Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-03-31
2002-05-07
Fredman, Jeffrey (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069100, C536S022100
Reexamination Certificate
active
06383753
ABSTRACT:
FIELD OF THE INVENTION
This invention generally relates to a novel cell cycle regulatory gene named Essential for Mitotic Growth 1 (Emg1) that is conserved in yeast, worms, mammals and plants. Additionally, this invention relates to a temperature sensitive mutation of Emg1, designated Emg1-1. Also, this invention relates to an Emg1 interactive protein, designated Emg-1-nuclear interacting protein-1 (ENIP1), and the respective coding sequence. Furthermore, this invention relates to cells and organisms that are made deficient in expression or made to over express these genes or proteins. Further still, drugs screens for compounds that are agonistic or antagonistic to Emg1, Emg1-1 and ENIP1 regulation of the cell cycle are claimed by this invention. Even further still, screens for Emg1, Emg1-1 and ENIP1 intra- and interspecific homologs as well as associated binding molecules are claimed by this invention.
BACKGROUND
In order for cells to proliferate and divide faithfully, cellular mechanisms have evolved to ensure proper progression through the eukaryotic cell division cycle. Expression of genes encoding proteins that promote passage through the cell cycle are tightly regulated and carefully coordinated to physiological and environmental cues such as growth promoting stimuli and growth arresting signals. Progression through the cell cycle requires the ordered synthesis, assembly and movement of macromolecular cell structures including proteins, membranes, and importantly, the genetic information encoded by the genomic DNA. Coordination of these processes involves the successive activation of specific regulatory cell cycle proteins known as cyclins and cyclin-dependent kinases which in turn orchestrate the transcription of genes whose products effect defined cellular programs for cell division. In addition to signals that promote progression, the cell cycle is also regulated by negative controls which prevent cell proliferation during circumstances in which damage to, or instability of, the genome is sensed. These surveillance systems, called checkpoints, prevent passage through the cell cycle until the cellular damage can be repaired; or in the event of irreparable damage, permanently stall cell growth and, in some cells, trigger the programmed cell death pathway. A different, programmed exit from the cell cycle, such as the process of terminal differentiation by specialized cells, also requires a coordinated response to specific physiological cues and activation of transcriptional events which lead to the cessation of proliferation. Thus cellular growth must balance positive and negative controls on cell cycle progression in order to ensure the replication of genetic material with high fidelity as well as the maintenance of cell-specific phenotypes.
The tight control over cell proliferation by both positive and negative regulators indicates the central importance of coordinating cellular proliferation with respect to physiological and environmental stimuli. The loss of this control, through genetic mutation of key regulatory components of the cell cycle or impaired sensing of external cues can lead to deregulated growth and disease states with devastating consequences for the cell, tissue and organism. One example is the progression from normal cells to cancer cells which is thought to require a number of genetic changes in proteins that normally control the cell cycle. Several lines of evidence including basic research in model systems such as yeast, correlations with the frequencies of mutation in growth regulatory genes from tumor biopsies, studies on tumor-causing viruses, and identification of the molecular basis for genetic predisposition to tumor development in patients with hereditary diseases have pointed to common molecular and genetic mechanisms which facilitate the evolution of cancer cells. Specifically, mutations that lead to decreased fidelity in the replication and repair of the genome, such as in genes that regulate cell cycle checkpoints, the activity of the cyclin and cyclin-dependent kinases, or transcription factors that coordinate cell cycle progression, are common means by which a cells lose genetic stability. Loss of genetic stability is a hallmark of tumor cells. Conversely, understanding the mechanisms by which cells can re-enter the cell cycle from a resting state, such as a terminally differentiated cell type, should provide targets for pro-proliferative therapies in cases where cellular growth is needed to repair tissue injury, such as in response to ischemic damage to the heart or liver regeneration or regeneration of nervous tissues, such as in Alzheimer's disease.
Therefore, what is needed is the identification of key regulatory genes that control cell proliferation as they may provide a means for the screening of compounds that are agonistic or antagonistic for mitosis. Such compounds, once identified, may be useful as diagnostic or therapeutic agents in the treatment of diseases characterized by unregulated cell growth.
SUMMARY OF THE INVENTION
The present invention generally comprises a novel, substantially purified oligonucleotide sequences from
S. cerevisiae,
mouse and human that encode for the newly discovered genes, Emg1, Emg1-1 and ENIP1. The expression of Emg1 and ENIP1 have been shown to be required for mitosis. The gene is highly conserved. For example, transfection of murine Emg1 into yeast expressing a vector containing a temperature sensitive loss of function (lof) Emg1 mutant (e.g., Emg1-1) overcomes the deficiency of the mutant. Additionally, for example, transfection of a vector expressing murine Emg1 into yeast harboring a null allele of Emg1 overcomes the growth deficiency of the yeast mutant. Mouse Emg1 is 50% homologous with yeast Emg1. Mouse and human Emg1 share 87% homology.
The present invention generally relates to compositions and methods of identifying and testing Emg1 pathway agonists and antagonists. The present invention is not limited by the method of the employed screen. In one embodiment, the present invention contemplates screening suspected compounds in a system utilizing transfected cell lines. In one embodiment, the cells may be transfected transiently. In another embodiment, the cells may be stably transfected. In yet another embodiment, translation products of the invention may be used in a cell-free assay system. Furthermore, in yet another embodiment, antibodies generated to the translation products of the invention may be used in immunoprecipitation assays. In still another embodiment yeast based assays incorporating transfected yeast (e.g., transiently or stability transfected yeast) may be used to screen for Emg1 agonists and antagonists. And in still another embodiment, transgenic animals may be generated with the transgene contained in a vector containing an inducible, tissue specific promotor or a restrictive promoter such as a temperature sensitive promoter. In still yet another embodiment, the vector may contain a temperature sensitive variant of Emg1, for example Emg1-1.
The invention also relates to methods to identify other members of the Emg1 pathway (e.g., binding partners). One such binding partner has already been identified as the Emg1-nuclear interacting protein-1 (ENIP1). ENIP1 is essential for cell growth. The present invention is not limited to the methods employed to identify Emg1 pathway constituents. In one embodiment, antibodies generated to translation products of the invention may be used in immunoprecipitation experiments to isolate novel Emg1 pathway constituents or natural mutations thereof. In another embodiment, the invention may be used to generate fusion proteins (e.g., Emg1-GST fusion proteins) that could also be used to isolate novel Emg1, Emg1-1 and ENIP1 pathway constituents or natural mutations thereof. In yet another embodiment, screens may be conducted using the yeast two-hybrid system using Emg1 as the bait. In yet another embodiment, screens may be conducted using affinity chromatography using Emg1 as the ligand.
The invention also relates to the production
Liu Phillip C. C.
Thiele Dennis J.
Chunduru Suryaprabha
Fredman Jeffrey
Medlen & Carroll LLP
Regents of the University of Michigan
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