected by spinning in the 45 Ti rotor at 40,000 RPM or 30 minutes.
  • the resulting pellet was resuspended using a B- pestle dounce in buffer H/0.0 (50 mM Hepes-KOH, pH 7.5, 1 mM EDTA, 1 mM EGTA, 5 mM Mg Acetate, 0.02% NP-40, 10% glycerol) and dialyzed versus H/0.15M KC1 (Buffer H with 0.15 M KC1 added).
  • Achromobacter protease I (Lysylendopeptidase: Wako) was added and incubated at 30 °C for 24 hrs. After digestion the samples were centrifuged and the supernatant was passed through an Ultrafree-MC filter (Millipore, 0.22 ⁇ m). The gel slices were then washed twice in 0.1 % TFA for one hour and the washes were recovered and filtered as above. All filtrates were combined and reduced to a volume suitable for injection on the HPLC using a speed-vac.
  • the digests were separated by reverse-phase HPLC (Hewlett-Packard 1090 system) using a Vydac C18 column (2.1x 250 mm, 5 ⁇ m, 300 angstroms) with an ion exchange pre-column (Brownlee GAX-013, 3.2x 15mm).
  • the peptides were eluted from the C-18 column by increasing acetonitrile concentration and monitored by their absorbance at 214, 280, 295, and 550 nm.
  • Amino acid sequencing of the purified peptides was performed on an automated sequencer (Applied Biosystems model 470) with on-line HPLC (Applied Biosystems model 1020A) analysis of PTH-amino acids.
  • ORC SUBUNIT CLONING ORCI: To clone the gene for the largest (120 kd) subunit of ORC, the following degenerate oligonucleoide primers 1201 and 1202 were synthesized based on the sequence of the first ORCI peptide. These oligos were used to perform PCR reactions using total yeast genomic DNA from the strain W303 a as target. A 48 base pair fragment was specifically amplified. This fragment was subcloned and sequenced. The resulting sequence encoded the predicted peptide indicating that it was the co ⁇ ect amplification product. A radioactively labeled form of the PCR product was then used to probe a genomic library of yeast DNA sequences resulting in the identification of two overlapping clones. Sequencing of these clones resulted in the identification of a large open reading frame that encoded a protein with a predicted molecular weight of 120 kd and that encoded all four of the ORCI peptide sequences.
  • ORC3 To clone the gene for the 62 kd subunit of ORC, the following degenerate oligonucleoide primers 621 and 624 were synthesized based on the sequence of the third peptide. These oligos were used to perform PCR reactions using total yeast genomic DNA from the strain W303 a as target. A 53 base pair fragment was specifically amplified. This fragment was subcloned and sequenced. The resulting sequence encoded the predicted peptide indicating that it was the co ⁇ ect amplification product. A radioactively labeled form of the PCR product was then used to probe a genomic library of yeast DNA sequences resulting in the identification of two overlapping clones.
  • ORC4 By comparing the sequnce of the ORC4 peptides to that of the known potentially protein encoding sequnces in the genbank database we found that a portion of the ORC4 coding sequence had been previously cloned in the process of cloning the adjacent gene. Using the information from the database we were able to design a perfect match oligo and use this to immediately screen a yeast library. Using this oligo as a probe of the same yeast genomic DNA library a lambda clone was isolated that contained the entire ORC4 gene. This gene encoded a protein of predicted molecular weight 56 kd and also all of the peptides derived from the peptide sequencing of the 56 kd subunit.
