WO2004005514A1

WO2004005514A1 - Human protooncogene 6 and protein encoded therein

Info

Publication number: WO2004005514A1
Application number: PCT/KR2002/001264
Authority: WO
Inventors: Jin Woo Kim
Original assignee: Jin Woo Kim
Priority date: 2002-07-04
Filing date: 2002-07-04
Publication date: 2004-01-15
Also published as: AU2002345414A1

Abstract

A human protooncogene having a base sequence of SEQ ID NO: 1 or a fragment thereof is overexpressed in various cancer tissues and can be used in diagnosing various cancers and an antisense gene complementary thereto can be used in treating cancers.

Description

HUMAN PROTOONCOGENE 6 AND PROTEIN ENCODED THEREIN

Field of Invention

The present invention relates to a novel protooncogene and protein encoded therein, which can be used in diagnosis of various cancers, preparation of transgenic animal, anti sense gene therapy and anticancer drug development.

Background of the Invention

Higher animals including man each carry approximately 100,000 genes, but only about 15% thereof is expressed, and characteristics of individual's biological process, e.g., genesis, differentiation, homeostasis, responses to stimuli, control of cell cell division cycle, aging and apoptosis (programmed cell death), are determined depending on which genes are expressed (Liang, P. and A. B. Pardee, Science 257: 967-971, 1992).

Pathogenic phenomena such as tumorigenesis are caused by gene mutation, which brings about changes in the mode of gene expression. Therefore, comparative studies of gene expressions in various cells have been conducted to provide bases for establishing viable approaches to the understanding of diverse biological phenomena.

For example, the mRNA differential display (DD) method suggested by

Liang and Pardee is effective in elucidating the nature of tumor suppressor genes, cell cycle-related genes and transcriptional regulatory genes that control apoptosis

(Liang, P. and A. B. Pardee supra). Further, the DD method has been widely used in examining the interrelationship of various genes in a cell.

It has been reported that tumorigenesis is caused by various genetic changes such as the loss of chromosomal heterozygosity, activation of oncogenes and inactivation of tumor suppressor genes, e.g., p53 gene (Bishop, J. M., Cell 64: 235-248, 1991; and Hunter, T., Cell 64: 249-270, 1991). Further, it has been reported that 10 to 30% of human cancer arises from the activation of oncogene through amplification of protooncogenes.

Therefore, the activation of protooncogenes plays an important role in the etiology of many tumors and there has existed a need to identify protooncogenes. The present inventor has endeavored to unravel the mechanism involved in the tumorigenesis of cervical cancer; and, has unexpectedly found that a novel protooncogene, Human Cervical Cancer 6 (HCC-6), is specifically overexpressed in cancer cells. This protooncogene can be effectively used in diagnosis, prevention and treatment of various cancers, e.g., leukemia, cervix, lymphoma, colon, lung and skin cancers.

Summary of the Invention

Accordingly, the primary object of the present invention is to provide a novel protooncogene and a fragment thereof. Other objects of the present invention are to provide: a recombinant vector containing said protooncogene or a fragment thereof and a microorganism transformed therewith; a protein encoded in said protooncogene and a fragment threrof; a kit for diagnosing cancer which comprising said protein or a fragment thereof; an antisense gene having a base sequence complementary to that of said protooncogene or a fragment thereof; and a process for treating or preventing cancer by using said antisense gene.

In accordance with one aspect of the present invention, there is provided a novel protooncogene having the nucleotide sequence of SEQ ID NO: 1 or a fragment thereof.

In accordance with another aspect of the present invention, there is provided a recombinant vector containing said protooncogene or a fragment thereof and a microorganism transformed with said vector.

In accordance with still another aspect of the present invention, there is provided a protein having the amino acid sequence of SEQ ID NO: 2 or a fragment thereof derived from said protooncogene or a fragment thereof.

