Award Abstract #0240998
Combined Micromechanical-Biochemical Study of Mitotic Chromosome Structure

NSF Org:	MCB Division of Molecular and Cellular Biosciences
Initial Amendment Date:	September 17, 2003
Latest Amendment Date:	August 2, 2007
Award Number:	0240998
Award Instrument:	Continuing grant
Program Manager:	Susan Porter Ridley MCB Division of Molecular and Cellular Biosciences BIO Directorate for Biological Sciences
Start Date:	October 1, 2003
Expires:	September 30, 2008 (Estimated)
Awarded Amount to Date:	$379794
Investigator(s):	John Marko john-marko@northwestern.edu (Principal Investigator)
Sponsor:	University of Illinois at Chicago 809 S MARSHFIELD RM 608 CHICAGO, IL 60612 312/996-9406
NSF Program(s):	GENES AND GENOME SYSTEMS
Field Application(s):
Program Reference Code(s):	BIOT,9183,1116
Program Element Code(s):	1112

ABSTRACT

This project is a study of the structure of the mitotic chromosome of animal cells. For this work the investigator will use a unique combination of micromanipulation and biochemical techniques. Biophysical force measurements will be used to assay biochemically-introduced structural changes in mitotic chromosomes. In this way the role of specific types of molecules in defining mitotic chromosome structure can be quantitatively studied. Preliminary results indicate that DNA itself provides the structural integrity of the folded mitotic chromosome. These results challenge the classical protein scaffold model of mitotic chromosome structure, and indicate the value of this approach in ruling out specific models of chromosome structure. The first objective of this project is further study of DNA connectivity in the mitotic chromosome in this way, and to extend these measurements to Xenopus chromosomes. Additional enzyme experiments will study the role of RNA and protein in mitotic chromosome structure. A second objective is to compare the physical properties of Xenopus chromosomes from cells with chromatids reconstituted using Xenopus egg extracts. A third objective is to characterize the interchromosome filaments observed when chromosomes are removed from cells during mitosis, and in particular to quantify their DNA content. The question of whether or not there is a contiguous protein skeleton inside the mitotic chromosome is a basic and open topic in cell biology.

Understanding the relation of reconstituted chromosomes to chromosomes in cells is extremely important given the general assumption that the former system is an accurate model of chromosomes. Finally, putting the question of existence of mitotic interchromosome filaments on solid ground will demand new thinking about many assumptions about mitotic chromosome structure. Broader impacts of this research will be the development of new biophysical techniques (nanonewton-scale force measurement, microfluidic enzymatic treatments) for the study of chromosome structure. Along with this, the proposed activities will focus on research education for Ph.D. students, combining physics and biology. There is currently a national shortage of young scientists with this kind of training. This project will therefore uniquely strengthen the scientific personnel and technique pools in this growth area.

PUBLICATIONS PRODUCED AS A RESULT OF THIS RESEARCH

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J. Yan, T.J. Maresca, D. Skoko, C.D. Adams, B. Xiao, M.O. Christensen, R. Heald, J.F. Marko.  "Micromanipulation studies of chromatin fibers in Xenopus egg extracts reveal ATP-dependent chromatin assembly dynamics.,"  Molecular Biology of the Cell,  v.18,  2007,  p. 464.

L.H. Pope, C. Xiong, J.F. Marko.  "Proteolysis of Mitotic Chromosomes Induces Gradual and Anisotropic Decondensation Correlated with a Reduction of Elastic Modulus and Structural Sensitivity to Rarely Cutting Restriction Enzymes,"  Molecular Biology of the Cell,  v.17,  2006,  p. 104.

Padinhateeri Ranjith, Jie Yan, John F. Marko.  "Nucleosome hopping and sliding kinetics determined from dynamics of single chromatin fibers in Xenopus egg extracts,"  Proc. Natl. Acad. Sci. USA,  v.104,  2007,  p. 13649.


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