Bud formation in yeast affords the opportunity to study simple patterns of spatial organization in a system with well developed genetics, molecular genetics, cell biology and biochemistry. Yeast cells can choose sites for budding in two different spatial patterns. a cells and alpha cells form buds in an axial pattern in which buds are initiated at the mother- daughter junction of the previous division. In the bipolar pattern, a/alpha cells are restricted to bud at their poles; the daughter cell (bud of the previous division) exhibits a strong tendency to bud away from its mother. The long term objective of this project is to understand at the molecular level the machinery responsible for producing the two patterns. Such an understanding requires determining the subcellular location of all proteins involved, knowing the interactions between components, and deciphering the mechanism by which each protein functions, be it a kinase, nucleotide-binding protein or some other type of molecule. Within the scope of this grant proposal, questions which will be addressed are the following: 1) What is the source of axial spatial information? It appears that BUD3 protein is central to furnishing axial information, since it assembles in the mother-bud neck i. e., axial positions. What proteins BUD3 assembles upon, what proteins BUD3 instructs and how BUD3 localization is temporally regulated will be explored. BUD4 , the other axial-specific gene, will be cloned, sequenced, and deleted from the genome. The expression of BUD4 will be examined as the possible basis of cell-type control of budding pattern, and antibodies will be raised to determine its subcellular location. Attempts will be made to identify and analyze additional axial- specific BUD genes. 2) What is the source of bipolar information? An extensive genetic screen will be performed to search for bipolar-specific genes. Interesting genes will be cloned, and analyzed with the goal of this granting period being to localize the products within the cell. 3) What is the basis of cell-type control? BUD4 transcription and the modification state of BUD proteins (especially BUD3) will be examined as the potential basis for cell-type control. The understanding of the mechanisms of budding pattern, that is derived from this work, is of general interest because formation of a bud involves the assembly of cell polarity towards the bud site, and because the plane of cell division directly follows the mother-bud axis. Orientation of an axis of cell polarity and regulation of cell division planes are recurring themes during the development and functioning of multicellular organisms. For example, killing of target cells by T-cells involves polarization of the T-cell towards its target, and specification of cell fates during mouse development is determined by the orientation of cell divisions.
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