The nuclear matrix (NM) is emerging as a crucial element in nuclear metabolism. It organizes chromatin, serves in RNA splicing and may participate in selecting DNA regions for transcription. Despite growing interest, methods of NM preparation have been largely arbitrary and harsh. We have systematically applied biochemical analysis and resinless section electron microscopy to arrive at an NM preparation closest to the in vivo structure. The NM proteins are completely distinct from those of chromatin with almost no cross contamination of fractions. In resinless sections the matrix structure and lamina appear intact. This more nearly native NM has many proteins lost in harsher preparation, many of which are highly cell type specific. Further fractionation reveals an underlying network of core filaments which contain all of the hnRNA and serve as a structural component of the NM. The research proposed here is to continue characterizing the proteins of the NM and core filaments using MAB libraries developed in the current grant period. In this way, some NM functions can be identified. The MABs are used for spatial localization by a method developed for gold beads in resinless sections for both interphase and mitotic cells. The MABs are also used to select cDNA clones from lambda gt11 expression libraries for molecular sequencing. We have report the preliminary characterization of antigens to 9 MABs as well as results using MABs, such as to HPV 16 E7 protein, obtained from other laboratories. We have completely sequenced two NM proteins and one from the core filaments. We have also identified several different behaviors of NM proteins at mitosis and are investigating these by stereoscopic immunogold staining.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA045480-06
Application #
3188557
Study Section
Special Emphasis Panel (SSS (01))
Project Start
1987-07-01
Project End
1995-06-30
Budget Start
1992-07-01
Budget End
1993-06-30
Support Year
6
Fiscal Year
1992
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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