Brief Description of the Drawings

The above and other objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with the accompanying drawings which respectively show;

Fig. 1 : the results of DDRT-PCR for CA245 expressed in normal cervix tissue, primary cervical cancer tissue, metastatic lymph node tissue and CUMC-6 cervical cancer cells;

Fig. 2A: the results of northern blot analysis for HCC-6 protooncogene expressed in cervical cancer tissues;

Fig. 2B: the results obtained with the same sample of Fig. 2A hybridized with β-actin;

Fig. 3A: the results of northern blot analysis for HCC-6 protooncogene expressed in normal human 12-lane multiple tissues;

Fig. 3B: the results obtained with the same sample of Fig. 3A hybridized with β-actin; Fig. 4A: the result of northern blot analysFis for HCC-6 protooncogene expressed in human cancer cell lines;

Fig. 4B: the result obtained with the same sample of Fig. 4A hybridized with β-actin;

Fig. 5: the sodium dodecyl sulfate (SDS)-PAGE results showing protein expression patterns before and after the IPTG induction in HCC-6 transformed E.coli cells.

Detailed Description of the Invention

The novel protooncogene of the present invention, designated human cervical cancer 6 (hereinafter "HCC-6 protooncogene"), consists of 899 base pairs and has the nucleotide sequence of SEQ ID NO: 1.

In SEQ ID NO: 1 , the open reading frame corresponding to base Nos. 174 to 854 (852-854: termination codon) is a full-length protein encoding region and the predicted amino acid sequence derived therefrom is shown in SEQ ID NO: 2 which consists of 226 amino acids (hereinafter "HCC-6 protein").

However, in consideration of the degeneracies of codons and the preferred codons in a specific organism wherein the protooncogene of the present invention is to be expressed, various changes and modifications of the DNA sequences of SEQ ID NO: 1 may be made, e.g., in the coding area thereof without adversely altering the amino acid sequence of the expressed protein, or in the non-coding area without adversely affecting the expression of the protooncogene. Therefore, the present invention also includes, in its scope, a polynucleotide having substantially the same base sequence as the inventive protooncogene, and a fragment thereof. As used herein, "substantially the same polynucleotide" refers to a polynucleotide whose base sequence shows 80% or more, preferably 90% or more, most preferably 95% or more homology to the protooncogene of the present invention.

The protein expressed in vertebrate mitochondria from the protooncogene of the present invention consists of 266 amino acids and has the amino acid sequence of SEQ ID NO: 2. However, when it is expressed in a prokaryotic cell, e.g., E. coli, protein synthesis is stopped at TGA termination codon (315-317 of SEQ ID NO: 1; tryptophane codon in mitochondria), which results in the production of HCC-6 protein consisting of 47 amino acids (1-47 of SEQ ID NO: 2) and having the molecular weight of about 5 kDa. However, various substitution, addition and/or deletion of the amino acid residues of protein may be performed without adversely affecting the protein's function. Further, a portion of the protein may be used when a specific purpose is to be fulfilled. These modified amino acids and fragments thereof are also included in the scope of the present invention. Therefore, the present invention includes, in its scope, a polypeptide having substantially the same amino acid sequence as the protein derived from the oncogene of the present invention and a fragment thereof. As used herein, "substantially the same polypeptide" refers to a polypeptide whose amino acid sequence shows 80% or more, preferably 90% or more, most preferably 95% or more homology to the amino acid sequence of SEQ ID NO: 2.

The protooncogene or the protein of the present invention can be obtained from human cancer tissues or synthesized using a conventional DNA or peptide synthesis method. Further, the gene thus prepared may be inserted to a conventional vector to obtain an expression vector, which may, in turn, be introduced into a suitable host, e.g., a microorganism such as an E. coli or yeast, or an animal cell such as a mouse or human cell. A transformed host may then be used in producing the inventive DNA or protein on a large scale. For example, E. coli DH5α was transformed with expression vector pCEV-LAC containing the inventive HCC-6 gene to obtain an E. coli transformant designated DH5α /HCC-6/pCEV-LAC which was deposited on April 10, 2001 with the Korean Collection for Type Cultures (KCTC) (Address: Korea Research Institute of Bioscience and Biotechnology (KRIBB), #52, Oun-dong, Yusong-ku, Taejon, 305-333, Republic of Korea) under the accession number, KCTC 0989BP, in accordance with the terms of Budapest Treaty on the International Recognition of the Deposit of Microorganism for the Purpose of Patent Procedure. In preparing a vector, expression-control sequences, e.g., promoter, terminator, self-replication sequence and secretion signal, are suitably selected and combined depending on the host cell used. The overexpression of the protooncogene of the present invention occurs not in normal cervical tissues but in cervical cancer tissues. This suggests that the protooncogene of the present invention induces the uterine cervical cancer. In addition to epithelial tissues such as cervical cancer tissue, the overexpression of the protooncogene of the present invention is also observed in various cancer tissues, e.g., leukemia, uterine, colon, lymphoma, and skin cancer tissues (see Figs. 2 and 4). Therefore, the protooncogene of the present invention is believed to be a factor common to all forms of cancers and it can be advantageously used in the diagnosis of various cancers and the production of a transgenic animal as well as in an antisense gene therapy.

