Cloning of the pmb gene encoding a basic amino acid transport protein in Neurospora crassa

Abstract

Neurospora crassa is known to possess at least six genetically and biochemically distinct amino acid transport systems; three constitutive amino acid permeases and three specialized amino acid permeases. The constitutive permeases include a neutral amino acid-specific system (N system), a basic amino acid-specific system (B system), and a general system (G system) that can transport neutral, basic, and acidic amino acids. We have cloned the pmb gene which codes for a component of the B system. A Neurospora crassa triple mutant (pmn:pmb:his-2 ) was constructed from previously described mutants. This strain is suitable for use as a host strain in a transformation procedure for selecting DNA from the wild type pmb locus. Protoplasts of the pmn:pmb:his-2 strain were prepared and transformed by sib selection with DNA fragments from cosmid library pools of wild type Neurospora crassa. One cosmid (pMOcosX-X7:5E) complemented the pmb mutant allele and was confirmed to be tightly linked to RFLP (Restriction Fragment Length Polymorphism) markers flanking the pmb locus on linkage group IV. A subclone of the pMOcosX-X7:5E cosmid, pB22-22 (3.2Kb), complemented the pmb mutant allele and was mapped with restriction enzymes. The subclone hybridizes to RNA transcripts 3.5Kb in size on Northern blot experiments. The subclone pB22-22 has been sequenced and the pmb portion of the sequence containing a promoter initiation site has been analyzed; it has two CAAT motifs at -166 and -155 when positions are given in base pairs (bp) from the A(+1) of the proposed translational start: a TATA motif is located at -123. There are two CT-rich regions; one is from -117 to -106 (l2bp length) and the other is from -50 to -23 (28bp length) containing 83% of and 75% of pyrimidine bases each. The promoter site of the pmb locus contains sequences for binding motifs of CPC-l (cross-pathway control protein) at upstream and downstream of the translation start point. This suggests that the pmb gene is regulated by the cpc-1 gene.