TRANSCRIPTION OF THE T(R)-DNA IN OCTOPINE CROWN GALL TUMORS (T-DNA, AGROBACTERIUM, TUMOR-INDUCING PLASMID)

VICTOR JOSEPH DIRITA, Purdue University

Abstract

Pathogenic strains of Agrobacterium tumefaciens, a Gram negative soil bacterium, cause crown galls at wound sites on dicotyledonous plants. The major virulence determinant of A. tumefaciens is a large plasmid known as the tumor inducing, or Ti, plasmid. Upon infection, a region of the Ti plasmid known as the T-DNA is transferred from A. tumefaciens to the plant cell where it is integrated into the nuclear genome. In addition to the oncogenes whose products disrupt the phytohormone balance in the plant cell, the T-DNA encodes the biosynthesis of novel sugar derivatives which can be utilized by the inciting strain of A. tumefaciens as a sole carbon source. The ability to utilize these compounds, termed opines, confers competitive advantage upon A. tumefaciens over other soil bacteria which are unable to catabolize opines. Tumors which synthesize the opine known as octopine frequently harbor two independent T-DNA's. T(,L)-DNA harbors the oncogenes and the octopine synthase gene. T(,R)-DNA encodes no function essential to tumorigenesis but does encode the biosynthesis of two opines, mannopine and agropine. The subject of this thesis is the nature of T(,R)-DNA transcription. Northern blotting and S1 nuclease analyses of the T(,R)-DNA from a tobacco octopine tumor line demonstrated that five polyadenylated transcripts originate within this T-DNA. Subsequent genetic experiments showed that three of these transcripts are involved in the biosynthesis of mannopine and agropine. A plasmid was constructed which allowed the analysis of the mannopine synthase gene (mas) promoter by Bal 31 deletion mutagensis. This construct contains mas upstream sequences cloned upstream of the coding sequences for neomycin phosphotransferase II (NPTII) from Tn5. Deletions which leave intact 318 bases upstream of the initiation of transcription are able to direct G418 resistant growth of sunflower crown gall tissue. A deletion which leaves 130 bases upstream leads to a G418('s) phenotype. It is possible to detect small amounts of the NPTII transcript (by S1 nuclease protection analysis) in the tissue containing the -130 deletion. NPTII mRNA is not detectable in a tumor line containing a deletion leaving only 57 bases upstream of mas/NPTII transcription initiation. These experiments define a minimum size of the mas promoter required for accurate transcription, 130 bases, and suggest that sequences between -130 and -318 are important for the efficiency of transcription.

Degree

Ph.D.

Subject Area

Molecular biology

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