Purification of Human Cytoplasmic Actins From .

Eukaryotic cells rely on actin to support cellular structure, motility, transport, and a wide variety of other cytoplasmic functions and nuclear activities. Humans and other mammals express six closely related isoforms of actin, four of which are found primarily in skeletal, cardiac, and smooth muscle tissues. The final two isoforms, β and γ, are found in non-muscle cells.

Purification of human β- and γ-actin from budding yeast.

Biochemical studies of human actin and its binding partners rely heavily on abundant and easily purified α-actin from skeletal muscle. Therefore, muscle actin has been used to evaluate and determine the activities of most actin regulatory proteins but there is an underlying concern that these proteins perform differently from actin present in non-muscle cells. To provide easily accessible and relatively abundant sources of human β- or γ-actin (i.

Classical Genetics with Saccharomyces cerevisiae.

The budding yeast Saccharomyces cerevisiae is an outstanding experimental model organism that has been exploited since the early part of the twentieth century for studies in biochemistry and genetics. It has been the premiere experimental system for modern functional genomics and continues to make important contributions to many areas of biology. Here we discuss its many virtues as an organism for classical genetic research.

The Gcn2 Regulator Yih1 Interacts with the Cyclin Dependent Kinase Cdc28 and Promotes Cell Cycle Progression through G2/M in Budding Yeast.

The Saccharomyces cerevisiae protein Yih1, when overexpressed, inhibits the eIF2 alpha kinase Gcn2 by competing for Gcn1 binding. However, deletion of YIH1 has no detectable effect on Gcn2 activity, suggesting that Yih1 is not a general inhibitor of Gcn2, and has no phenotypic defect identified so far. Thus, its physiological role is largely unknown.

Using two-hybrid interactions to identify separation-of-function mutations.

Protein functions within cells frequently require they interact physically with a number of partner proteins to coordinate the appropriate biochemical processes. Mutational analysis has been quite useful for analyzing how the loss of a gene or protein impacts cell function or more specifically particular pathways. However, the genetics approach to studying gene function can be limited by not having the right mutations; for example because the mutant allele ablates all function, as is the case for deletion (null) alleles and most temperature-sensitive alleles.