False and true positives in arthropod thermal adaptation candidate gene lists

Genetica - Genome-wide studies are prone to false positives due to inherently low priors and statistical power. One approach to ameliorate this problem is to seek validation of reported candidate...     

Purification and characterization of rat liver glutaminase

Phosphate-dependent glutaminase (EC 3.5.1.2) from livers of starved rats was purified about 400-fold to near homogeneity. The specific activity of the final pool was more than 30 U/mg protein. For the rapid quantification of the enzyme activity a simple and sensitive assay, based on the determination of the produced ammonia with an o-phthalaldehyde reagent, was developed which avoids massive dilution of the samples. The enzyme preparation involved extraction of the enzyme from sonified isolated mitochondria after treatment with a brief hypotonic shock followed by ammonium sulphate precipitation, ion-exchange and hydroxyapatite chromatography. A major improvement was the stabilization of the enzyme by chymostatin protecting it from degradation by a protease of presumably lysosomal origin. In the presence of chymostatin or leupeptin the half-life of glutaminase in a crude mitochondrial preparation subsequent to mild treatment with digitonin could be increased to more than 200 h. The relative molecular mass of the protein (Mr 170,500) was estimated by sucrose gradient ultracentrifugation. The molecular mass of the subunits (Mr 57,000) was determined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. These results suggest a protein composed of three subunits of identical molecular mass. The molecular data clearly differentiate liver glutaminase from the phosphate-dependent glutaminase present in kidney.     

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Molecular markers residing close to the Rhg4 locus conferring resistance to soybean cyst nematode race 3 on linkage group A of soybean

 The restriction fragment length polymorphism (RFLP) clone pBLT65 is a 450-nt soybean cDNA encoding a portion of the bifunctional enzyme aspartokinase-homoserine dehydrogenase (AK-HSDH). pBLT65 maps within 3.5 cM of the i locus, conferring a pigmented seed coat, on linkage group A; hence, it is closely linked to the Rhg ₄ locus conferring resistance to race 3 of the soybean cyst nematode. From this useful RFLP we developed a PCR reaction yielding polymorphic bands for use in marker-assisted breeding programs to select progeny containing the Rhg ₄ allele. The polymorphic bands were sequenced to determine the cause of the polymorphisms. Using primers 548 and 563, PCR amplification of DNA from the soybean cultivar Peking (Rhg ₄ ) yielded three DNA fragments, 1a (1160 bp), 1b (1146 bp) and 3 (996 bp). Amplification of DNA from the cultivar Kent (rhg ₄) yielded DNA fragments 2 (1020 bp), 3 (996 bp) and 4 (960 bp). Fragments 1a, 1b, 2 and 4 were also polymorphic between the soybean lines PI 290136 and BARC-2(Rj ₄ ). A segregating population of 80 F₂ and F₃ plants derived from the cross PI 290136×BARC-2 (Rj ₄ ) was used to confirm the map position of the PCR polymorphisms near the i locus, and hence the Rhg ₄ locus on linkage group A. The nucleotide sequences of fragments 1b, 3 and 4 were determined. Large and small deletions in the intronic region were responsible for the size differences of the different fragments, whereas the exon was well conserved. Received: 8 January 1998 / Accepted: 15 July 1998