  • ORC5 To clone the gene for the 53 kd subunit of ORC, the following degenerate oligonucleoide primers 535 and 536 were synthesized based on the sequence of the first ORC5 peptide. These oligos were used to perform PCR reactions using total yeast genomic DNA from the strain W303 a as target. A 47 base pair fragment was specifically amplified. This fragment was subcloned and sequenced. The resulting sequence encoded the predicted peptide indicating that it was the co ⁇ ect amplification product. A radioactively labeled form of the PCR product was then used to probe a genomic library of yeast DNA sequences resulting in the identification of a single lambda clone.
  • ADDRESSEE FLEHR, HOHBACH, TEST, ALBRITTON & HERBERT
  • B STREET: 4 Embarcadero Center, Suite 3400
  • TCGTCCTTTA AATTATTACA ATAAACTGTT TTCTGAAACT GCAAATAAAA ATGAACTGTA 1080 TCTCACTGCA GAATTAGCCG AATTGCAGCT ATTTAACTTT ATCAGGGTTG CCAACGTAAT 1140 GGATGGAAGC AAATGGGAAG TATTGAAAGG AAATGTCGAT CCAGAAAGAG ACTTTACAGT 1200 TCGTTATATT TGTGAGCCGA CTGGGGAGAA ATTTGTGGAC ATTAATATTG AGGATGTCAA 1260 AGCTTACATA AAGAAAGTGG AGCCAAGGGA AGCCCAGGAA TATTTGAAAG ATTTAACACT 1320
  • Asp Glu Gin Gly Asn lie lie Asp Gly Gly Gin Lys Arg Leu Arg Arg 20 25 30
  • Lys Ala Tyr lie Lys Lys Val Glu Pro Arg Glu Ala Gin Glu Tyr Leu 195 200 205
  • Lys Asp Leu Thr Leu Pro Ser Lys Lys Lys Glu lie Lys Arg Gly Pro 210 215 220
  • GAGCTCAACA CCACCATTGA GAACGTAGAA TTTCAATTTT TAAGCTGATT CTCTTTCTGC 60
  • AAA AGG GTT GAC CCA CAT GGA GAA AGA CAA CTG AGA AGA ATT CAT TCA 929 Lys Arg Val Asp Pro His Gly Glu Arg Gin Leu Arg Arg He His Ser
  • AAA AAA ATG TTT CCC CAG TAT TGG TTT GAA TTG ACT CAA GGA TTC TCC 1745 Lys Lys Met Phe Pro Gin Tyr Trp Phe Glu Leu Thr Gin Gly Phe Ser 300 305 310
  • GTA CCC TAC ACG TAT GCG GAA CTT GAA AAA CTT CTG AAA ACC GTT TTA 2657 Val Pro Tyr Thr Tyr Ala Glu Leu Glu Lys Leu Leu Lys Thr Val Leu 605 610 615
  • AATTGAATAT AACCAATTTT AATCTGATAG AATTATATCA TAATTTGCTT ATTGGCAAAC 1980 TAGACTCCTA TCTAGATCGT TGGTCAGCAT GTAAAGAGTA TAAGGATCGG CTTCATTTTG 2040 AACCCATTGA TACAATTTTT CAAGAGCTAT TTACTTTGGA CAACAGAAGT GGATTACTTA 2100 CCCAGTCGAT TTTCCCTTCT TACAAGTCAA ATATCGAAGA TAACTTACTA AGTTGGGAGC 2160 AGGTGCTGCC TTCGCTTGAT AAAGAAAATT ATGATACTCT TTCTGGAGAT TTGGATAAAA 2220
  • MOLECULE TYPE peptide (xi ) SEQUENCE DESCRIPTION : SEQ ID NO: 6 :
  • TACTCTCCTT TCTACCAGGT ATTCTAACTC TATTATATAA TTAAAAAAAA AATAACCATA 540 TATTTTGTAT TAAGTTTCAT ACATGTGTTC AAGTGTATTT TTGGATTTAT CATTTTTCTA 600
  • AACTACTAAT ATCGGTAATA TTCAAAAGAA GAAGCATGAC TATAAGCGAA GCTCGTCTAT 780 CACCGCAAGT CAATCTTCTC CCAATAAAGA GGCACTCAAA CGAAGAGGTA GAGGAGACTG 840 CAGCGATTCT AAAAAAGCGT ACTATAGATA ATGAAAAGTG TAAAGACAGC GACCCTGGTT 900 TTGGTTCCCT TCAAAGAAGG TTACTGCAGC AACTTTATGG CACACTTCCT ACGGACGAAA 960 AGATAATCTT CACATATTTA CAAGATTGTC AACAAGAGAT CGATAGAATC ATTAAACAAT 1020
  • GCTGCACAAC GAAATTAAAT ATCTTGGAAT ATTTAGAAAA GAGGGTAAAG AGTAGATTTT 1560
  • AAAAGCGCCC TACTGTATGG AAAAACAATG AATGAGGAGA CTGAACGGCG CAAAATTGTT 2220
  • CTGTGTATTT CTTTGTTCTT TGCCGTTGTT TACGTTAGTA AGAAATCGGC ATTGAAAAAA 360

