Mitosis is a basic biological process of profound biomedical importance. Many medically important conditions result form or include disorders of chromosome behavior in mitosis or the related process of meiosis. While there is an excellent knowledge of the microscopically visible events of mitosis, such as chromosome movement, spindle formation and spindle behavior, we are virtually ignorant of the molecular mechanisms underlying these events. Microtubule integrity and function are essential for proper mitosis but we do not know how or why. Our research is directed towards genetically analyzing the molecular aspects of a subset of mitotic processes, (i.e., those relating to the function of mitotic spindle microtubules). Our goal has been and will continue to be the recovery and analysis of mutations in the genes encoding polypeptides (other than tubulin) that are components of isolated microtubules and of the mitotic spindle in Drosophila melanogaster. Our approach is interdisciplinary in nature and it begins by identifying and isolating the """"""""microtubule-associated proteins"""""""" from cultured Drosophila melanogaster cells. We then produce monoclonal or polyclonal antibodies recognizing these polypeptides and characterize the antibodies with respect to the antigen they recognize and its structural distribution in Drosophila cells. Those antibodies that recognize proteins found in the mitotic spindle are then used as probes to isolate the genes encoding these proteins by screening a recently developed random shear genomic library constructed in the expressing vector lambda gtll. The isolated genes are characterized and used a probes for in situ hybridization to polytene chromosomes to determine the map position of the mitotic spindle protein genes. We then utilize our knowledge of the location of these genes by using the standard techniques of Drosophila genetics to recover mutations in these loci. Ultimately, analysis of the phenotypes of these mutations will reveal the in vivo function of the mitotic microtubule-associated proteins and the role of microtubules in mitosis. Thus far, we have identified an Mr 205,000 protein (or family of proteins) that is a component of isolated microtubules and of the mitotic spindle and cytoplasmic microtubules. We have isolated and mapped its coding sequences and have found that it is likely to be a member of a small multi-gene family. The bulk of the current proposal is to finish the molecular analysis of this gene family and to begin the recovery and analysis of mutations in its members. Additionally we will continue our program of protein identification, gene isolation, mapping and ultimately classical genetic analysis.
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