Nevertheless, the clear correlation we observe between the formation of a Ssa1p-PAp complex and the amelioration

of incompatibility-like phenotypes in yeast poses questions about whether Hsp70 proteins play a role in escape and in modulating incompatibility during the sexual cycle in N. crassa and other organisms. Finally, due to its essential function and evolutionary conserved structure, type I RNRs represent attractive drug targets. Indeed, the development of peptide inhibitors that disrupt the quaternary structure or activity of RNR is a field that may present a relatively safe and efficacious chemotherapeutic strategy [57]. Ironically, inherent within the N. crassa large subunit of RNR already lies the potential for a strain-specific antibiotic-like activity, as manifested by the growth inhibition of cells resulting from nonself fusions in N. crassa. The trans-species GF120918 inhibitory activity of PAp in yeast further suggests that the un-24 incompatibility domain may present insights into a broad-spectrum antimicrobial peptide that can be targeted to selected species or strains. Conclusions We have described a novel nonself recognition domain located in the C-terminus of UN-24. Our results GSK2118436 solubility dmso demonstrated that the PA, but not the OR, C-terminus

retains activity when expressed in S. cerevisiae. We demonstrate that low-level expression of PA(p) results in several incompatibility-like cytologies, an increase in cell size and the formation of a complex consisting of yeast Rnr1p and PA(p). These phenomena are resolved when PA(p) is expressed at high level, where an apparent complex between Ssa1p and PA(p) forms. Results from our study indicate that yeast can be used to investigate nonself recognition systems. Furthermore, our study shows that Hsp70 proteins can alleviate incompatibility, which may suggest their involvement in the escape process or in the sexual cycle of N. crassa. Finally, given the unique Chloroambucil trans-species activity of the PA(p) protein, and the ubiquitous

and evolutionarily well conserved target, RNR, it would be interesting to determine if variations of this protein have applications as chemotherapeutic agents. Methods Manipulation of N. crassa strains and molecular genetic methods The N. crassa strains used [with Oakridge (OR) alleles at all undesignated het loci] were: C2(2)-1 (un-24 PA het-6 PA thr-2 a) and C9-2 (un-24 OR het-6 OR het-c PA thr-2 a). DNA cloning was done with plasmids pCB1004 [58], which contains the hygromycin phosphotransferase (hph) selectable marker conferring hygromycin resistance, and pCR2.1 (Invitrogen, Carlsbad, CA). PCR reactions were performed with Taq DNA polymerase (New England Biolabs, Mississauga, ON) or iProof DNA polymerase (BioRad, Mississauga, ON) according to the manufacturer’s recommendations. Oligonucleotide primer sequences are available upon request. All constructs were 4SC-202 in vitro sequenced and verified as error-free.