A diagnostic method that can be performed using the protooncogene of the present invention may comprise, for example, the steps of hybridizing nucleic acids separated from the body fluid of a subject with a probe containing the protooncogene of the present invention or a fragment thereof, and determining whether the subject has the protooncogene by using a conventional detection method in the art. The presence of the protooncogene may be easily detected by labeling the probe with a radioisotope or an enzyme. Therefore, a cancer diagnostic kit containing the protooncogene of the present invention or a fragment thereof is also included in the scope of the present invention. A transgenic animal produced by introducing the protooncogene of the present invention into a mammal, e.g., mice, is also included in the scope of the present invention. In producing such a transgenic animal, it is preferred to introduce the inventive protooncogene to a fertilized egg of an animal before the 8-cell stage. The transgenic animal can be advantageously used in screening for carcinogens or anticancer agents such as chemotherapeutic drugs.

The present invention also provides an antisense gene, which is useful in a gene therapy. As used herein, the term "an antisense gene" means a polynucleotide comprising a base sequence which is fully or partially complementary to the sequence of the mRNA which is transcribed from the protooncogene having the base sequence of SEQ ID NO: 1 or a fragment thereof, said nucleotide being capable of preventing the expression of the open reading frame (ORF) of the protooncogene by way of attaching itself to the protein- binding site of mRNA.

The present invention also includes within its scope a process for treating or preventing cancer in a subject by way of administering a therapeutically effective amount of the inventive antisense gene thereto.

In the inventive antisense gene therapy, the antisense gene of the present invention is administered to a subject in a conventional manner to prevent the expression of the protooncogene. For example, the antisense oligodeoxynucleotide (ODN) is mixed with a hydrophobicized poly-L-lysine derivative by electrostatic interaction in accordance with the method disclosed by Kim, J. S. et al. (J. Controlled Release 53: 175-182, 1998) and the resulting mixed antisense ODN is administered intravenously to a subject.

The present invention also includes within its scope an anti-cancer composition comprising the antisense gene of the present invention as an active ingredient, in association with pharmaceutically acceptable carriers, excipients or other additives, if necessary. The pharmaceutical composition of the present invention is preferably formulated for administration by injection.

The amount of the antisense gene actually administered should be determined in light of various relevant factors including the condition to be treated, the chosen route of administration, the age and weight of the individual patient, and the severity of the patient's symptoms.

The protein expressed from the inventive protooncogene may be used in producing an antibody useful as a diagnostic tool. The antibody of the present invention may be prepared in the form of a monoclonal or polyclonal antibody in accordance with any of the methods well known in the art by using a protein having the amino acid sequence of SEQ ID NO: 2 or a fragment thereof. Cancer diagnosis may be carried out using any of the methods known in the art, e.g., enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich assay, immunohistochemical staining, western blot or immunoassay blot on polyacrylic gel, to assess whether the protein is expressed in the body fluid of the subject. Therefore, a cancer diagnostic kit containing the protein having the amino acid sequence of SEQ ID NO: 2 or a fragment thereof is also included in the scope of the present invention.

A continuously viable cancer cell line may be established by using the protooncogene of the present invention, and such a cell line may be obtained, for example, from tumor tissues formed on the back of a nude mouse by injecting fibroblast cells transformed with the protooncogene of the present invention. The cell lines thus prepared may be advantageously used in searching for anti- cancer agents.

