Functional Analysis Of Sfi1p In The Sporulation Process Of Saccharomyces Cerevisiae

As the microtubule organizing center of yeast cells, the spindle pole body (SPB) plays an essential role in cell division and maintenance of genome stability, and therefore has attracted a great deal of attention and an enormous research efforts in elucidating its structure feature and function in the field of cell biology. Sfi1p is a newly identified SPB component of Saccharomyces cerevisiae. Structural characterization and functional analysis of Sfi1p will not only shed new light on our current understanding about yeast SPB, but also aids investigation of its homologues, and hence the MTOC in higher eukaryotes.In this work, we created two temperature-sensitive(ts) alleles of SFI1. Using these mutants, we investigate the function of Sfi1p in the sporulation process of Saccharomyces cerevisiae.SFI1 ts alleles, sfi1-778 and sfi1-791, were generated by error-prone PCR. Both alleles were integrated individually into the SFI1 locus of the haploid wild-type strain, W303-1A, using pop-in/pop-out gene replacement strategy. The mutant carrying the sfi1-778 allele was designated as LJ778 and the one carrying sfi1-791 allele as LJ791. Diploid strains homozygous for sfi1-778 or sfi1-791 were constructed by transforming HO-bearing plasmid into LJ778 and LJ791, and the resulting diploids were named as LJ778D and LJ791D respectively.At restrictive temperature, both the LJ778 and LJ791 mutants showed large-budded morphology, similar to SFI1 null mutant, suggesting a possible G2/M arrest. To verify this possibility, the microtubule and nuclear appearance of the LJ778 and LJ791 strain were studied by florescence microscopy under restrictive temperature. We found that most of the large-budded cells had a single nucleus at the neck. Tub1p-GFP localization of spindle microtubules showed that 90% of the large-budded LJ778 and LJ791 cells were unable to assemble mitotic spindles in that they contained either short monopolar nuclear spindles or microtubules emanating from the spindle pole body, which appeared as a single brightly-stained spot. These results indicated that at restrictive temperature, the LJ778 and LJ791 mutant arrested at G2/M transition.In addition, a small fraction of the large-budded cells contained a single nucleus at random position in the mother cells and some of them were binucleated, indicating...