Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1998-09-10
2003-11-11
Caputa, Anthony C. (Department: 1642)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S004000, C435S007100, C435S184000, C435S975000, C530S350000, C530S387100
Reexamination Certificate
active
06645748
ABSTRACT:
FIELD OF THE INVENTION
The invention provides isolated nucleic acid and amino acid sequences of Xenopus CENP-E (XCENP-E), antibodies to XCENP-E, methods of screening for CENP-E modulators using biologically active CENP-E, and kits for screening for CENP-E modulators.
BACKGROUND OF THE INVENTION
Segregation of genetic material during mitosis is mediated by the microtubules of the mitotic spindle (see, e.g., McIntosh, in
Microtubules
, pp. 413-434 (Hyams & Lloyd, eds., 1994). During mitosis, chromosomes are dynamically attached to spindle microtubules via the kinetochore, which is a structure located at the centromere of the chromosome. Kinetochores are involved in coordinating chromosome movement via microtubule assembly and disassembly. The kinetochore and its component proteins thus play an important role in the dynamics of mitosis.
Spindle microtubules have a defined polarity, with their slow-growing, “minus” ends anchored at or near the spindle pole, and their dynamic, fast-growing “plus” ends interacting with chromosomes (McIntosh, et al.,
J. Cell Biol
. 98:525-533 (1984)). During prometaphase, chromosomes establish interactions with the fast-growing plus ends of microtubules via the kinetochore. Chromosomes then undergo a series of microtubule-dependent movements, culminating in alignment at the metaphase plate, equidistant from the two spindle poles. This process is called “congression.” However, the molecular mechanisms underlying chromosome congression are poorly understood (see, e.g., Rieder, et al.,
J. Cell Biol
. 124:223-33 (1994)). A major question has been whether any kinetochore-associated microtubule motors play an important role in congression.
The two predominant and opposing forces are currently thought to be responsible for chromosome movement during congression: (1) an anti-poleward polar force associated with regions of high microtubule density near the spindle poles, and (2) a poleward force generated at the kinetochore (Khodjakov, et al.,
J. Cell Biol
. 135:315-327 (1996); Waters, et al.,
J. Cell Sci
. 109:2823-2831 (1996); reviewed in Rieder, et al.,
Int. Rev. Cytol
. 79:1-57 (1982); Mitchison, et al.,
Annu. Rev. Cell Biol
. 4:527-49 (1988); Rieder, et al.,
J. Cell Biol
. 124:223-33 (1994)).
Studies in vitro have demonstrated the presence of both plus and minus end-directed microtubule motor activities on kinetochores that may be responsible for these chromosome movements (Mitchison, et al.,
J. Cell Biol
. 101:766-77 (1985); Hyman, et al.,
Nature
351:206-211 (1991)). The outstanding issue, however, has been the identity of the molecules at the kinetochore which act as motors and generate the force for chromosome movement.
In general, both genetic and biochemical approaches have demonstrated crucial roles for microtubule motors in spindle assembly, spindle pole separation, and regulation of spindle microtubule dynamics. These motors include Eg5, CHO1/MKlp1, ncd, cut7, bimC, CIN8, KIP1, KAR3, Xklp2, XKCM1, and XCTK2 (Sawin, et al.,
Nature
359:540-543 (1992); Blangy, et al.,
Cell
83:1159-1169 (1995); Sawin, et al.,
J. Cell Biol
. 112:925-940 (1991); Nislow, et al.,
J. Cell Biol
. 111:511-522 (1990); Endow, et al.,
J. Cell Sci
. 107:859-867 (1994); Hagan, et al.,
Nature
347:563-566 (1990); Hagan, et al.,
Nature
356:74-76 (1992); Enos, et al.,
Cell
60:1019-1027 (1990); Hoyt, et al.,
J. Cell Biol
. 118:109-120; Roof, et al.,
J. Cell Biol
. 118:95-108 (1992); Saunders, et al.,
Cell
70:451-458 (1992), Boleti, et al.,
J. Cell. Biol
. 125:1303-1312; Walczak, et al.,
Cell
84:37-47 (1996); Walczak, et al.,
J. Cell Biol
. 136:859-70 (1997)). Two kinesin superfamily members, Xenopus Xklp1 and Drosophila nod localize to chromosome arms. With the exception of these two chromatin-associated motors, which are thought to mediate polar ejection forces, none of these other proteins have been implicated directly in congression or in chromosome movement during other phases of mitosis (Theurkauf, et al.,
J. Cell Biol
. 116:1167-1180 (1992); Afshar, et al.,
Cell
81:129
, Cell
81:128-138 (1995); Vernos, et al.,
Trends in Cell Biol
. 5:297-301 (1995)).
