Title

Isolation and Characterization of the P-glycoprotein Gene From the Dimorphic Fungus Mucor racemosus

Date of Award

2004

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Glenn Shearer

Advisor Department

Biological Sciences

Abstract

Genomic clones for the P-Glycoprotein gene were isolated from the dimorphic zygomycete Mucor racemosus (Mr ). One of the clones, A6.4, was selected for further study. The A6.4 clone insert was subcloned into a pGEM 11 vector. The pGEM 11 insert was sequenced. Sequencing resulted in a 7.3 Kb DNA fragment. Analysis of the fragment revealed a putative open reading frame of 5.3 Kb with 6 exons that ranged in length from 142 bp to 1226 bp, and 5 introns that ranged in length from 54 bp to 66 bp. The predicted amino acid sequence for this gene shares a high degree of homology with several other P-Glycoprotein (PG) genes in other eukaryotes including over 40% conserved amino acids with the human multidrug resistance gene. The putative coding sequence of the Mucor P-Glycoprotein (MPG) is interrupted by five introns possessing GT/AG splice junctions consistent with splice junctions and internal consensus sequences found in other filamentous fungi. The putative MPG further includes the highly conserved Walker A and B motifs, as well and the signature motif. All of these motifs have been implicated in ATP interaction. Primer extension analysis revealed a transcriptional start site 27 bases upstream of the putative translation initiation codon. Analysis of the 5 ' - and 3' flanking regions of the MPG revealed several putative regulatory elements and polyadenylation sites respectively. Restriction analysis of the Mr genomic DNA has shown the MPG gene to be present in the Mr genome in single copy. Quantitative PCR demonstrated that the MPG gene was upregulated approximately 3 fold after growth inhibition induced by Cycloheximide (CHX), Trichodermin (TDM), Amphotericine B (AMB) or leucine starvation.