Student Theses and Dissertations

Date of Award

1991

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Chua Laboratory

Abstract

The cauliflower mosaic virus 35S promoter is composed of several cis-elements, each of which confers a different expression pattern in transgenic tobacco. One such cis-element, activation sequence (as)-1 (-83 to -63) mediates preferential expression in root. The element contains a tandem repeat of the motif TGACG, that is crucial for as-1 function and binding of a tobacco nuclear factor, activation sequence factor (ASF)-1. Two other TGACG-containing cis-elements from plant promoters were identified by a computer assisted search of a gene bank. These cis-elements, hex-1 from the wheat histone H3 promoter and nos-1 from the nopaline synthase promoter of T-DNA, also bind ASF-1. Another tobacco nuclear factor, designated hex-i-specific binding factor (HSBF), binds only to hex-1, A tobacco cDNA library was screened for DNA-binding proteins using hex-1as a binding probe and two types of clones encoding hex-1-binding proteins were isolated. Based on their binding specificities to as-1, hex-1, and nos-1, the encoded DNA-binding proteins, named TGA1a and TGA1b, appear to correspond to ASF-1 and HSBF, respectively. Both TGA1a and TGA1b are bZIP proteins. The TGA1a mRNA level is much higher in root than in leaf. TGA1a can function as an as-1-specific transcription activator in a HeLa cell in vitro system as well as a wheat germ in vitro system. In both systems, TGA1a stimulates transcription by increasing the number of preinitiation complexes. When microinjected into cotyledon cells of transgenic tobacco plants carrying a promoter linked to as-I, TGA1a can induce the expression of the promoter. Judging from these results, TGA1a is likely to be the factor that is responsible for the as-1 function in vivo. Such a factor is present in leaf cells in a limiting concentration. Although the bZIP domain of TGA1a is sufficient for the specific binding to its target site, another domain (DS domain) increases the apparent DNA-binding affinity by stabilizing the dimeric form of TGA1a. The N-terminal region of TGA1a, which is enriched in acidic residues, appears to be essential for transactivation in vivo. However, when assayed in a HeLa cell in vitro system, most parts of the protein except the bZIP domain seem dispensable for activity.

Comments

A thesis submitted to the faculty of the Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

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