Analysis of the DNA sequence of the putative ABC transporter NCU09975 in Neurospora crassa strains carrying acriflavin resistance markers.

Abstract

Genomic DNA sequence was determined for the putative Neurospora crassa ABC transporter NCU09975 from several different classical mutant strains including several acriflavin resistant mutants. The sensitivity of these strains to acriflavin was tested. While the open reading frame NCU09975 has multiple polymorphisms in strains sequenced for other purposes, none of the acriflavin resistant classical mutants tested had polymorphisms in the NCU09975 coding region or in the 195 bases upstream of the translation start site. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This regular paper is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol59/iss1/4 Fungal Genetics Reports 59, 2012. 26 Analysis of the DNA sequence of the putative ABC transporter NCU09975 in Neurospora crassa strains carrying acriflavin resistance markers. Aric E. Wiest, Sarah Koch and Kevin McCluskey. Fungal Genetics Stock Center, University of MissouriKansas City, Kansas City, MO 94110 USA Fungal Genet Reports 59: 26 29 Genomic DNA sequence was determined for the putative Neurospora crassa ABC transporter NCU09975 from several different classical mutant strains including several acriflavin resistant mutants. The sensitivity of these strains to acriflavin was tested. While the open reading frame NCU09975 has multiple polymorphisms in strains sequenced for other purposes, none of the acriflavin resistant classical mutants tested had polymorphisms in the NCU09975 coding region or in the 195 bases upstream of the translation start site. Introduction While years of mutagenesis and subsequent characterization in Neurospora crassa produced one of the most densely saturated genetic maps (Perkins et al. 2001), many of the classical mutants are not yet associated with open reading frames from the genome sequence (Galagan et al. 2003). Among these are acriflavin resistant mutants which have been assigned to seven different loci. A subset of these genes also confer resistance to multiple related drugs, including acridine orange, malachite green, and aminotriazole (Akiyama and Nakashima 1996). Moreover some acriflavin resistance genes are dominant while others are recessive. Because we wanted to find a dominant selectable marker for transformation of Neurospora and because the abc-3-like ORF NCU09975 had a large number of polymorphisms in its primary sequence among a group strains subject to whole genome sequence (McCluskey et al. 2011) we undertook to characterize this locus in strains carrying the acriflavin resistance markers acr-1 and Acr-3. We additionally validated the acriflavin sensitivity in the classical mutant strains carrying polymorphisms in NCU09975. Materials and methods Strains were cultured on Vogel's minimal medium (Vogel 1956) using standard practices (Davis and De Serres 1970) (Table 1). Table 1. Strains and their characteristics FGSC number Genotype Marker Location Acriflavin sensitivity 2489 mat-A IL Sensitive 1215 Acr-3 mat-a IL IL Resistant 1209 Acr-3 mat-A IL IL Resistant 875 acr-1 mat-a IL IL Sensitive 305 amyc mat-A IL IL Sensitive 1363 smco-1 mat-A I IL Sensitive 3921 tng mat-A IIL; IL Sensitive 7022 fld IVR; IL Sensitive Published by New Prairie Press, 2017 Fungal Genetics Reports 59, 2012. 27 Acriflavin was dissolved in water and filter sterilized prior to addition to sterile culture medium. It was stored in the dark and fresh stocks were prepared regularly. Acriflavin sensitivity testing was carried out by pipetting 10 ul of a freshly prepared suspension of conidia and hyphal fragments in water (approximately 10 3 cfu/ ml) onto the surface of agar solidified medium in 10 x 75 mm glass culture tubes. Results were logged after two, four and ten days. Genomic DNA was extracted from 2 3 day old liquid shake cultures using the ZR Fungal DNA kit (Zymo Research, Irvine, CA). NCU09975 DNA was amplified as shown in Figure 1, using primers specific for this ORF (Table 2). Primers 1F and 5R were used together to amplify a 2848 base fragment including the start codon and 195 upstream bases. Primers 7F and 11R were used to amplify a 2878 base fragment including the stop codon and 667 bases downstream (Figure 1). These large fragments which overlap by 370 bases were directly sequenced at the UMKC School of Biological Sciences core facility using an Applied Biosystems 3100 Genetic Analyzer (Foster City, USA). Sequences were aligned and analyzed using Sequencher (Gene Codes Corporation, Ann Arbor, USA).