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Urology & Nephrology (AREA)
  • Analytical Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Veterinary Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Communicable Diseases (AREA)
  • Cell Biology (AREA)
  • Virology (AREA)
  • General Engineering & Computer Science (AREA)
  • Pathology (AREA)
  • Toxicology (AREA)

Abstract

On décrit des gènes de l'origine du complexe de réplication d'ADN (ORC), des peptides recombinés d'ORC et des procédés permettant d'identifier des protéines de liaison à l'ADN, ainsi que l'utilisation des compositions en cause. Des vecteurs et cellules qui comprennent de tels gènes d'ORC servent à produire ces peptides recombinés d'ORC qui sont particulièrement utiles dans le criblage d'agents, sélectifs pour l'ORC, qui permettent le diagnostic, le pronostic ou le traitement de maladies, notamment des infections fongiques ou des maladies néoprolifératives. Les procédés décrits permettent d'identifier un gène codant une protéine associé, directement ou non, à une séquence d'ADN déterminée, et ils consistent à transformer une banque d'expression de protéines hybrides en une souche reporter, cette banque comprenant des séquences codant des protéines, fusionnées avec un domaine d'activation de transcription exprimé de façon constitutive et la souche reporter comprend un gène reporter doté d'une copie au moins d'une séquence d'ADN sélectionnée située dans sa région promotrice. On détecte et récupère des clones qui expriment le produit de transcription ou de traduction du gène reporter.
PCT/US1994/014563 1993-12-16 1994-12-16 Genes et proteines de complexe de replication, et procedes associes WO1995016694A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU13734/95A AU700405B2 (en) 1993-12-16 1994-12-16 Origin of replication complex genes, proteins and methods
EP95904929A EP0733057A4 (fr) 1993-12-16 1994-12-16 Genes et proteines de complexe de replication, et procedes associes
JP7516984A JPH09506768A (ja) 1993-12-16 1994-12-16 複製開始点複合体遺伝子、蛋白および方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US16847993A 1993-12-16 1993-12-16
US08/168,479 1993-12-16

Publications (1)

Publication Number Publication Date
WO1995016694A1 true WO1995016694A1 (fr) 1995-06-22

Family

ID=22611645

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1994/014563 WO1995016694A1 (fr) 1993-12-16 1994-12-16 Genes et proteines de complexe de replication, et procedes associes

Country Status (5)

Country Link
EP (1) EP0733057A4 (fr)
JP (1) JPH09506768A (fr)
AU (1) AU700405B2 (fr)
CA (1) CA2178965A1 (fr)
WO (1) WO1995016694A1 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997041153A1 (fr) * 1996-05-02 1997-11-06 Cold Spring Harbor Laboratory Genes regulant la replication de l'adn
WO1998039483A1 (fr) * 1997-03-04 1998-09-11 Ventana Genetics, Inc. Methodes d'identification de sequences d'acide nucleique codant des agents qui influent sur des phenotypes cellulaires
WO1999024563A1 (fr) * 1997-11-07 1999-05-20 Iconix Pharmaceuticals, Inc. Procede de caracterisation de cibles genetiques de remplacement
US6074819A (en) * 1996-05-02 2000-06-13 Cold Spring Harbor Laboratory DNA replication-regulating genes
US6281347B1 (en) 1997-09-10 2001-08-28 O'donnell Michael Human origin of replication complex genes and uses thereof
US6361954B1 (en) 1996-05-02 2002-03-26 Cold Spring Harbor Laboratory Methods of immunoassay for human CDC6
US6566057B1 (en) 1997-02-14 2003-05-20 Deltagen Proteomics, Inc. Methods and compositions for peptide libraries displayed on light-emitting scaffolds
US6623922B1 (en) 1997-02-14 2003-09-23 Deltagen Proteomics Methods for identifying, characterizing, and evolving cell-type specific CIS regulatory elements

