Purification and Characterization of OTF-l, A Transcription Factor Regulating Cell Cycle Expression of a Human Histone H2b Gene: Demonstration of its Functional Identity with NF-III, a Factor Required for Efficient Initiation of Adenovirus DNA Replication

Author

Colin F. Fletcher

Date of Award

1988

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Roeder Laboratory

Abstract

An octamer-binding transcription factor, OTF-l, which stimulates transcription of a human histone H2b gene, has been purified from HeLa nuclear extracts. This purification was achieved through the use of DNA affinity chromatography, and the factor was unambiguously identified by renaturation of activity following SDS-polyacrylamide gel electrophoresis. The purified factor retained the ability to efficiently stimulate H2b transcription in a reconstituted in vitro system. This effect was dependent on an intact octamer element and was observed in the absence of other H2b promoter elements (except the TATA motif). This activity is absent from nuclear extracts prepared from cells synchronized in G2. However, the apparent mass and binding activity of the factor are unchanged in Sand G2. Because this factor can stimulate transcription in a G2 extract, we suggest that the modulation of activity is due to either in vivo constraints on binding or covalent (or other) modification(s). We have demonstrated that this factor is identical, by a number of criteria, to NF-llI. Therefore, OTF-l is able to stimulate the initiation of replication of adenovirus DNA. This effect was shown to be dependent on the presence of an intact NF-llI binding site in the adenovirus origin of replication. Finally, preliminary data suggesting that we have isolated a cDNA clone for this factor are presented and discussed.

Comments

A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Recommended Citation

Fletcher, Colin F., "Purification and Characterization of OTF-l, A Transcription Factor Regulating Cell Cycle Expression of a Human Histone H2b Gene: Demonstration of its Functional Identity with NF-III, a Factor Required for Efficient Initiation of Adenovirus DNA Replication" (1988). Student Theses and Dissertations. 381.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/381