quantification-lipid-abundance-evaluation-lipid-distribution

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in <em>Caenorhabditis elegans</em> by Nile Red and Oil Red O Staining

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved ...

examination-mitotic-meiotic-fission-yeast-nuclear-dynamics

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

University of Southern California

The replication fork protection complex (FPC) coordinates multiple processes that are crucial for unimpeded passage of the replisome through various barriers and difficult to replicate areas of the genome. We examine the function of Swi1 and Swi3, fission yeast's primary FPC components, to elucidate how replication fork stability contributes to DNA integrity in meiosis. We report that destabilization of the FPC results in reduced spore viability, delayed replication, changes in recombination, and chromosome missegregation in meiosis I and meiosis II. These phenotypes are linked to accumulation and persistence of DNA damage markers in meiosis and to problems with cohesion stability at the centromere. These findings reveal an important connection between meiotic replication fork stability and chromosome segregation, two processes with major implications to human reproductive health.

Destabilization of the replication fork protection complex disrupts meiotic chromosome segregation.

Tetrad dissection is a powerful tool in yeast genetics that allows the analysis of products of a single meiosis. With just a few tetrads, it is possible to determine linkage, identify unique phenotypes associated with double mutants, or assess specific meiotic defects. Strains are crossed on nitrogen-limiting medium for 3 days. With the help of a micromanipulator, ripe asci are isolated to spots 5 mm apart on a YES plate. Incubation at 36 °C for about 3-5 h is necessary for the ascus walls to break down. Once the spores are released, they are individually placed in a row containing four tetrad products, separated by 5 mm. The spores are then put in the appropriate temperature for the cross until colonies form, and phenotypes are assessed by replica plating or microscopic analysis.

Tetrad Dissection in Fission Yeast.

Random spore analysis (RSA) is a tool that allows for the screening of a large number of meiotic products. It requires only a limited effort, and is often the method of choice for constructing strains with unambiguous genotypes. It is also useful to identify the frequency of rare events. Strains are crossed on a nitrogen-limiting medium for three days. Mated cells are observed under the microscope to check for the presence of ripe asci. To release spores from their ascus, a sample of the cross is taken from the mating plate and resuspended in an enzyme solution overnight at 25-29 °C. Spores are then counted using a hemocytometer before plating an appropriate number. Incubation at the appropriate temperature follows until colonies form.

Wilber Escorcia

Leonard Davis School of Gerontology,
Program in Molecular and Computational Biology

University of Southern California

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Wilber Escorcia

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Leonard Davis School of Gerontology,Program in Molecular and Computational Biology

University of Southern California

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Leonard Davis School of Gerontology,
Program in Molecular and Computational Biology