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992013091A1 (fr) * 1991-01-18 1992-08-06 Oncogene Science, Inc. Procede de modulation par transcription de l'expression genique d'oncogenes et de genes supprimant les tumeurs

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992013091A1 (fr) * 1991-01-18 1992-08-06 Oncogene Science, Inc. Procede de modulation par transcription de l'expression genique d'oncogenes et de genes supprimant les tumeurs

Non-Patent Citations (12)

* Cited by examiner, † Cited by third party
Title
BIOCHEMISTRY, Volume 29, issued 1990, HEIGER-BERNAYS et al., "Effects of the Antitumor Drug cis-Diamminedichloroplatinum(II) and Related Platimun Complexes on Eukaryotic DNA Replication", pages 8461-8466. *
CELL, Volume 65, issued 31 May 1991, IRIE et al., "SGV1 Encodes a CDC28/cdc2-Related Kinase Required for a G Alpha Subunit-Mediated Adaptive Response to Phereomone in S. Cerevisiae", pages 785-795. *
CYTOCHEMISTRY, Volume 12, issued 1991, HOFFMAN et al., "A New Class of Reversible Cell Cycle Inhibitors", pages 26-32. *
EMBL GENE SEQUENCE LISTING, Accesssion Number M60416 M36724, issued 19 May 1992, CLARK et al. *
EMBO JOURNAL, Volume 12, Number 10, issued 1993, COPPELECCHIA et al., "A New Yeast Translation Initiation Factor Suppresses a Mutation in the elF-4A RNA Helicase", pages 4005-4011. *
J. BIOLOGICAL CHEMISTRY, Volume 262, Number 21, issued 25 July 1987, MATSUDAIRA, "Sequence from Picomole Quantities of Proteins Electroblotted onto Polyvinylidene Difluoride Membranes", pages 10035-10038. *
J. MOLECULAR BIOLOGY, Volume 183, issued 1985, LATHE, "Synthetic Oligonucleotide Probes Deduced from Amino Acid Sequence Data: Theoretical and Practical Considerations", pages 1-12. *
NATURE, Volume 357, issued 14 May 1992, BELL et al., "ATP-Dependent Recognition of Eukaryotic Origins of DNA Replication by a Multiprotein Complex", pages 128-134. *
NATURE, Volume 357, issued 14 May 1992, DIFFLEY et al., "Protein-DNA Interactions at a Yeast Replication Origin", pages 169-172. *
NATURE, Volume 366, issued 4 November 1993, MICKLEM et al., "Yeast Origin Recognition Complex is Involved in DNA Replication and Transcriptional Silencing", pages 87-89. *
PROC. NAT. ACAD. SCI., USA, Volume 86, issued March 1989, GOULD et al., "Use of the DNA Polymerase Chain Reaction for Homology Probing: Isolation of Partial cDNA or Genomic Clones Encoding the Iron-Sulfur Protein of Succinate Dehydrogenase from Several Species", pages 1934-1938. *
See also references of EP0733057A4 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5851821A (en) * 1996-05-02 1998-12-22 Cold Spring Harbor Laboratory DNA Replication-regulating genes
US6074819A (en) * 1996-05-02 2000-06-13 Cold Spring Harbor Laboratory DNA replication-regulating genes
WO1997041153A1 (fr) * 1996-05-02 1997-11-06 Cold Spring Harbor Laboratory Genes regulant la replication de l'adn
US6361954B1 (en) 1996-05-02 2002-03-26 Cold Spring Harbor Laboratory Methods of immunoassay for human CDC6
US6566057B1 (en) 1997-02-14 2003-05-20 Deltagen Proteomics, Inc. Methods and compositions for peptide libraries displayed on light-emitting scaffolds
US6623922B1 (en) 1997-02-14 2003-09-23 Deltagen Proteomics Methods for identifying, characterizing, and evolving cell-type specific CIS regulatory elements
US5955275A (en) * 1997-02-14 1999-09-21 Arcaris, Inc. Methods for identifying nucleic acid sequences encoding agents that affect cellular phenotypes
US6579675B2 (en) 1997-02-14 2003-06-17 Deltagen Proteomics, Inc. Methods for identifying nucleic acid sequences encoding agents that effect cellular phenotypes
WO1998039483A1 (fr) * 1997-03-04 1998-09-11 Ventana Genetics, Inc. Methodes d'identification de sequences d'acide nucleique codant des agents qui influent sur des phenotypes cellulaires
AU745827B2 (en) * 1997-03-04 2002-04-11 Deltagen Proteomics, Inc. Methods for identifying nucleic acid sequences encoding agents that affect cellular phenotypes
US6281347B1 (en) 1997-09-10 2001-08-28 O'donnell Michael Human origin of replication complex genes and uses thereof
US6322973B1 (en) 1997-11-07 2001-11-27 Iconix Pharmaceuticals, Inc. Surrogate genetics target characterization method
WO1999024563A1 (fr) * 1997-11-07 1999-05-20 Iconix Pharmaceuticals, Inc. Procede de caracterisation de cibles genetiques de remplacement

