Characterization and Isolation of the Human Misato Gene Homologue

Abstract

Misato is a tubulin-like protein in Drosophila melanogaster, which is involved in chromosome segregation. Defects at the misato locus of Drosophila lead to massive nondisjunction and consequent cell death during embryonic development. It is proposed that this novel gene plays an essential role in cell division and may be involved in the organization of the spindle apparatus. The primary purpose of this project is to isolate and characterize the human homologue of the misato gene and explore possible functions of its gene product. We identified a 2.4 kb clone from a human heart cDNA library using a probe made from a human cDNA from the EST database that demonstrated significant homology with the misato protein. The clone we isolated had an open reading frame of 555 amino acid residues and has an overall amino acid sequence similarity of 45% with 29% identity to the misato protein of Drosophila. Both proteins share two regions of sequence similarity with tubulin, the larger one of which lies between two highly conserved regions that are components of the GTP-binding site in tubulin. Within this tubulin-like motif, there is a peptide similarity of 69% with 60% identity between misato and the clone we isolated. This region is also conserved in a gene product that was isolated from Arabidopsis thaliana, which shares an overall homology of 47% with misato. Northern Blot analysis determined that the human gene is expressed in several tissues. High expression levels are found in testis, pancreas, brain, and heart. The sequence information will be implemented in the prokaryotic expression of the gene in order to ultimately generate monoclonal antibodies, which will be used to localize the protein in tissues and cells. We predict that the function of misato will have some correlation to cell division in humans, particularly in the formation of the spindle apparatus, and may therefore have a role in cancer. This association may make it possible to develop diagnostic tools to improve the recognition of genetic defects and neoplastic growth.