| Title | Construction of Multiple Mutant Strains by Mating Procedures for the Cloning of pmn and pmb Genes Encoding Amino Acid Permeases in Neurospora crassa |
|---|---|
| Author | Han, Hyo Young * · Min, Kyung Hee¹ |
| Address | Department of Biologym Sookmyung Women's University; ¹Research Center for Molecular Microbiology, Institute for Molecular Biology and Genetics, Seoul National University |
| Bibliography | Journal of Microbiology, 33(2),142-145, 1995, |
| DOI | |
| Key Words | mating, pmn:pmb:his-2, para-fluoro-phenylalanine, canavanine, Neurospora crassa, positive selection |
| Abstract | The pmb gene encoding a basic amino acid transport protein in Neurospora crassa could be cloned by using a mutant strain defective in pmb gene as a host strain, using a negative selection on the media containing amino acid analogue canavanine. To select positive transformants of the genes for cloning, an auxotrophic marker (his-2) was added to a pmb mutant strain by mating ; a triple mutant (pmn : pmb : his-2) was constructued by crossing a strain defective in basic amino acid transport system (# 1683-bat um 535 "A") to a double mutant strain defective in neutral amino acid transport and histidine production (mitr6r : his-2 "a"). Crossing was performed on synthetic crossing (SC) media containing histidine. The pmn : pmb and pmn :pmb : his-2 strains were selected among the progeny colonies from crosses on plates containing 50㎍/㎖ para-fluoro-phenylalanine (PFPA), 200㎍/㎖ canavanine, and 500㎍/㎖ histidine. The selected colonies were cultured on minimal media with or without histidine for discarding pmn : pmb strain, because the pmn : pmb : his -2 strain grows only on histidine containing media. The pmn :pmb : his-2 strain selected can be used as a host strain for the cloning of the pmb and the pmn genes from a Neurospora genomic library by means of positive selections. |
| Download PDF | Eng_330210_142-145p.pdf |