A ras-1 bd Mauriceville strain for mapping mutations in Oak Ridge ras-1 bd strains

Abstract

We describe the construction of a Neurospora crassa Mauriceville strain carrying the ras-1bd mutation marked by the bacterial hygromycin resistance gene, hph (new FGSC # 10156). This strain is valuable for mapping mutations in Oak Ridge strains that carry the bd mutation. 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/vol53/iss1/9 30 Fungal Genetics Newsletter A ras-1 Mauriceville strain for mapping mutations in Oak Ridge ras-1 strains bd bd A. Kaitlyn Beasley*, Teresa M. Lamb*, Wayne K. Versaw and Deborah Bell-Pedersen* *Center for Biological Clocks Research and Department of Biology, Texas A&M University, College Station, TX 77843 Fungal Genetics Newsletter 53:30-33 We describe the construction of a Neurospora crassa Mauriceville strain carrying the ras-1 mutation marked by the bacterial bd hygromycin resistance gene, hph (new FGSC # 10156). This strain is valuable for mapping mutations in Oak Ridge strains that carry the bd mutation. The bd mutation has been a benefit to circadian rhythm research as it slows down wild type growth rates and sharpens the conidial banding pattern on racetubes (Sargent et al., 1966; Bell-Pedersen et al., 2005). Recent work (Belden et al., 2006) has shown that the bd mutation lies in the ras-1 gene. Mutations that affect circadian banding patterns are typically isolated in strains carrying the ras-1 allele. Mapping such mutations by the CAPS method (Jin et al., in press) requires that the mutant phenotype be followed in bd a cross to a wild type Mauriceville strain. Assuming non-linkage, only half of the progeny will be bd, and thus readily scorable. Insertion of the ras-1 allele into the Mauriceville parent renders all progeny useful for segregation analysis. Furthermore, bd marking ras-1 with the hygromycin resistance gene, hph, permits a simple drug resistance test to determine linkage to ras-1. bd The strategy for gene replacement was similar to published split marker gene deletion strategies (Catlett et al., 2003; Colot et al., 2006). However, rather than deleting the wild type gene, we replaced it with the bd allele and inserted the hph gene in the 3'UTR at position + 225 from the stop codon (Figure 1). Figure 1. Schematic diagram of ras-1 genetic manipulations. Regions of homology are indicated by vertical hatch marks; there are 763 bp of homology between the split hph fragments, 3223 bp of homology at the 5' end, and 2582 bp of homology at the 3' end. Pvu II and Xho I sites are marked to show the differences between the wild type (wt) genomic (g) locus and the proper ras1 -hph integrant. The dashed line indicates a gap where the hph gene is integrated and the dotted lines indicate flanking genomic bd DNA that remains unchanged. The replacement fragments were generated in two steps. The first step involved amplification of ras-1 genomic regions from the bd Oak Ridge bd strain (FGSC 1858). The 5' region was amplified using primers ras1 F1 and ras1-hyg R1, while the 3' region was amplified using primers hyg-ras1 F2 and ras1 R2. The second step was to generate hybrid pieces that contained a portion of the bacterial hph gene fused to either 5' or 3' regions of ras-1 (Figure 1). The 5' split marker fragment was generated by amplification bd of the 5' region of ras-1 and the complete hph gene, with primers ras1 F1 and YG2. The 3' split marker fragment was generated bd by amplification of the 3' region of ras-1 and the complete hph gene, with primers HY2 and ras1 R2. The complete hph gene bd was obtained by amplification of pBP15 (a gift from D. Ebbole) with primers M13F2 and M13R2. The pBP15 plasmid contains a 1.4kb HpaI PtrpC-hph fragment from pCB1003 (Carroll et al., 1994) subcloned into the EcoRV site of pBluescript II SK Published by New Prairie Press, 2017