Evaluation of nascent RNA as a criterion of chromatin fractionation

Date Issued
August 1, 1980
Author(s)
Townes, Tim M.
Advisor(s)
Stuart Riggsby
Additional Advisor(s)
Jeffrey Becker
Robert Miller
Joseph Fuhr
Wen Yang
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Abstract
Separation of chromatin into transcriptionally active and inactive fractions is a goal which has been pursued for many years. Many methods have been proposed and have been shown to be successful based on one or more criteria for activity. One of the most widely applied criterion has been the presence of nascent RNA chains in certain fractions following pulse-labeling with labeled RNA precursor. The applicability of this criterion has been questioned by the finding that exogenously added RNA binds selectively to a chromatin fraction which co-sediments with putatively nascent RNA in sucrose gradient fractionation schemes. Chapters I and II of Part I describe the partitioning of non-nascent RNA in two other fractionation schemes, the DNAse II-MgCl2 and ECTHAM-cellulose procedures, respectively.

The results presented in Chapter I demonstrate that free RNA, which is either added exogenously or generated endogenously, binds to the same fraction that is in fact enriched in actively transcribed DNA sequences. Therefore, although this fraction may indeed by enriched in transcriptionally active chromatin, the "nascent RNA" assay is not a reliable indicator of such activity.

The results presented in Chapter II demonstrate that free RNA does not selectively bind to "active" chromatin generated by the ECTHAM-cellulose procedure. However, free RNA itself binds to the resin and elutes at the same pH as chromatin which has many of the structural features expected for an active fraction. Therefore, the nascent RNA assay is also an unreliable indicator of activity for the ECTHAM-cellulose procedure.

The work described in Part I, Chapters I and II grew out of a project in which chromatin fractionation was to be used as the first step in the isolation of molecules which regulate transcription. Although the project as originally proposed was not completed, Chapters I, II and III of Part II describe the preparation of reagents which are essential in assays for specific regulatory activity.

Degree
Doctor of Philosophy
Major
Microbiology
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