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Biochemical Genetics (v.40, #11-12)
Genetic Selection for Modulators of the MAP Kinase and β-Catenin Growth-Control Pathways in Mammalian Cells by William Wheatley; Sanghee Yoo; Michael Pierce; Matthew Rebentisch; Mark Endo; Isaac Peterson; Mark Stump; Ken McCormack; Miguel Garcia-Guzman; Alexander Kamb (pp. 359-378).
Transdominant genetic selections can yield protein fragment and peptide modulators of specific biochemical pathways. In yeast, such screens have been highly successful in targeting the MAP (mitogen-activated protein) kinase growth-control pathway. We performed a similar type of selection aimed at recovery of modulators of the mammalian MAP kinase cascade. Two pathway activators were identified, fragments of the TrkB and Raf-1 kinases. In a second selection directed at the β-catenin growth-control pathway, three different clones encoding cadherin fragments were recovered. In neither selection were peptide inhibitors observed. We conclude that some transdominant selections in mammalian cells can readily yield high-penetrance protein fragments, but may be less amenable to isolation of peptide inhibitors.
Keywords: transdominant genetics; MAP kinase; β-catenin; peptide; Raf; TrkB; cadherin
Inorganic Pyrophosphatase: A New Polymorphic Allozyme Locus in Pacific Salmon by David R. Kuligowski; Gary A. Winans (pp. 379-385).
Genetic polymorphism of inorganic pyrophosphatase was investigated in 2799 individuals in four species of Pacific salmon: chinook salmon (Oncorhynchus tshawytscha), coho salmon (O. kisutch), kokanee (O. nerka), and steelhead(O. mykiss), using horizontal starch gel electrophoresis. This enzyme system appears to be an isolocus system with electrophoretically indistinguishable allozymes encoded by two loci (PP-1,2*) expressed in retinal tissue. Mendelian inheritance was observed with a breeding study in three family crosses. Population variability in four species was characterized in 44 populations from the U.S. Pacific coast. Three alleles were found in chinook salmon; two alleles each were found in coho salmon, kokanee, and steelhead. Chinook salmon and kokanee populations differed enough with respect to PP-1,2* frequencies that this isolocus is useful for genetic stock identification in these species.
Keywords: inorganic pyrophosphatase; genetic polymorphism; isolocus; electrophoresis; Pacific salmon
Genetic Polymorphism of Antithrombin III, Haptoglobin, and Haemopexin in Wild and Domestic European Rabbits by M. Branco; N. Ferrand (pp. 387-393).
Genetic polymorphism of European rabbit (Oryctolagus cuniculus) plasma proteins antithrombin III, haptoglobin, and haemopexin was investigated by means of isoelectric focusing in free and immobilized pH gradients followed by immunoblotting. The study of two wild and one domestic populations led to the recognition of six alleles of antithrombin III and haptoglobin, and five alleles of haemopexin.
Keywords: European rabbit; Oryctolagus cuniculus ; antithrombin III; haptoglobin; haemopexin; hybrid isoelectric focusing; genetic polymorphism
Examination of Ethanol Responsive Liver and Brain Specific Gene Expression, in the Mouse Strains with Variable Ethanol Preferences, Using cDNA Expression Arrays by Brenda C. Murphy; Tillie Chiu; Michelle Harrison; Raihan K. Uddin; Shiva M. Singh (pp. 395-410).
Strains of mice that differ in voluntary alcohol consumption (VAC) are valuable models for the identification of genes involved in the complex etiology of alcohol effects and alcoholism. These mice offer a novel approach to the identification of strain-specific ethanol responsive (SSER) genes in tissues directly involved in alcohol metabolism and preference. We assessed mRNA from the liver and brain from male mice representing C57BL/6J, BALB/c, A/J, and DBA/2J strains following ethanol treatment (chronic ethanol fed liquid diet for 14 days or acute i.p. injection at two doses; 4 g/kg or 8 g/kg), using an expression array containing 588 genes (Clontech #7741–1). The results have identified NADPH cytochrome P450 oxidoreductase, insulin-like growth factor binding protein-1, glutathione S-transferase Mu 1, and cathepsin L as ethanol responsive genes in the liver. Further, we have established that IkB-α and clusterin genes in the brain are ethanol responsive, but only at the lower dose of the ethanol challenge. Although a number of other genes showing subtle (<;2X) differences across strains and treatment combinations were reproducible in repeated blots, they were not confirmed by still evolving independent technologies of gene specific mRNA quantitation. The results demonstrate that comparative expression studies are an efficient approach to discover interacting gene networks that underlie the etiology of complex phenotypes including response to alcohols.
Keywords: acute/chronic ethanol; mouse; liver; brain; cDNA expression arrays
Molecular Analysis of the rDNA ITS-1 Intergenic Spacer in Drosophila mulleri, D. arizonae, and Their Hybrids by Milla Alves Baffi; Carlos Roberto Ceron (pp. 411-421).
We analyzed the ITS-1 spacer region of the rDNA in Drosophila mulleri and D. arizonae, two sibling species belonging to the mulleri complex (repleta group) and in hybrids obtained in both cross directions. In spite of several previous studies showing the incompatibility of crosses involving D. arizonae females and D. mulleri males, we were able to obtain hybrids in this direction. Complete ITS-1 region was amplified using primers with homology at the 3′-end of the 18S rDNA and the 5′-end of the 5.8S rDNA genes. Our data demonstrated that D. mulleri and D. arizonae can be differentiated as they present a difference in length for the ITS-1 region. The amplified fragment for this region in D. mulleri has a length of 600 bp, whereas in D. arizonae this fragment is about 500 bp. It was also observed that male and female hybrids obtained in both cross directions present two amplified fragments, confirming the location of the ribosomal cistrons in the X chromosomes and microchromosomes of both parental species.
Keywords: ribosomal DNA; ITS-1 spacer; Drosophila mulleri ; D. arizonae ; interspecific hybrids
Genetic Polymorphism of Apolipoprotein C Polypeptide in Native Asian Sheep by Kenji Tsunoda; Shoji Watanabe; Junichi Sato; Keizo Sato (pp. 423-432).
The frequency distributions of apolipoprotein C alleles in native Asian sheep (Bengal, Kagi, Lampuchhre, Vietnamese, Myanmar, Baruwal, Bhyanglung, Khalkhas, and Kuwait) were determined using a one-dimensional polyacrylamide gel isoelectric-focusing immunoblotting technique. Among these Asian sheep, the genetic locus consisted mainly of two common APOC*1 and APOC*3 alleles. The Baruwal sheep most frequently possessed APOC*1 (0.9881), showing significant differences from all other Asian sheep in allele frequency (0.7195–0.9032, P < 0.02). Compared with European sheep tested previously, these Asian sheep had significantly higher frequencies of APOC*1 (0.7828 and 0.7127 averages, P < 0.0005) and APOC*3 (0.2162 and 0.11 averages, P < 0.0001). In particular, APOC*2 was detected at an extremely low frequency (0.0025) in the Khalkhas sheep only, and was uncommon in all Asian sheep (0.001 average), in contrast with the 0.1773 average of European sheep (P < 0.00001). This finding suggests that APOC*2 is exceedingly rare in Asian sheep. With the exception of triglycerides and VLDL cholesterol in the Vietnamese sheep, no phenotypic effects on plasma lipid and lipoprotein levels in the Asian sheep tested were seen.
Keywords: apolipoprotein C; genetic polymorphism; isoelectric focusing immunoblotting; plasma lipoprotein; native Asian sheep