Evolutionary analysis of heterochromatin protein compatibility by interspecies complementation in Saccharomyces.

The genetic bases for species-specific traits are widely sought, but reliable experimental methods with which to identify functionally divergent genes are lacking. In the Saccharomyces genus, interspecies complementation tests can be used to evaluate functional conservation and divergence of biological pathways or networks. Silent information regulator (SIR) proteins in S. bayanus provide an ideal test case for this approach because they show remarkable divergence in sequence and paralog number from those found in the closely related S. cerevisiae. We identified genes required for silencing in S. bayanus using a genetic screen for silencing-defective mutants. Complementation tests in interspecies hybrids identified an evolutionarily conserved Sir-protein-based silencing machinery, as defined by two interspecies complementation groups (SIR2 and SIR3). However, recessive mutations in S. bayanus SIR4 isolated from this screen could not be complemented by S. cerevisiae SIR4, revealing species-specific functional divergence in the Sir4 protein despite conservation of the overall function of the Sir2/3/4 complex. A cladistic complementation series localized the occurrence of functional changes in SIR4 to the S. cerevisiae and S. paradoxus branches of the Saccharomyces phylogeny. Most of this functional divergence mapped to sequence changes in the Sir4 PAD. Finally, a hemizygosity modifier screen in the interspecies hybrids identified additional genes involved in S. bayanus silencing. Thus, interspecies complementation tests can be used to identify (1) mutations in genetically underexplored organisms, (2) loci that have functionally diverged between species, and (3) evolutionary events of functional consequence within a genus.

A simple method for DNA isolation from clotted blood extricated rapidly from serum separator tubes.

BACKGROUND: After clinical laboratory tests have been performed, it can be difficult to obtain DNA without further patient involvement. Although the blood clot remaining within the serum-separation tube after serum collection is a source of DNA, recovery of the clot from the tube is a significant challenge. METHOD: We devised a method to efficiently remove clotted blood from the serum-separation gel and extract DNA from clotted whole blood samples, obtaining maximum yield of the DNA without DNA contamination by the separation gel. The method involved centrifugation of the sample in the inverted original 10-mL collection tube to displace the separation gel for easy isolation of the blood clot and shearing of the blood clot by centrifugation through a 20-gauge wire mesh cone at 2000 g in a swinging-bucket rotor. After erythrocyte lysis and proteinase-K digestion of the fragmented clot, DNA was precipitated with isopropanol in the presence of glycogen. RESULTS: The mean amount of DNA obtained from a 4-mL clotted blood sample prepared by this method was 37.1 microg for clots processed soon after collection, with a reduction to 0.439 microg for clots stored for 1 month before extraction. The quality of the DNA was comparable to that extracted directly from whole blood, and it was found to be suitable for PCR-mediated analysis. CONCLUSION: We have formulated a method that overcomes the difficulties of safely extricating a blood clot from serum-separation tubes, allowing rapid DNA extraction for the purposes of genetic investigation.