Distribution, abundance, and genetic diversity of Clinostomum spp. metacercariae (Trematoda:Digenea) in a modified Ozark stream system.

Land-use alterations can have profound influences on faunal distributions, including host-parasite relationships. Yellow grub trematodes ( Clinostomum spp.) have complex life cycles involving 3 hosts: a snail, a fish or amphibian, and a bird. Here, we analyze the distribution, prevalence, intensity, abundance, and genetic diversity of encysting metacercariae of Clinostomum spp. in salamanders and fishes throughout an aquatic system that includes a natural Ozark stream and man-made ponds. We found Clinostomum sp. infecting permanently aquatic Oklahoma salamanders ( Eurycea tynerensis ; 56% prevalence) and larval grotto salamanders ( Eurycea spelaea ) immediately downstream from a man-made pond. However, Clinostomum sp. did not infect any salamanders in the spring that supplies this pond, or in sections farther downstream (~0.5 and 2 km). Metacercariae of Clinostomum sp. were present in ~90% of introduced largemouth bass ( Micropterus salmoides ) in the man-made pond adjunct to the stream. Morphological examination and phylogenetic analyses based on the mitochondrial gene cytochrome oxidase 1 ( Co1 ) and the nuclear ribosomal gene 18S show that fishes and salamanders at this site are primarily infected with Clinostomum marginatum . There is a relatively high degree of mitochondrial haplotype diversity in C. marginatum at this site but no consistent genetic difference between parasites in largemouth bass from the man-made pond and those in salamanders from the stream. Based on the microgeographic distribution and relationships of metacercariae of C. marginatum at this site, we hypothesize that the adjunct man-made pond has created an ecological situation that brings the cercariae of this parasite into contact with novel stream salamander hosts.

Electroeluting DNA fragments.

Purified DNA fragments are used for different purposes in Molecular Biology and they can be prepared by several procedures. Most of them require a previous electrophoresis of the DNA fragments in order to separate the band of interest. Then, this band is excised out from an agarose or acrylamide gel and purified by using either: binding and elution from glass or silica particles, DEAE-cellulose membranes, "crush and soak method", electroelution or very often expensive commercial purification kits. Thus, selecting a method will depend mostly of what is available in the laboratory. The electroelution procedure allows one to purify very clean DNA to be used in a large number of applications (sequencing, radiolabeling, enzymatic restriction, enzymatic modification, cloning etc). This procedure consists in placing DNA band-containing agarose or acrylamide slices into sample wells of the electroeluter, then applying current will make the DNA fragment to leave the agarose and thus be trapped in a cushion salt to be recovered later by ethanol precipitation.

Glycerol-3-phosphate acyltransferases gat1p and gat2p are microsomal phosphoproteins with differential contributions to polarized cell growth.

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial step in the synthesis of all glycerolipids. It is the committed and rate-limiting step and is redundant in Saccharomyces cerevisiae, mammals, and plants. GPAT controls the formation of lipid intermediates that serve not only as precursors of more-complex lipids but also as intracellular signaling molecules. Saccharomyces cerevisiae possesses two GPATs, encoded by the GAT1 and GAT2 genes. Metabolic analysis of yeast lacking either GAT1 or GAT2 indicated partitioning of the two main branches of phospholipid synthesis at the initial and rate-limiting GPAT step. We are particularly interested in identifying molecular determinants mediating lipid metabolic pathway partitioning; therefore, as a starting point, we have performed a detailed study of Gat1p and Gat2p cellular localization. We have compared Gat1p and Gat2p localization by fluorescence microscopy and subcellular fractionation using equilibrium density gradients. Our results indicate Gat1p and Gat2p overlap mostly in their localization and are in fact microsomal GPATs, localized to both perinuclear and cortical endoplasmic reticula in actively proliferating cells. A more detailed analysis suggests a differential enrichment of Gat1p and Gat2p in distinct ER fractions. Furthermore, overexpression of these enzymes in the absence of endogenous GPATs induces proliferation of distinct ER arrays, differentially affecting cortical ER morphology and polarized cell growth. In addition, our studies also uncovered a dynamic posttranslational regulation of Gat1p and Gat2p and a compensation mechanism through phosphorylation that responds to a cellular GPAT imbalance.