Regulation of paramecium variable surface antigen expression
Abstract
Paramecium tetraurelia can express many variable surface antigens, but only one is present on the cell surface at any given time. Previous experiments demonstrated that a chimeric gene containing the 5′ upstream sequence and 5′ coding region of B51 attached to the A51 coding region was co-expressed with the B51 gene on the cell surface. Utilizing this co-expression system, a frameshift mutation was created near the 3′ end of the chimeric gene to study the role of the surface antigen itself in regulation of mutual exclusion. The results showed that the transcription of the chimeric gene is down regulated when its corresponding antigen is not present on the cell surface. This provides the first molecular evidence of transcriptional self-regulation of variable surface antigens. Although the 5′ coding region is required for co-expression on the cell surface, internal deletion constructs of the B51 antigen gene show that the 5′ upstream sequence is sufficient to regulate mutual exclusion. A construct consisting of the B51 upstream sequence attached to the GFP coding region was transcribed in B51 + cells but not in A51+ cells. A chimeric gene that contains the B51 upstream region and the A51 coding region was transcribed in transformed cells that expressed the B51 antigen. These data suggest that the cis-element regulating mutually exclusive transcription for B51 is located in the upstream sequence. A model incorporating these and previous results is presented. Additional experiments investigated the significance of the conserved cysteine periods of variable surface antigens. Internal deletions of the A51 protein were constructed and analyzed. These experiments demonstrated that alteration of the cysteine periods and removal of up to two whole cysteine periods near the N-terminus do not affect the synthesis of the protein on the cell surface. In contrast, deletions near the C-terminus revealed a small region that is essential for display of the protein on the cell surface. These studies demonstrate the flexibility of the cysteine repeats and the usefulness of internal deletion analysis in identifying critical segments of the protein.
Degree
Ph.D.
Advisors
Forney, Purdue University.
Subject Area
Molecular biology
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