The advancement of molecular biology in general, and of basic cancer research in particular, is often limited by the sensitivity of detection of biological substances of interest, and also by the paucity of general methods for detecting and mapping protein-protein and protein-DNA interactions in situ (in intact cells). We have recently developed three new and generally applicable approaches to these problems: (1) An """"""""indirect-labeling"""""""" method for post-separation autoradiographic detection of nucleic acids and proteins using neutron activation; (2) A method for mapping in vivo DNA-protein contacts in chromosomes using formaldehyde treatment of intact cells [1]; (3) A method for protein nearest neighbor analysis in vivo. Having shown the feasibility of these three approaches, we propose to further develop and refine each of them.
Our specific aims i n the proposed studies are: 1) To increase the sensitivity of our """"""""indirect-labeling"""""""" method for neutron-mediated detection of nucleic acids and proteins. The sensitivity of the current, initial version of the neutron method is comparable to that of silver staining techniques for proteins, and is thus at least six orders of magnitude below the theoretical sensitivity of the method. Increased sensitivity of the indirect labeling neutron method should eventually allow for instance, post-separation detection and quantitation of low- to medium-abundance proteins from single mammalian cells. Analysis of tumor cell heterogeneity using O'Farrel two-dimensional protein maps derived from individual tumor cells, detection of specific proteins from individual differentiated cells, detection and analysis of minor cell cycle-regulated proteins in single cells are among the many future applications of this new approach. 2) To develop procedures to allow the mapping of formaldehyde-induced in vivo DNA-protein crosslinks at nucleotide resolution, thus permitting in vivo footprint analysis of DNA-protein interactions. Nucleosome mapping in vivo is one of the applications of this new approach. Our initial version of the formaldehyde technique [1] is designed to map in vivo DNA-protein contacts at the resolution provided by restriction endonuclease mapping. 3) To develop a general method for protein nearest neighbor analysis in vivo. The results of the proposed studies should be of immediate utility for molecular biological studies on cancer as well as for many other fields of biochemical research.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA043309-04
Application #
3185487
Study Section
Biochemistry Study Section (BIO)
Project Start
1986-07-07
Project End
1991-06-30
Budget Start
1989-07-01
Budget End
1990-06-30
Support Year
4
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Type
Schools of Arts and Sciences
DUNS #
City
Cambridge
State
MA
Country
United States
Zip Code
02139
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