Also Published As

Publication number Publication date
AU1373495A (en) 1995-07-03
CA2178965A1 (fr) 1995-06-22
EP0733057A4 (fr) 1998-08-19
EP0733057A1 (fr) 1996-09-25
JPH09506768A (ja) 1997-07-08
AU700405B2 (en) 1999-01-07

Similar Documents

Publication Publication Date Title
Xiao et al. CSE1 and CSE2, two new genes required for accurate mitotic chromosome segregation in Saccharomyces cerevisiae
US6171781B1 (en) NF-AT polypeptides and polynucleotides
Loeb et al. The yeast nuclear import receptor is required for mitosis.
Elion et al. FUS3 encodes a cdc2+/CDC28-related kinase required for the transition from mitosis into conjugation
CA2263744A1 (fr) Procede permettant d'identifier des genes codant des proteines possedant une activite favorisant la proliferation cellulaire
US6207374B1 (en) Tuberous sclerosis 2 gene and uses thereof
JPH08506480A (ja) 新種蛋白質分離のための相互作用を用いる補捉システム
US5260200A (en) Isolated DNA encoding an insulin receptor substrate
AU700405B2 (en) Origin of replication complex genes, proteins and methods
US6476193B1 (en) NLK1 protein and NLK1 protein complexes
CA2234417A1 (fr) Kinase humaine serique regulee par les glucocorticoides, cible pour les maladies renales chroniques
US6753158B1 (en) Assays, agents, therapy and diagnosis relating to modulation of cellular DNA repair activity
WO1994025607A1 (fr) Procede de dosage pour medicaments qui retablissent la fonction de la proteine mutante regulatrice de la conductance membranaire de la mucoviscidose (cftr)
WO2001064913A2 (fr) Proteines de cycle cellulaire associees au rad9, compositions et procedes d'utilisation
WO1997012617A9 (fr) Proteines impliquees dans le ciblage du centre de transfert de peptidyle, et procedes et agents therapeutiques correspondants
JP2002512041A (ja) 緑内障および前眼部形成不全の診断および予後判定のためのｆｒｅａｃ３遺伝子における新規変異
WO1993001205A1 (fr) Tyrosine-kinase
JP2000511408A (ja) 新規治療
Zhang et al. Amino acid residues in Std1 protein required for induction of SUC2 transcription are also required for suppression of TBPΔ57 growth defect in Saccharomyces cerevisiae
JP2005509447A (ja) 疾患の治療および／または予防のための、ｍａｄ２変異を有するターゲティング細胞
CA2451250A1 (fr) Modulation de l'interaction entre l'amyloide a serique et tanis et agents utiles a cet effet
JP2005509425A (ja) 疾患の治療におけるｍｅｃ１関連遺伝子の変異
WO2001051509A2 (fr) Proteine 140 associee a shc (sap-140)
McCright The identification and characterization of B56, a new family of protein phosphatase 2A regulatory subunits
EP1045926A2 (fr) Famille de genes possedant une activite de modulation de la transformation

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2178965

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 1995904929

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1995904929

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1995904929

Country of ref document: EP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载