Title

The isolation and characterization of conjugation-specific genes in the hypotrichous ciliated protozoan, {\it Euplotes crassus\/}

Date of Completion

January 1996

Keywords

Biology, Molecular|Biology, Microbiology

Degree

Ph.D.

Abstract

The hypotrichous ciliated protozoan Euplotes crassus undergoes extensive DNA excision during the macronuclear development in conjugation. One of the primary interests of our laboratory has been to isolate these components and reconstruct an in vitro DNA splicing system. My thesis research was aimed in the identification of genes that are specifically expressed during macronuclear development by differential hybridization screening in an effort to isolate some of the components of DNA excision machineries.^ Three developmental specific cDNA clones: conZA7, conZA8, and conZB1 were isolated. Quantitative Northern Blot analyses showed that at their mRNAs are very abundant during conjugation. Cytological analyses indicated that high level of transcripts persisted through the commencement of macronuclear development suggesting that the products of these genes might play a role in these events. Southern Blot analyses showed that all three genes have their corresponding macronuclear gene counterparts.^ Macronuclear genes of conZA7, conZA8, and conZB1 have all been cloned and sequenced. conZA7 and conZA8 code for novel proteins whereas conZB1 codes for a replacement variant histone H3 protein. ZB1 protein could potentially change the configuration of the genome during macronuclear development. An Internal Eliminated Sequence was identified within the last exon of the micronuclear conZA7 gene indicating that these conjugation-specific genes were transcribed from the old fragmenting macronucleus. These data suggest that the old macronucleus plays a contradictory role during conjugation. While it is being destoried, it is also required for the expression of conjugation-specific genes. ^