A candidate for powering chromosome movement in mitosis is centromere-associated protein-E (CENP-E), a member of the kinesin superfamily of microtubule motor proteins. Human CENP-E has been cloned and is an integral component of the kinetochore (Yen, et al.,
Nature
359:536-539 (1992); Yao, et al.,
The microtubule motor CENP
-
E is an integral component of kinetochore corona fibers that link centromeres to spindle microtubules
(manuscript)). CENP-E localizes to kinetochores throughout all phases of mitotic chromosome movement (early prometaphase through anaphase A) (Yen, et al.,
Nature
359:536-539 (1992); Brown, et al.,
J. Cell. Biol
. 125:1303-1312 (1994); Lombillo, et al.,
J. Cell Biol
. 128:107-115 (1995)).
Previous efforts have suggested a role for CENP-E in mitosis. Microinjection of a monoclonal antibody directed against CENP-E into cultured human cells delays anaphase onset (Yen, et al.,
EMBO J
. 10:1245-1254 (1991)). Anti-CENP-E antibody injection into maturing mouse oocytes induces arrest at the first reductional division of meiosis (Duesbery, et al.,
Proc. Natl. Acad. Sci. USA
(in press, 1997)). Antibodies against CENP-E block microtubule depolymerization-dependent minus end-directed movement of purified chromosomes in vitro (Lombillo, et al.,
J. Cell Biol
. 128:107-115 (1995)).
However, these experiments have not demonstrated the precise role of CENP-E in mitosis, nor have they shown the activity of CENP-E, in particular any motor activity. Recently, CENP-E was reported to be associated with minus end-directed microtubule motor activity, raising the possibility that CENP-E might be responsible for poleward kinetochore movements (Thrower, et al.,
EMBO J
. 14:918-926 (1995)). However, biologically active CENP-E has never been isolated, neither from naturally occurring nor recombinant sources.
SUMMARY OF THE INVENTION
The present invention provides for the first time biologically active CENP-E and surprisingly demonstrates, contrary to previous reports, that CENP-E is a motor that powers chromosome movement toward microtubule plus ends. Using immunodepletion and antibody addition to Xenopus egg extracts, the present invention further demonstrates that CENP-E plays an essential role in congression. The present invention also provides for the first time the nucleotide and amino acid sequence of isolated Xenopus CENP-E.
In one aspect, the invention provides an isolated, biologically active CENP-E protein, wherein the CENP-E protein has the following properties: (i) at least one activity selected from the group consisting of plus end-directed microtubule motor activity, ATPase activity, and microtubule binding activity; and (ii) the ability to specifically bind to polyclonal antibodies generated against CENP-E. In one embodiment, the CENP-E protein has an average molecular weight of about 300-350 kDa.
In one embodiment, the CENP-E protein has an amino acid sequence having at least 34%, or alternatively at least 45%, or alternatively at least 55% sequence identity with a XCENP-E motor domain of SEQ ID NO:1. Alternatively, CENP-E has at least 60%, 65% or 70% sequence identity with a XCENP-E motor domain of SEQ ID NO:1. In an alternative embodiment, the CENP-E has 70%, or alternatively 75%, or alternatively 80%, or alternatively 85%, or alternatively 90% or alternatively 95% amino acid sequence identity to a Xenopus CENP-E core motor domain as measured using a sequence comparison algorithm. In an alternative embodiment, the CENP-E protein has an amino acid sequence of SEQ ID NO:1.
In another embodiment provided herein, the CENP-E protein is encoded by a nucleic acid sequence having at least 70% sequence identity with SEQ ID NO:2. In another aspect of the present invention, the CENP-E protein is encoded by a nucleic acid which hybridizes under high stringency to a nucleic acid having a sequence complementary to that of SEQ ID NO:2.
In one embodiment, the CENP-E protein is from a hum
Cleveland Don W.
Goldstein Lawrence S. B.
Sakowicz Roman
Wood Kenneth W.
Holleran Anne L.
The Regents of the University of California
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