Function of an Unconventional Myosin in Tetrahymena

Cuny Brooklyn College, Brooklyn NY

Investigators

Abstract

Dr. Gavin's laboratory has identified a novel unconventional myosin, Myo1p, in the model organism Tetrahymena thermophila, a ciliated protozoan. Approximately 2.7 kb of MYO1 have been cloned and sequenced thus far. To uncover the functions of Myo1p, targeted gene disruption was used to create a Tetrahymena strain that lacked a functional MYO1 gene. Transformed MYO1 cells had a reduced rate of food vacuole formation, and the macronucleus frequently failed to elongate properly during amitotic cell division resulting in grossly unequal segregation of DNA to progeny. In order to test additional hypotheses for Myo1p function in endocytosis and nuclear segregation, a full-length MYO1 sequence will have to be acquired and new reagents developed. This one-year project involves the completion of MYO1 cloning and sequencing and the creation of expression constructs for GFP-actin, GFP-tubulin, and GFP-Myo1p fusion proteins. PCR-based library screening of both genomic DNA and cDNA libraries will be used to complete the cloning and sequencing of MYO1. Screening is in progress and will be continued over the next year. In addition, RACE will be used to confirm the 5' and 3' ends of the cloned MYO1. Two different approaches involving standard recombinant protocols will be used to create GFP constructs for expression in Tetrahymena. In one approach, GFP-tagged genes (MYO1, actin, or tubulin) will be linked to pVGF-1, a rDNA-based replicative vector. Another approach for creating GFP constructs that will be used is to insert a GFP tagging construct at the 5' end of the gene sequence of interest. The functions and structure-function relationships of conventional myosins, such as skeletal muscle myosin, have been the subject of intense study for many years, and are reasonably well understood. In contrast, the various unconventional non-muscle myosins have only recently begun to attract serious research attention, and our understanding of the roles they play in the life and functionality of different types of cells is only slowly beginning to emerge. This project is expected to add to both the knowledge base of unconventional myosins and to our understanding of the biology of single-celled organisms such as the ciliated protozoan used in these studies.