CIF150/hTAFII150 is necessary for cell cycle progression

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid

Reexamination Certificate

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C435S375000, C435S091100, C536S023100, C536S024300, C536S024500, C530S300000, C530S350000

Reexamination Certificate

active

06174679

ABSTRACT:

TECHNICAL AREA OF THE INVENTION
The invention relates to the area of cell cycle progression. More particularly, the invention relates to proteins which regulate cell cycle progression.
BACKGROUND OF THE INVENTION
Alterations in the regulation of mitosis or cell cycle progression play an important role in diseases such as neoplasia and anemia. Manipulation of genes involved in regulating the cell cycle can be used to prevent or treat these diseases. Detections of mutations in cell-cycle regulatory genes can also be used to detect neoplastic cells and genetic predispositions to neoplasias. Thus, there is a need in the art for the identification of cell cycle regulator genes which can be used in methods of diagnosing, prognosing, and treating neoplasia and other diseases in humans and other mammals.
SUMMARY OF THE INVENTION
It is an object of the invention to provides reagents and methods for regulating mitosis or cell cycle progression in human cells and for treating disorders related to alterations in mitosis or cell cycle progression. These and other objects of the invention are provided by one or more of the embodiments described below.
One embodiment of the invention is an isolated and purified subgenomic polynucleotide which encodes a protein comprising an amino acid sequence which is at least 85% identical to the nucleotide sequence shown in SEQ ID NO:2. Percent identity is determined using a Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 1.
Another embodiment of the invention is an isolated and purified subgenomic polynucleotide which comprises at least 11 contiguous nucleotides selected from the nucleotide sequence shown in SEQ ID NO:1.
Yet another embodiment of the invention is a construct which comprises a promoter and a polynucleotide segment encoding a human CIF150/hTAF
II
150 protein as shown in SEQ ID NO:2. The polynucleotide segment is located downstream from the promoter. Transcription of the polynucleotide segment initiates at the promoter.
Still another embodiment of the invention is a host cell comprising a construct. The construct comprises a promoter and a polynucleotide segment encoding a human CIF150/hTAF
II
150 protein as shown in SEQ ID NO:2.
Another embodiment of the invention provides a homologously recombinant cell having incorporated therein a new transcription initiation unit. The new transcription initiation unit comprises an exogenous regulatory sequence, an exogenous exon, and a splice donor site. The transcription initiation unit is located upstream of a coding sequence of a CIF150/hTAF
II
150 gene. The exogenous regulatory sequence directs transcription of the coding sequence of the CIF150/hTAF
II
150 gene.
Even another embodiment of the invention provides a method to aid in the diagnosis or prognosis of neoplasia in a human. Expression of a first CIF150/hTAF
II
150 gene in a first tissue of a human suspected of being neoplastic is compared with expression of a second CIF150/hTAF
II
150 gene in a second tissue of a human which is normal. The second CIF150/hTAF
II
150 gene has the coding sequence shown in SEQ ID NO:1. Increased expression of the first CIF150/hTAF
II
150 relative to the second CIF150/hTAF
II
150 gene indicates neoplasia in the first tissue.
Yet another embodiment of the invention provides a method to aid in the diagnosis or prognosis of neoplasia in a human. A human CIF150/hTAF
II
150 gene, mRNA, or protein in a first tissue suspected of being neoplastic is compared with a second human CIF150/hTAF
II
150 gene, mRNA, or protein in a second tissue which is normal. The second CIF150/hTAF
II
150 gene has the coding sequence shown in SEQ ID NO:1. A difference between the first and second genes, mRNAs, or proteins in the second tissue indicates neoplasia in the first tissue.
Still another embodiment of the invention provides a method to aid in detecting a genetic predisposition to neoplasia in a human. A CIF150/hTAF
