Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving transferase
Reexamination Certificate
2000-06-01
2003-07-15
Monshipouri, M. (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving transferase
C435S194000, C435S006120, C435S252300, C435S320100, C435S325000, C536S023100, C536S023200
Reexamination Certificate
active
06593098
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to agents that may be used to regulate mitosis in eucaryotic cells. Specifically, novel human BUB genes and their encoded proteins are disclosed. These genes and proteins may be used as targets for the development of novel agents that inhibit aberrant cellular proliferation in tumor cells.
BACKGROUND OF THE INVENTION
Several publications are referenced in this application in parentheses in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these publications is incorporated by reference herein.
The production of two viable and equivalent daughter cells in mitosis requires that events leading to cell division proceed in a carefully ordered fashion. Among other tightly regulated events, replicated sister chromosomes must be properly segregated, one to each daughter cell. For this reason, mitosis cannot be allowed to proceed if the genome has not been fully replicated or if chromosomes are not properly attached to a fully assembled mitotic spindle. Mechanisms required for ensuring the dependency of cell division on completion of such prerequisite steps have been termed mitotic checkpoints (52).
This checkpoint mechanism directly monitors the spatial position of chromosomes within the spindle and applies this information to regulate the activities of proteins that induce chromosome separation and allow the cell to exit mitosis and complete cell division (2, 3, 4). The mitotic checkpoint has sufficient sensitivity to detect the presence of a single unaligned chromosome amidst tens of chromosomes that are aligned (5). Furthermore, this checkpoint will block the cell in mitosis by delaying the onset of anaphase for many hours so that the unaligned chromosome has ample opportunity to establish the spindle connections that allow alignment at the spindle equator. Studies in yeast, insect cells and vertebrate cells show that this mitotic checkpoint monitors the kinetochore as a means to determine whether chromosomes have achieved metaphase alignment. As the kinetochore is the chromosomal site for microtubule attachment and contains molecular motors that specify various aspects of chromosome movement (6, 7), the checkpoint is likely to recognize biochemical signals at the kinetochore that differ between unaligned and aligned chromosomes (4). Consistent with this possibility, laser ablation of a single unattached kinetochore of a monopolar chromosome will abrogate the checkpoint arrest and the chromosomes aligned at the equator will separate as the cell enters anaphase (8). These experiments also demonstrate that checkpoint delay is mediated by an inhibitory signal from the unattached kinetochore. Interestingly, there is a reproducible lag of approximately 20 minutes from the time that the last chromosome becomes aligned till the onset of anaphase (8). This lag may define the time required to complete the biochemical reactions that are necessary for turning off the checkpoint induced block and the activation of the cyclosome/APC complex that specifies anaphase onset through ubiquitin-mediated proteolysis (9). To date, the nature of the signal that the kinetochore emits when it is not properly aligned has not yet been elucidated.
Kinetochores generate motive force through interactions with microtubules. For a bipolar attached chromosome, the tendency for each of the two oppositely-faced kinetochores to move towards their respective poles generates tension between the kinetochore pair. This contrasts with an unattached or a monopolar chromosome whereby little or no tension is generated at the kinetochores.
Elegant micromanipulation experiments performed on the trivalent sex chromosomes in the mantis spermatocyte (10) suggested that kinetochore tension serves as the signal which is detected by the mitotic checkpoint apparatus. During meiosis I, the two X chromosomes pair with a single Y chromosome. On occasion, one of the X's fails to pair and its mono-orientation delays anaphase. If kinetochore tension was supplied by using a microneedle to pull the unattached kinetochore in the direction of the opposite pole, the block to anaphase was lifted and all the chromosomes that were at the spindle equator separated and moved poleward. Again, there was a reproducible time-lag of approximately 15 minutes between application of kinetochore tension on the unaligned X chromosome and separation of the chromosomes aligned at the equator.
At a biochemical level, the inhibitory signal emitted from the unattached kinetochore is likely due to the specific phosphorylation of kinetochore proteins whose identities remain unknown. This hypothesis arises from the observation that a monoclonal antibody 3F3/2 (11), specific for undefined phosphoproteins, recognized differentially expressed phosphoepitopes at kinetochores in PtK cells (12). 3F3/2 was isolated from antibodies generated against mitotic frog extracts that had been incubated with ATP-&ggr;S so that proteins would be selectively thio-phosphorylated by mitotic kinases (11). Although the precise identity of the 3F3/2 epitope is unknown, it recognizes a small subset of undefined proteins that are phosphorylated in mitosis. It is unknown whether proteins that contain the 3F3/2 epitope overlap with the more familiar MPM-2 phosphoepitope that also appears primarily in mitosis (13). The connection between 3F3/2 phosphorylation and the mitotic checkpoint was based on the early observation that 3F3/2 staining was invariably more prominent at the kinetochore of unaligned chromosomes in PtK cells. In contrast, staining was lost or greatly diminished at the kinetochores of chromosomes that were aligned at the spindle equator. If mAb 3F3/2 was microinjected into PtK cells, the kinetochore-bound antibodies did not interfere with chromosome alignment but they failed to separate even though they were aligned at the spindle equator (14). Thus, the 3F3/2 phosphoepitope(s) at the kinetochore are probably not involved with kinetochore motility. Consistent with the idea that unaligned kinetochores emit a signal that blocks the onset of anaphase, the persistence of the 3F3/2 phosphorylated proteins at the kinetochore of aligned chromosomes, as a result of their association with the injected 3F3/2 antibody, was believed to maintain the checkpoint delay.
An important advance towards resolving the biochemical mechanism by which the checkpoint detects kinetochore tension was made by showing that tension regulated 3F3/2 phosphorylation (18). In a series of micromanipulation experiments using grasshopper spermatocytes, it was shown that when a chromosome was experimentally displaced from the metaphase plate, the intensity of 3F3/2 staining was increased over the levels found at aligned kinetochores. The same observation was made in unmanipulated cells, where the absence of tension resulting from the erroneous attachment of both kinetochores to the same pole produced intense 3F3/2 staining at kinetochores. Importantly, when tension was experimentally introduced by pulling one of the kinetochores of the maloriented chromosome, 3F3/2 staining was lost at the kinetochore under tension while bright staining was still detected at the sister kinetochore that was not under tension. The combined data strongly suggest that the phosphorylation states of kinetochore proteins are regulated by the amount of tension that is exerted at kinetochores. The issue of how 3F3/2 phosphoproteins (and other b)biochemical changes) at the kinetochore are detected is unknown.
As discussed above, during cell division, quality control mechanisms monitor critical events, such as DNA replication, cell growth, and chromosome segregation. In most cases, these checkpoint systems will override the underlying cell-cycle machinery and block advancement into the next stage of the cell cycle until certain requirements are met in the current cell cycle stage. Although key regulators of mitotic progression such as cyclinB/cdc2kinase and the cyclosome/Anaphase Promoting Complex (APC) have been identified and charact
Chan Gordon
Jablonski Sandra
Yen Timothy
Dann Dorfman Herrell & Skillman P.C.
Fox Chase Cancer Center
Monshipouri M.
Rigaut Kathleen D.
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