Complementation of un-16 and the development of a selectable marker for transformation of Neurospora crassa

Although nearly sixty temperature-sensitive lesions have been mapped in Neurospora crassa, most of their functions have not been identified. These loci are called unknown (un). As part of an effort to identify the open reading frame associated with one of these, we undertook to walk to un-16 using the complementation of temperature-sensitivity as a selection. Cosmids complementing un-16 were identified and the un-16 gene was subcloned. DNA sequence analysis of un-16 revealed that it encodes the highly conserved S9 protein of the 40S ribosomal subunit. This gene has proven useful as a selectable marker and may provide a simple mechanism for the controlled alteration of protein synthesis in N. crassa. Temperature-sensitive mutants can be very useful tools for various studies, because they allow temperature-controlled inactivation of essential cellular processes such as DNA, RNA and protein syntheses. An extensive set of temperature-sensitive N. crassa mutants is available from the Fungal Genetics Stock Center (FGSC); however, only a few of these mutants have been studied in depth (Perkins et. al., 2001). To enhance the usefulness of this potentially valuable collection of mutants, we have begun to clone and define the respective mutations in some of these strains. Direct selection of complementing cosmids offers a straight forward approach to identifying the underlying lesion associated with temperature-sensitive mutants in a number of organisms (for example, see Osherov et al, 2000). This is made simpler when the mutation being studied has been mapped on the genome. We have undertaken to complement temperature-sensitive mutants using cosmids mapped on the genome by the Neurospora genome program (Galagan et al. 2003)