The following Examples are given for the purpose of illustration only, and are not intended to limit the scope of the invention. Example 1 : Cultivation of tumor cells and separation of total RNA

(Step 1) Cultivation of tumor cells

For differential display of mRNA, normal cervical tissues, untreated primary cervical cancer tissues and metastatic common iliac lymph node tissues were obtained from cervical cancer patients who underwent radical hysterectomy. The human cervical cancer cell line used in the differential display method was CUMC-6 cell line described by Kim et al, (Gynecol. Oncol. 62: 230-240, 1996). Cells obtained from the above-described tissues and CUMC-6 were maintained on Waymouth's MB 752/1 medium (Gibco) supplemented with 2 mM/£of glutamine, 100 IU/m# of penicillin, 100 βglv of streptomycin, and 10% of fetal bovine serum (Gibco). Only the cell suspensions with greater than 95% viability, as assessed by trypan blue dye exclusion described by Freshney ("Culture of Animal Cells: A Manual of Basic Technique" 2nd Ed., A. R. Liss, New York, 1987) were used in the present experiments.

(Step 2) Isolation of total RNA and differential display of mRNA

Total RNAs were extracted from normal cervical tissues, primary cervical cancer tissues, metastatic common iliac lymph node tissues and CUMC-6 cells obtained in Step 1 using a commercial system (RNeasy total RNA kit) provided by Qiagen (Qiagen Inc., Germany) and the removal of DNA contaminants from the RNAs was accomplished using Message clean kit (GenHunter Corp., Brookline, MA).

Example 2 : Differential display reverse transcription (DDRT)-PCR

Differential display reverse transcription was performed in accordance with the reverse transcription-polymerase chain reaction (RT-PCR) method described by Liang and Pardee (1992), supra, with minor modifications.

First, reverse transcription was carried out using 0.2 μg each of the total RNAs obtained in Step 2 of Example 1 and one of the three primers, i.e., H-T11G, H-T11C, or H-T11A, as anchored oligo-dT primers (RNAimage kit, GenHunter Cor., MA, USA).

Then PCR was conducted using the same anchored 3' primer of SEQ ID NO: 3 and the arbitrary 5' primer of SEQ ID NO: 4 (H-AP24 primer of RNAimage primer sets 1-4, H-AP 1-32) in the presence of 0.5 mM [α-³⁵S]- labeled dATP (1200 Ci/mmol). The PCR thermal cycle was repeated 40 times, the cycle being composed of: 95 °C for 40 sec, 40 °C for 2 min and 72 °C for 40 sec, and finally the reaction was carried out at 72 ^°C for 5 min. PCR-amplified fragments were resolved in 6% polyacrylamide sequencing gels. Differentially expressed fragments were identified by inspection of autoradiograms.

The band of fragment CA245 cDNA of size 147 bp (nucleotide Nos. 705- 851 of SEQ ID NO: 1), were excised from the dried sequencing gel. The cDNAs were eluted by boiling for 15 min and reamplified with the same primer pairs and PCR conditions as used in the above amplification step except that no [α-³⁵S]-labeled dATP and 20 μM dNTPs were used.

Example 3 : Cloning

The reamplified CA245 PCR product obtained as above was inserted into the pGEM-T Easy vector using TA cloning system (Promega, USA) in accordance with the manufacturer's instructions.

(Step l) Ligation

2 βi of the reamplified CA245 PCR product obtained in Example 2, 1 f of pGEM-T easy vector (50 ng), 1 ≠ of T4 DNA ligase 10X buffer solution and 1 βi of T4 DNA ligase (3 weiss units/ i; Promega, USA) were charged into a 0.5 mi tube and distilled water was added thereto to a final volume of 10 βi.

The ligation reaction mixture was incubated overnight at 14^°C .

(Step 2) TA cloning transformation

TA cloning transformation was performed using the following protocol.

