Cell death caused by excision of centromeric DNA from a chromosome in Saccharomyces cerevisiae.

If genetically modified organisms (GMOs) are spread through the natural environment, it might affect the natural environment. To help prevent the spread of GMOs, we examined whether it is possible to introduce conditional lethality by excising centromeric DNA from a chromosome by site-specific recombination in Saccharomyces cerevisiae as model organism. First, we constructed haploid cells in which excision of the centromeric DNA from chromosome IV can occur due to recombinase induced by galactose. By this excision, cell death can occur. In diploid cells, cell death can also occur by excision from both homologous chromosomes IV. Furthermore, cell death can occur in the case of chromosome V. A small number of surviving cells appeared with excision of centromeric DNA, and the diploid showed greater viability than the haploid in both chromosomes IV and V. The surviving cells appeared mainly due to deletion of a recombination target site (RS) from the chromosome.

The relationship between chromosomal positioning within the nucleus and the SSD1 gene in Saccharomyces cerevisiae.

Eukaryotic cells are characterized by very large chromosomal DNAs efficiently packed within the nucleus. To identify the mechanism of chromosomal packaging based on the uniqueness of the centromere region in Saccharomyces cerevisiae, we isolated the HCH6 mutant, which shows 2.5-fold higher efficiency of site-specific recombination between the CEN5 and HIS3 loci than the wild-type CH53 strain. This mutant also displayed defects in cell integrity at high temperature. The SSD1 gene was perhaps responsible for this defect. The efficiency of site-specific recombination was decreased by the introduction of SSD1 in HCH6 cells and increased by disruption of SSD1 in the wild-type cells. Furthermore, the distances between the CEN5 and HIS3 loci and between the CEN5 locus and the spindle pole body (SPB) indicated that disrupting SSD1 caused a loss of the anchoring of the CEN5 locus near SPB. These results suggest Ssd1p-dependent cross-talk between chromosomal positioning within the nucleus and the positioning of cellular components within the cell.

Lipase-catalyzed asymmetric synthesis of desprenyl-carquinostatin A and descycloavandulyl-lavanduquinocin.

An asymmetric synthesis of the core carbazole structure, 6-desprenyl-carquinostatin 3 and 6-descycloavandulyl-lavanduquinocin 3, toward a total synthesis of carquinostatin A (1) and lavanduquinocin (2), has been established. Lipase QLM (Meito) catalyzed enantioselective acetylation of the racemic alcohol 6 gave the (-)-acetate 7 and the (+)-alcohol 6 with high enantioselectivity. The absolute stereochemistry of the (-)- and (+)-alcohol 6 have been determined to be R- and S-configurations, respectively, by the advanced Mosher method. In the same manner, the (-)-acetate 13 and the (+)-alcohol 12 have been obtained from the racemic alcohol 12. The (R)-(-)-acetate 13, derived from the (R)-(-)-acetate 7, was the same as the (-)-acetate 13, which has been determined to be (R)-configuration. Oxidation of the (R)-(-)-acetate 13 followed by hydrolysis afforded (R)-(-)-6-desprenyl-carquinostatin [and (R)-(-)-6-descycloavandulyl-lavanduquinocin] 3. In addition, oxidation of the (S)-(+)-alcohol 12 provided (S)-(+)-3, which is the enantiomer of 6-desprenyl-carquinostatin A (R)-(-)-3.

Isolation and characterization of triacylglycerol-secreting mutant strain from yeast, Saccharomyces cerevisiae.

To establish the molecular bases for development of a microbiological system approaching excretive fermentation of useful lipids, a mutant strain that accumulates lipids in the medium was isolated from the laboratory yeast Saccharomyces cerevisiae. Following the mutagenesis to strain YP1, a long chain fatty acid utilizer with ethylmethane sulfonate, the mutant strain, STG1, was selected from about 80,000 colonies. The analysis of extracellular lipids and the monitoring of leakage of intracellular proteins indicated that strain STG1 secreted lipids containing triacylglycerols into the extracellular space without cell lysis. Genetic studies clarified that this mutation was recessive and was complemented by wild-type genomic DNA fragments. STG1 was considered to be a good tool for elucidation of the molecular mechanism for transmembrane lipid transport.

Region specificity of chromosome III on gene expression in the yeast Saccharomyces cerevisiae.

A single copy of a reporter gene cassette, such as PGKP-lacZ-LEU2 (promoter-reporter-marker gene) cassette, was inserted into one of 32 positions along chromosome III in Saccharomyces cerevisiae with an interval of approximately 10 kb. The amounts of translational gene product (beta-galactosidase) synthesized by the cassette-transformed cells were then determined. The region specificity in chromosome III could be demonstrated in gene expression: two higher-expressed regions (hot regions), 133 and 199 (MAT) regions, and seven lower-expressed regions (cold regions). For the steady and high production of polypeptide, foreign gene products, by yeast, we would like to state that we hope for an insertion of the artificially prepared gene cassette [(promoter)-(foreign gene)-(marker gene) ] into a hot region, such as 199 (MAT) region of chromosome III.