Jonathan M. Phillips, Ph.D.

Dissertation research performed under the guidence of Dr. David G. Kaufman

ABSTRACT
    The nuclear matrix is the primary site of DNA metabolism including replication, repair, and transcription. Little is known about specific associations of DNA sequences with nuclear matrix and even less is known about how, or even if, these associations fluctuate with the cell cycle. Human chromosome 16 cosmid grids were used as a model to investigate associations of specific DNA sequences with the nuclear matrix during G0 and S phases of the cell cycle. DNA from normal human fibroblasts that replicated early in S phase (ER-DNA), and DNAse I resistant matrix associated regions (MARs) that were associated with nuclear matrix in G0 (G0-MARs) and in early S phase (S-MARs) were obtained. Chromosome 16 grids were probed with ER-DNA and 9 cosmids were selected for Southern hybridization analysis using G0- and S-MARs probes. Of 76 fragments, 1.3% hybridized strongly to G0-MARs, 11% hybridized strongly to S-MARs, 59% hybridized to both G0- and S-MARs, and 28% did not hybridize. Five cosmid fragments containing both G0- and S-MARs and one fragment negative for both G0- and S-MARs were cloned and sequenced. No MARs consensus binding sequence was elucidated. To determine the similarity of G0-MARs and S-MARs at a whole cosmid level and with a larger sample size, 384 arbitrarily selected chromosome 16 cosmids were probed with G0- and S-MARs. About 4% of the cosmids preferentially hybridized to G0-MARs, 0.3% preferentially hybridized to S-MARs, and 96% hybridized to both G0- and S-MARs. G0- and S-MARs were analyzed by semi-arbitrarily primed polymerase chain reaction (SAP-PCR). Although most segments of DNA amplified by SAP-PCR are associated with nuclear matrix during both G0 and S phases (57%), there are segments that appear to be uniquely associated during G0 (28%) or during early S phase (15%). Together, these data suggest that most DNA sequences associated with the nuclear matrix in G0 are also associated in early S phase. However, these data also suggest that there are subsets of specific sequences that are associated with nuclear matrix in either G0 or early S phase, but not both.