II
150 gene, mRNA, or protein in a fetal tissue of a human is compared with a wild-type human CIF150/hTAF
II
150 gene, mRNA, or protein. The wild-type CIF150/hTAF
II
150 gene has the coding sequence shown in SEQ ID NO:1. A difference between the CIF150/hTAF
II
150 gene, mRNA, or protein in the fetal tissue of the human and the wild-type human CIF150/hTAF
II
150 gene, mRNA, or protein indicates a genetic predisposition to neoplasia in the human.
Even another embodiment of the invention provides a method of screening test compounds for the ability to interfere with the binding of a CIF130 protein to a CIF150/hTAF
II
150 protein. A test compound is contacted with a CIF150/hTAF
II
150-binding domain of a CIF130 protein and a CIF130-binding domain of a CIF150/hTAF
II
150 protein. The CIF130 protein has the amino acid sequence shown in SEQ ID NO:4. The CIF150/hTAF
II
150 protein has the amino acid sequence shown in SEQ ID NO:2. The CIF130-binding domain binds to the CIF150/hTAF
II
150-binding domain in the absence of the test compound. The amount of at least one of the CIF130- or CIF150/hTAF
II
150-binding domains which is bound or unbound is determined in the presence of the test compound. A test compound which decreases the amount of bound CIF130- or CIF150/hTAF
II
150-binding domains or which increases the amount of unbound CIF130- and CIF150/hTAF
II
150-binding domains is a potential inducer of mitosis or cell cycle progression.
Yet another embodiment of the invention provides a method of screening test compounds for the ability to interfere with the binding of a CIF130 protein to a CIF150/hTAF
II
150 protein. A cell is contacted with a test compound. The cell comprises two fusion proteins. A first fusion protein comprises (1) a CIF150/hTAF
II
150-binding domain of a CIF130 protein and (2) either a DNA binding domain or a transcriptional activating domain. A second fusion protein comprises a CIF130-binding domain of a CIF150/hTAF
II
150 protein. The CIF130 protein has the amino acid sequence shown in SEQ ID NO:4. The CIF150/hTAF
II
150 protein has the amino acid sequence shown in SEQ ID NO:2. The CIF130-binding domain binds to the CIF150/hTAF
II
150-binding domain. If the first fusion protein comprises a DNA binding domain, then the second fusion protein comprises a transcriptional activating domain. If the first fusion protein comprises a transcriptional activating domain, then the second fusion protein comprises a DNA binding domain. The interaction of the first and second fusion proteins reconstitutes a sequence-specific transcription activating factor. The cell also comprises a reporter gene comprising a DNA sequence to which the DNA binding domain specifically binds. Expression of the reporter gene is measured. A test compound which decreases the expression of the reporter gene is a potential inducer of mitosis or cell cycle progression.
Even another embodiment of the invention provides a method of increasing expression of a gene. A promoter region of the gene is contacted with a CIF150/hTAF
II
150 protein as shown in SEQ ID NO:2. The promoter region of the gene comprises a CIF150/hTAF
II
150 binding element. The CIF150/hTAF
II
150 binding element comprises a nucleotide sequence 5′-Py X G A G A/C A/Py-3′ (SEQ ID NO:7). Expression of the gene is thereby increased.
Still another embodiment of the invention is an antisense oligonucleotide as shown in SEQ ID NO:5.
Yet another embodiment of the invention provides an isolated and purified subgenomic polynucleotide which comprises 5′-Py X G A G A/C A/Py-3′ (SEQ ID NO:7).
The present invention thus provides the art with reagents and methods of affecting human mitosis or cell cycle progression and treating disorders associated with alterations in mitosis or cell cycle progression.


REFERENCES:
patent: WO 94/17087 (1994-08-01), None
Anderson, Nature 392/supp.: 25-30, Apr. 1998.
Reynolds et al., Molecular Medicine Today, pp. 25-31, Jan. 1999.
Wodnar-Filipowicz et al., EMBL Database, Accession No. K01334,Proc. Natl. Acad. Sci. USA 81:2295-2297, 1984.
Kaufmann et al., “CIF, an

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