E. coli JM109 (Promega, WI, USA) was incubated in 10 mi of LB broth (Bacto-trypton 10 g, Bacto-yeast extract 5 g, NaCl 5 g) until the optical density at 600 nm reached about 0.3 to 0.6. The cultured mixture was kept at 0^°C for 10 minutes and centrifuged at 4,000 rpm at 4^°C for 10 min. The supernatant was removed and cells were harvested. The harvested cell pellet was exposed to 10 mi of 0.1 M CaCl₂ at 0^°C for 30 min to 1 hour to obtain competent cells. The resultant was centrifuged at 4,000 rpm at 4^°C for another 10 min and the collected cells were suspended in 2 mi of 0.1 M CaCl₂ at 0°C .

200 βi of the competent cell suspension was transferred to a microfuge tube and 2 βi of the ligation product obtained in Step 1 of Example 3 was added thereto. The mixture was incubated in a water bath at 42 °C for 90 sec and rapidly cooled to 0^°C . Added thereto was 800 βi of SOC medium (Bacto- trypton 2.0 g, Bacto-yeast extract 0.5 g, 1 M NaCl 1 mi, 1 M KC1 0.25 mi, TDW 97 mi, 2 M Mg²⁺ 1 nι!, 2 M glucose 1 mi) and the mixture was incubated at 37 "C for 45 min at 220 rpm in a rotary shaking incubator.

LB agar plates containing ampicillin were prepared by spreading 25 βi of X-gal (40 mg/ i stock in dimethylformamide) on top of agar with a glass spreader. 25 βi of the transformed cells thus obtained was spread thereon and the plates were incubated at a 37 ^°C incubator overnight. White colonies were loaded on an LB agar plate containing ampicillin and transformed E. coli, i.e., JM109/CA245 were selected and incubated in a terrific broth (TDW 900 mi, Bacto-trypton 12 g, Bacto-yeast extract 24 g, glycerol 4 ml, 0.17 M KH₂P0₄, 0.72 N K₂HP0₄ 100 mi).

Example 4 : Separation of recombinant plasmid DNA

The CA245 plasmid DNA of the transformed E. coli was separated by employing Wizard™ Plus Minipreps DNA Purification Kit (Promega, USA) in accordance with the manufacturer's instructions.

A portion of the plasmid DNA thus separated was treated with ECoRI enzyme and subjected to gel electrophoresis to confirm the insertion of CA245 gene in the plasmid.

Example 5 : Sequence Analysis of DNA

The CA245 PCR product obtained in Example 2 was subjected to PCR in accordance with the conventional method and the cloned, reamplified CA245 PCR fragments were subjected to sequence analysis according to the dideoxy chain termination method using a Sequenase version 2.0 DNA sequencing kit (United states Biochemical, Cleveland, OH) in accordance with the manufacturer's instructions. The base sequence of the DNA corresponds to nucleotide Nos. 705-851 in

SEQ ID NO: 1 and is designated "CA245".

The differential display reverse transcription polymerase chain reaction (DDRT-PCR) of the 147 bp cDNA fragment, i.e., CA245 obtained above was carried out using the 3' anchored primer H-T11A of SEQ ID NO: 3 and the 5' arbitrary primer H-AP24 of SEQ ID NO: 4 and resolved by electrophoresis. Identification of altered gene expression by DD in the primary cervical cancer, metastatic lymph node tissue and CUMC-6 cells is shown in Fig 1. As can be seen in Fig. 1, the 147 bp cDNA fragment, i.e., CA245 was expressed in the cervical cancer, metastatic tissue and CUMC-6 cervical cancer cells but not in the normal tissue.

Example 6 : Full length cDNA sequence analysis of the HCC-6 protooncogene

A bacteriophage λgtl l human lung embryonic fibroblast cDNA library (Miki, T. et al., Gene 83: 137-146, 1989) was screened by plaque hybridization with ³²P-labeled CA245 as a probe. The full-length HCC-6 cDNA clone, containing a 899 bp insert in pCEV-LAC vector was obtained from the human lung embryonic fibroblast cDNA library.

HCC-6 clone inserted into λpCEV vector (Miki, T et al., supra) was excised out of the phage in the form of the ampicilline-resistant pCEV-LAC phagemid vector (Miki, T. et al., supra) by Not I cleavage. To make a HCC-6 plasmid DΝA, pCEV-LAC vector containing HCC-6 gene was ligated with T4 DΝA ligase and ligated clone was transformed into E. coli DH5α.

