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

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Title:Cloning of the pmb gene encoding a basic amino acid transport protein in Neurospora crassa
Authors:Han, Hyo-Young
Date Issued:1993
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.
Description:Thesis (Ph. D.)--University of Hawaii at Manoa, 1993.
x, 140 leaves, bound ill. 29 cm
Rights:All UHM dissertations and theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission from the copyright owner.
Appears in Collections: Ph.D. - Biomedical Sciences

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