Neurospora crassa mutant hunts by various groups have identified albino (al) mutants that map to the al-1 - al-2 region on the right arm of linkage group I. The cloning of the al-1 and al-2 genes (Schmidhauser et al. 1990 Mol. Cell. Biol. 10:5064-5070, Schmidhauser et al. 1994 J. Biol. Chem. 269:12060-12066) allows assignment of locus to the above mutants by DNA mediated transformation. Analysis of phytoene desaturases from different organisms indicates at least three types of enzymes as defined by the number of desaturation steps carried out (Sandmann 1994 J. Plant Physiol. 143:444-447). The N. crassa phytoene desaturase, the al-1 gene product, introduces four double bonds converting phytoene to lycopene. Of the three intermediates in this reaction sequence two are colored. The occurrence of visibly distinguishable albino alleles in N. crassa has been noted (Perkins 1989 Fungal Genetics Newsl. 36:63). Assignment of locus to N. crassa albino alleles represents a first step in the functional characterization of N. crassa carotenogenic loci.
Our aim was a rapid assignment of albino mutations to the al-1 or al- 2 locus. Albino mutant strains used in this study were obtained from the Fungal Genetics Stock Center. DNA-mediated transformations were performed that should identify albino mutations as alleles of al-1, al-2 or unidentified loci. Competent spheroplasts were prepared according to the protocol of Royer and Yamashiro (1992 Fungal Genetics Newsl. 39:76-79). Spheroplasts were transformed with plasmid pSV50 DNA and either an al-1+ plasmid DNA, pTJS342, or an al-2+ plasmid DNA, pTJS542. Transformants were selected on Vogel s FGS media supplemented with benomyl at a final concentration of 1.5 µg/ml. Transformants that broke the surface of the agar were scored as white, indicating no albino complementation; or yellow or orange, indicating albino complementation. For all twenty strains tested complementation was observed following co-transformation with DNA containing a specific functional albino gene (Table 1).
Table 1. Assignment of locus to N. crassa albino alleles
Allele and Assigned mating type FGSC # locus 85201 a 381 al-1 7-32 A 912 al-1 1500-08 A 1138 al-1 1500-09 A 1139 al-1 1500-010 A 1140 al-1 1500-011 A 1141 al-1 1500-012 A 1142 al-1 1500-013 A 1143 al-1 JH9698 a 801 al-1 RES6 A 2152 al-1 Y256M231 A 909 al-1 Y602 a 797 al-1 Y2170 a 796 al-1 Y2171 a 795 al-1 alC a 800 al-1 alM a 798 al-1 alS A 827 al-2 B102 a 799 al-2 CN1 a 1107 al-2 RES100SUE A 2154 al-2
DNA-mediated transformation of albino mutants provides an easy and specific assay for locus. Genetic crossing is more time consuming due, in part, to the close proximity of the al-1 and al-2 loci. Sixteen strains or 80% of those tested in this study are alleles of al-1. The FGSC has 30 albino mutants in which the mutation had previously been assigned to locus. Among these 30 albino mutants 18 are al-1 alleles (including the former age-3) and 12 are al-2 alleles. The al-1 and al-2 transcription units are approximately equal in size. al-2 mutants accumulate steroids while al-1 mutants accumulate the colorless carotenoid phytoene (Goldie and Subden 1973 Biochem. Genet. 10:275-284). However, no difference in viability between different albino mutants and wild-type strains has been noted. Goldie and Subden (1973, ref. cit.) found that the RES6 strain accumulates phytoene suggesting a defect in al-1. Our results confirm their observation.
Chad Walter and Harn-Cherng Shiue participated in this work, which was supported by the National Institutes of Health (T.J.S.)