The transformed E. coli DH5α /HCC-6/pCEV-LAC thus obtained was deposited on April 10, 2001 with the Korean Collection for Type Cultures (KCTC)( Address: Korea Research Institute of Bioscience and Biotechnology (KRIBB), #52, Oun-dong, Yusong-ku, Taejon, 305-333, Republic of Korea) under the accession number, KCTC 0989BP, in accordance with the terms of Budapest Treaty on the International Recognition of the Deposit of Microorganism for the Purpose of Patent Procedure. The full sequence of HCC-6 consists of 899 bp which is identified in SEQ

ID NO: 1.

In SEQ ID NO: 1, the full open reading frame of the HCC-6 protooncogene of the present invention corresponds to nucleotides Nos. 174 to 854 and is predicted to encode amino acid sequence shown in SEQ ID NO: 2 which consists of 266 amino acids.

Example 7 : Northern blot analysis of the HCC-6 gene in various cells Total RNAs were extracted from normal cervical tissues, cervical cancer tissues and cervical cancer cell lines (CaSki and CUMC-6) as in Example 1.

To determine the level of HCC-6 gene expression, 20 βg denatured total RNAs from each tissue or cell lines were electrophoresed through 1% formaldehyde agarose gel and transferred to nylon membranes (Boehringer- Mannheim, Germany). The blots were hybridized with a ³²P-labeled random- primed HCC-6 full cDNA probe which was prepared using a Rediprime II random prime labeling system (Amersham, England). The northern blot analysis was repeated twice and the results were quantified by densitometry and normalized with β-actin.

Fig. 2A shows the results of northern blot analyses for HCC-6 gene expressed in normal cervical tissues, cervical cancer tissues and cervical cancer cell lines (CaSki and CUMC-6). As can be seen in Fig. 2A, the transcription level of HCC-6 is high in the cervical cancer tissues and cancer cell lines (CaSki (ATCC CRL 1550) and CUMC-6), but very low or undetactable in the normal cervical tissues.

Fig. 3A shows the results of northern blot analyses for HCC-6 gene expressed in normal human 12-lane multiple tissues; brain, heart, skeletal muscle, colon, thymus, spleen, kidney, liver, small intestine, placenta, lung and leukocyte tissues (Clontech). Fig. 3B shows the results obtained with the same samples hybridized with a β-actin probe to confirm mRNA integrity. As can be seen in Fig. 3A, HCC-6 mRNA (~1.0 kb) is weakly present or absent in many normal tissues. Fig. 4A shows the results of northern blot analyses for HCC-6 gene expressed in human cancer cell lines; HL-60, HeLa, K-562, MOLT-4, Raji, SW480, A549 and G361 (Clontech). Fig. 4B shows the results obtained with the same samples hybridized with a β-actin probe to confirm mRNA integrity. As can be seen in Fig. 4A, HCC-6 is transcribed at a high level in the promyelocytic leukaemia HL-60, HeLa cervical cancer cell, chronic myelogenous leukaemia K- 562, lymphoblastic leukaemia MOLT-4, Burkitt's lymphoma Raji, SW480 colon cancer cell, A549 lung cancer cell, and G361 melanoma cell.

Example 8: Determination of the size of the protein expressed after the transfection of E. Coli with HCC-6 protooncogene

A full-length HCC-6 protooncogene of SEQ ID NO: 1 was inserted into the multiple cloning site of pGEX-4T-3 vector (Amersham Pharmacia) and the resulting pGEX-4T-3/HCC-6 vector was transfected into E. coli BL21 (ATCC 47092). Glutathione S transferase (GST) is inserted at the front of the multiple cloning site of pGEX-4T-3 vector. The transfected E. coli was incubated using an LB broth medium in a rotary shaking incubator, diluted by 1/100, and incubated for 3 hours. 1 mM isopropyl β-D-thiogalacto-pyranoside (IPTG, Sigma) was added thereto to induce the protein synthesis.

