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KMID : 0578319950050050442
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Molecules and Cells
1995 Volume.5 No. 5 p.442 ~ p.447
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Sib-selection and Restriction Fragment Length Polymorphisms for Cloning the pmb Gene in Neurospora crassa
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Han, Hyo-Young
Lee, Myeong-sok/Min, Jyung-Hee
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Abstract
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The pmb gene encoding a basic amino acid transport protein in Neurospora crassa has been cloned by using a mutant strain defective in the pmb gene as a host strain. 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 constructed by crossing a strain defective in basic amino acid transport system with a double mutant strain defective in neutral amino acid transport and histidine production. The protoplasts of the pmn:pmb:his-2 strain were prepared and transformed with DNA fragments from genomic library DNA pools of wild type N. crassa by sib-selection. One cosmid (pMOcosX-X7:5E) complementing the pmb mutant allele was selected by means of positive selection and confirmed to be the pmb^(+) gene by restriction fragment length polymorphism (RFLP) procedures, where the pMOcosX-X7:5E was tightly linked to RFLP markers flanking the pmb locus on linkage group Ⅳ. The DNA of pMOcosX-X7:5E was amplified and used for the restriction mapping, for the production of deletion cloning sets, and for sequencing the pmb gene.
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