Mammalian checkpoint genes and proteins

Details Mammalian checkpoint genes and proteins Mammalian checkpoint genes and proteins

1997-09-05

2001-04-17

Arthur, Lisa B. (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S320100, C435S252100, C435S091200, C536S023100, C536S024300

Reexamination Certificate

active

06218109

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to mammalian proteins and gene sequences involved in cellular responses to DNA damage. In particular, the present invention provides checkpoint genes and proteins.
BACKGROUND OF THE INVENTION
The proper development of a multicellular organism is a complex process that requires precise spatial and temporal control of cell proliferation. Cell proliferation in the embryo is controlled via an intricate network of extracellular and intracellular signaling pathways that process growth regulatory signals. This signaling network is superimposed upon the basic cell cycle regulatory machinery that controls particular cell cycle transitions.
Cell cycle checkpoints are regulatory pathways that control the order and timing of cell cycle transitions, and ensure that critical events such as DNA replication and chromosome segregation are completed with high fidelity. For example, proliferating eukaryotic cells arrest their progression through the cell cycle in response to DNA damage. This arrest is critical to the survival of the organism, as failure to repair damaged DNA can result in the formation and transfer of mutations, damaged chromosomes, cancer, or other detrimental effects. The mechanism responsible for monitoring the integrity of the organism's DNA and preventing the progression through the cell cycle when DNA damage is detected is referred to as the “DNA damage checkpoint.”
In response to DNA damage, cells activate a checkpoint pathway that arrests the cell cycle, in order to provide time for repair, and induces the transcription of genes that facilitate the needed repair. In yeast, this checkpoint pathway consists of several protein kinases including phosphoinositide (PI)-kinase homologs hATM (human), scMec1 (
Saccharomyces cerevisiae
), spRad3 (
Schizosaccharomyces pombe
), and protein kinases scDun1 (
Saccharomyces cerevisiae
), scRad53 (
Saccharomyces cerevisiae
), and spChk1 (
Schizosaccharomyces pombe
) (See e.g., S. Elledge, Science 1664 [1996]).
Indeed, the ability to coordinate cell cycle transitions in response to genotoxic and other stressors is critical to the maintenance of genetic stability and prevention of uncontrolled cellular growth. Loss of a checkpoint gene leads to genetic instability and the inability of the cells to deal with genomic insults such as those suffered as a result of the daily exposure to ultraviolet radiation. The loss of negative growth control and improper monitoring of the fidelity of DNA replication are common features of tumor cells. When checkpoints are eliminated (e.g., by mutation or other means), cell death, infidelity in chromosome transmission, and/or increased susceptibility to deleterious environmental factors (e.g., DNA-damaging agents) result. A variety of abnormal cells arising due to infidelity during mitoses have been detected in humans, including aneuploidy, gene amplification, and multipolar mitoses (See, L. H. Hartwell and T. A. Weinert, Science 246:629 [1989]).
Accordingly, elucidation of checkpoint function, as well as the disruption of checkpoint function, will further the understanding of the process of cellular transformation (i.e., the conversion of normal cells to a state of unregulated growth), as well as cell differentiation and organismal development.
SUMMARY OF THE INVENTION
The present invention provides mammalian proteins and gene sequences involved in cellular responses to DNA damage. In particular, the present invention provides Chk1 genes and proteins.
In one embodiment, the present invention provides the nucleotide sequence set forth in SEQ ID NO:3. In alternative embodiments, the present invention provides SEQ ID NO:3, wherein it further comprises 5′ and 3′ flanking regions. In yet another embodiment, the sequence further comprises intervening regions. In a further embodiment, the present invention also provides a polynucleotide sequence which is complementary to SEQ ID NO:3 or variants thereof. In a preferred embodiment, the present invention provides a vector comprising the nucleotide sequence of claim
1
. The present invention also provides host cell(s) containing the vector of claim
4
.
The present invention also provides a purified Chk1 protein encoded by the nucleotide sequence of claim
1
, as well as a purified protein comprising the amino acid sequence set forth in SEQ ID NO:1. In addition, the present invention provides fusion proteins comprising a least a portion of the human Chk1 protein, as well as non-Chk1 protein sequences. It is not intended that the fusion proteins of the present invention be limited to any particular portion of the Chk1 portion or any particular non-Chk1 protein sequences. In preferred embodiments, the fusion the Chk1 protein portion of the fusion protein comprises at least a portion of SEQ ID NO:1. In an alternative embodiment, the non-Chk1 protein sequence comprises an affinity tag. In particularly preferred embodiment, the affinity tag comprises a histidine tract.
In yet another embodiment, the present invention provides the sequence set forth in SEQ ID NO:4. In an alternative embodiment, the nucleotide sequence further comprises 5′ and 3′ flanking regions. In another alternative embodiment, the nucleotide sequence further comprises intervening regions. In yet another embodiment, the present invention provides a polynucleotide sequence that is complementary to SEQ ID NO:4 or variants thereof.
The present invention also provides a vector comprising the nucleotide sequence set forth in SEQ ID NO:4. In one preferred embodiment, the present invention provides a host cell containing the vector comprising this nucleotide sequence.
The present invention further provides a purified Chk1 protein encoded by the nucleotide sequence of SEQ ID NO:4. In yet another embodiment, the present invention provides a purified protein comprising the amino acid sequence set forth in SEQ ID NO:2.
The present invention also provides fusions proteins comprising at least a portion of the murine Chk1 protein and a non-Chk1 protein sequence. It is not intended that the fusion proteins of the present invention be limited to any particular portion of the Chk1 portion or any particular non-Chk1 protein sequences. In preferred embodiments, the fusion the Chk1 protein portion of the fusion protein comprises at least a portion of SEQ ID NO:2. In an alternative embodiment, the non-Chk1 protein sequence comprises an affinity tag. In particularly preferred embodiment, the affinity tag comprises a histidine tract.
The present invention also provides methods for detecting Chk1 protein. In one embodiment, the method comprises the steps of providing in any order: a sample suspected of containing the Chk1 protein; an antibody capable of specifically binding to a Chk1 protein; mixing the sample and the antibody under conditions wherein the antibody can bind to the Chk1 protein; and detecting the binding. In one alternative embodiment, the sample comprises one or more cells suspected of containing Chk1 protein. In yet another embodiment, the cells contain an abnormal Chk1 protein. In a further embodiment, the cells are selected from the group consisting of human cells and murine cells.
The present invention also provides antibodies capable of recognizing at least a portion of human and/or murine Chk1 protein. In one embodiment, the present invention provides an antibody, wherein the antibody is capable of specifically binding to at least one antigenic determinant on the proteins encoded by an amino acid sequence selected from the group comprising SEQ ID NOS:1, 2, 7, 8, 9, and 10. In one preferred embodiment, the antibody is a polyclonal antibody, while in an alternative embodiment, the antibody is a monoclonal antibody.
The present invention also provides methods for producing antibodies comprising the steps of providing in any order: an antigen comprising at least a portion of Chk1 protein; and an animal having immunocompetent cells; and exposing the animal to the Chk1 protein under conditions such that the imm

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