The transfected E. coli cells in the culture were disrupted by sonication and subjected to gel electrophoresis using 12% sodium dodecyl sulfate (SDS) before and after the IPTG induction. Fig. 5 shows the SDS-PAGE results, which exhibit a protein expression pattern of the E. coli BL21 strain, transfected with pGEX-4T-3/HCC-6 vector. After the IPTG induction, a significant protein band was observed at about 31 kDa. This 31 kDa fused protein contained GST protein of about 26 kDa and HCC-6 protein of about 5.

While the embodiments of the subject invention have been described and illustrated, it is obvious that various changes and modifications can be made therein without departing from the spirit of the present invention which should be limited only by the scope of the appended claims.

IMJUΛlTsT riUiA l ϊ ON Tllli IM KHKVTIONAI. KIVOd t lON C 1- TUT DEIYfel l or

YOB. TUB UKK Γ OF IΆ I Ϊ- T rt x'huuiw INTERNATIONAL FORM

RECEIPT IN TIIE CASE OF AN ORIGINAL DEPOSIT issued pursuant lo Rule 7 1

O • KIM, Jin Woo πyundae Λμt H8-804, Λpgυjυng dong, Kangna -jju, Seoul 1.T5 110, Repυblit. of Knrea

I I KNTmCATtON OF THE MICROORGANISM

Accession number given by the

Identification reference tfivcn by the INTERNATIONAL DFPOS1TA RY DEPOSITOR AUTHORITY:

Kscherichia coli DH5@/HCC -6/pCE V -L AC KCTC 0989BP

D . SCIENTIFIC DESCRIPTION ΛNIVOR ΓRQI'OSED TAXCWOMIC DESIGNATION

The itiiciODiganism identified under I above was accompanied by.

I x 1 a scientific description

[ ] ,ι pnjposed tHXonomic designation

(Mark with a crohS where applicable)

M RFCKIPT AND ACCEPTANCE

Tins International DcμυsitaiΥ Authority accepts the microorganism identified under 1 above, which was received bv it on April 10 2001.

IV REGEiri OF REQUEST FOR CONVERSION

The nm rciorgamsm identified under 1 above was received by this International Dtpositaiy Authoiity on and a request tu convert the original depαsit to a deposit the Hυ . cst Treaty was received by it on

V INTERNATIONAL DEPOSITARY AUTHORITY

N,iiii ' Korean Collection for Type Cultures Sιgnature(.s) of pcrson(s) having (he power to represent the InterniiLion.il Depositary Authority of authorized ff" MKS)'

Λddiess Korea Research Institute of I Jioscicnce wύ Biotechnology (KRIBB) s*r>2, Oun-donff, YusoriH-ku,

.5 Taejoπ 305 i-U, BΛE, Kyuπjj Sook, Direi tor Rcpubl" of Korea Date: April 16 2001

I mm UP/4 (KCTC form 17) ' ⁽ill | C-

Claims

What is claimed is:

1. A human cervical cancer protooncogene having the base sequence of SEQ ID NO: 1 or a fragment thereof.

2. The protooncogene or fragment of claim 1 , which is a fragment having a base sequence corresponding to base Nos. 174 to 854 of SEQ ID NO: 1.

3. A protein having the amino acid sequence of SEQ ID NO: 2 or a fragment thereof.

4. A vector comprising the protooncogene or fragment of claim 1.

5. A microorganism transformed with the vector of claim 4.

6. The microorganism of claim 5, which is E. coli DH5α/HCC-6 (Accession No: KCTC 0989BP).

7. A process for preparing the protein or fragment of the claim 3 comprising culturing the microorganism of claim 5 or 6.

8. A kit for diagnosing cancer which comprises the protooncogene or fragment of claim 1 or 2.

9. A kit for diagnosing cancer which comprises the protein or fragment of claim 3.

10. An antisense gene having a base sequence which is complementary to the sequence of the full or partial mRNA transcribed from the protooncogene or fragment of claim 1 or 2 and being capable of binding the mRNA to inhibit the expression of said protooncogene or fragment.

11. A composition for preventing or treating cancer which comprises a therapeutically effective amount of the antisense gene of claim 10 and a pharmaceutically acceptable carrier.