A cold-sensitive mutant of Neurospora crassa obtained using tritium-suicide enrichment that is conditionally defective in the biosynthesis of cytoplasmic ribosomes

Abstract

The purpose of this investigation was to use tritium-suicide enrichment with a mutagenized population of wild-type Neurospora crassa to isolate cold-sensitive mutants with conditional defects in the production of cytoplasmic ribosomes. Eighty-six cold-sensitive mutant strains were obtained following tritium-suicide enrichment using [5- 3H]uridine. Zone sedimentation analysis of cytoplasmic ribosomes produced by the strains at 10°C (the nonpermissive temperature) indicated that one strain, PJ31562, is defective in the accumulation of ribosomal subunits at that temperature. The properties of strain PJ31562 are: (1) At 10°C the growth rate is 28 times slower than at 25°C, whereas the factor for the wild type is 5.1. At 25°C the mutant's growth rate is 90% that of the wild type. (2) At 10°C the mutant accumulates the two ribosomal subunits, 60 and 37 S, in markedly disproportionate amounts apparently as a result of the underproduction of, or an instability of, the 17 S ribosomal RNA component of the small ribosomal subunit. At 25°C the mutant strain still exhibits a disproportionality in ribosomal subunit accumulation but to a much lesser degree than at 10°C. (3) Genetic studies have shown that a single nuclear gene is responsible for both the cold sensitivity and ribosome biosynthesis defect of strain PJ31562. The mutation involved is located in linkage group IV and appears to be closely linked to, and not allelic with, the cold-sensitive mutation carried by strain PJ30201 which has been shown previously to exhibit a similar phenotype with respect to ribosomal subunit accumulation, and which defines the crib-1 locus. Thus tritium-suicide enrichment can be used to isolate cold-sensitive mutants of Neurospora among which a relatively low frequency have conditional defects in ribosome production.