Gene mapping of a mouse coat mutation has been investigated. First, 100 10-bp random primers were used to amplify DNA, but the mutation could not be located by this method because there were no correlation between the...Gene mapping of a mouse coat mutation has been investigated. First, 100 10-bp random primers were used to amplify DNA, but the mutation could not be located by this method because there were no correlation between the amplified products and coat phenotypes. Second, by using ldh1 , Car2, Mup1 , Pgb1 , Hbb, Es1O, Es1, Mod1, Gdc1 , Ce2, Es3 as genetic markers, linkage test crosses (two-point test) consisting of intercrossing uncovered BALB/c mice (homozygotes) to CBA/N and C57BL/6 mice with normal hair and backcrossing the heterozygotes of the F1 to the uncovered BALB/c mice were made. It was soon evident that the mutation was linked to Es3 on chromosome 11. Furthermore, three-point test was made by using Es3 and D11 Mit8 (a microsatellite DNA) as genetic markers. The result showed that the mutation was linked to Es3 with the percentage recombination of (7.89 ± 2.19) %, and linked to D11Mit8 with the percentage recombination of (26.30 ± 3.57)%. The percentage recombination between Es3 and D11Mit8 was展开更多
Of ICE protease family, CPP32 (apopain or Yama) plays a central role in different apoptotic pathways. To study the molecules regulating CPP32, yeast two_hybrid system was used to identify proteins (peptides) that inte...Of ICE protease family, CPP32 (apopain or Yama) plays a central role in different apoptotic pathways. To study the molecules regulating CPP32, yeast two_hybrid system was used to identify proteins (peptides) that interact with CPP32. First, the CPP32 gene was cloned into plasmid vector pGBT9. The resulting recombinant plasmid was designated as pGBT9/CPP. The pGBT9 /CPP plasmid was transformed into the yeast strain HF7C, then the leukemia library was introduced. The transformation mixture was plated on medium lacking Trp, Leu and His in the initial screen. Colonies growing on the selection medium were further assayed for β galactosidase activity. Within 42 Trp +Leu +His + colonies only 5 turned blue in the presence of X_Gal. Plasmid DNA from 5 positive yeast colonies was prepared respectively and used to transform HB101 by electroporation. The transformation mixture was plated on medium lacking Leu to be selected for the library plasmid. Finally, only one library plasmid, designated as pY1, was determined to be truly positive by retransformation of pGBT9/CPP and the library plasmid into HF7C. The inserted cDNA of pY1 encodes a peptide of 15 amino acids, suggesting that it may be the domain interacting with CPP32.展开更多
文摘Gene mapping of a mouse coat mutation has been investigated. First, 100 10-bp random primers were used to amplify DNA, but the mutation could not be located by this method because there were no correlation between the amplified products and coat phenotypes. Second, by using ldh1 , Car2, Mup1 , Pgb1 , Hbb, Es1O, Es1, Mod1, Gdc1 , Ce2, Es3 as genetic markers, linkage test crosses (two-point test) consisting of intercrossing uncovered BALB/c mice (homozygotes) to CBA/N and C57BL/6 mice with normal hair and backcrossing the heterozygotes of the F1 to the uncovered BALB/c mice were made. It was soon evident that the mutation was linked to Es3 on chromosome 11. Furthermore, three-point test was made by using Es3 and D11 Mit8 (a microsatellite DNA) as genetic markers. The result showed that the mutation was linked to Es3 with the percentage recombination of (7.89 ± 2.19) %, and linked to D11Mit8 with the percentage recombination of (26.30 ± 3.57)%. The percentage recombination between Es3 and D11Mit8 was
文摘Of ICE protease family, CPP32 (apopain or Yama) plays a central role in different apoptotic pathways. To study the molecules regulating CPP32, yeast two_hybrid system was used to identify proteins (peptides) that interact with CPP32. First, the CPP32 gene was cloned into plasmid vector pGBT9. The resulting recombinant plasmid was designated as pGBT9/CPP. The pGBT9 /CPP plasmid was transformed into the yeast strain HF7C, then the leukemia library was introduced. The transformation mixture was plated on medium lacking Trp, Leu and His in the initial screen. Colonies growing on the selection medium were further assayed for β galactosidase activity. Within 42 Trp +Leu +His + colonies only 5 turned blue in the presence of X_Gal. Plasmid DNA from 5 positive yeast colonies was prepared respectively and used to transform HB101 by electroporation. The transformation mixture was plated on medium lacking Leu to be selected for the library plasmid. Finally, only one library plasmid, designated as pY1, was determined to be truly positive by retransformation of pGBT9/CPP and the library plasmid into HF7C. The inserted cDNA of pY1 encodes a peptide of 15 amino acids, suggesting that it may be the domain interacting with CPP32.
