TABLE 1

palC mutations isolated in this work

AllelePhenotype pH 8.0Nucleotide change(s)Change in proteinMutant protein
palC114(1)tsT43A,T44GF15SF15S
palC86(2)tsT44CF15SF15S
palC103(2)tsT61CS21PS21P
palC185(1)A193insCGTTCTCTCCGCAI65fs1–64 + 5
palC146(1)+/−−T194A,T195AI65KI65K
palC95(2)++/−T202CY68HY68H
palC158(1)+/−−T202AY68NY68N
palC98(2)tsG301A,T302GV101RV101R
palC111(1)C306T,C307TQ103stop1–102
palC155(1)tsΔT320–G322L107Q,ΔE108L107Q,ΔE108
palC93(2)tsT331CW111RW111R
palC83(2)tsT419AI140KI140K
palC127(1)T438AY146stop1–145
palC134(1)C461AS154stop1–153
palC151(1)ΔC461S154stop1–153
palC97(2)+/−T481G,A482T,T483GY161VY161V
palC99(2)a+/−T481GY161DY161D
palC91(2)+/−A566TH189LH189L
palC181(1)+T567GH189QH189Q
palC100(2)a+/−T567AH189QH189Q
palC149(1)C570G,G571AS190stop1–189
palC178(1)C581GS194stop1–193
palC141(1)ΔA588–A600P196fs1–196 + 22
palC164(1)a+/−T653G,A654GL218RL218R
palC143(1)T668GL223stop1–222
palC129(1)ΔA701D234fs1–233 + 51
palC82(2)atsG703AD235ND235N
palC81(1)tsG703A,G718AD235N,A240TD235N,A240T
palC92(2)+/−A728CQ243PQ243P
palC148(1)ΔT747D249fs1–248 + 36
palC150(1)A748G,G755A,G756AK250E,W252stopK250E,1–251
palC85(2)+/−G755CW252SW252S
palC94(2)+/−G755CW252SW252S
palC84(2)tsT813GC271WC271W
palC88(2)tsA945TR315SR315S
palC80(1)G961AG321DG321D
palC144(1)ΔG984G328fs1–328 + 38
palC116(1)ΔT1093S365fs1–364 + 2
palC160(1)ΔC1114R372fs1–371 + 8
palC117(1)A1135TK379stop1–378
palC177(1)G1159TE387stop1–386
palC104(1)T1181GL394stop1–393
palC96(1)aT1181AL394stop1–393
palC89(2)+A1193TD398VD398V
palC112(1)C1204TR402stop1–401
palC153(1)T1336GL427stop1–426
palC179(1)T1336AL427stop1–426
palC159(1)A1337insGAI428fs1–427 + 29
palC90(2)+/−C1371T,C1372TP439FP439F
palC107(1)+/−C1371T,C1372TP439FP439F
palC162(1)ΔG1378–C1380ΔR442ΔR442
palC113(1)+/−G1381AR442HR442H
palC87(2)+/−−T1407AY451NY451N
palC131(1)+/−T1419insTTS455fs1–454 + 2
palC102(2)+/−T1423GL456RL456R
  • Novel palC mutations were selected after UV mutagenesis of diploid R pabaA1 yA2 areAr18/biA1 areAr3 palC40 inoB2 fwA1 (see Clutterbuck 1993 for gene symbols) on minimal medium (Cove 1966) pH 6.5 with 1% glucose and 5 mm GABA. Mutants were screened for reduced growth on pH 8 medium (Cove 1976) to distinguish acidity-mimicking mutants. Acidity-mimicking mutant diploids were haploidized on benlate-containing Aspergillus complete medium (Cove 1966; Hastie 1970) lacking inositol and with NaH2PO4 added to 1 m. Haploid isolates were phenotype tested and categorized by their growth on pH 8 medium (Cove 1976), which is the most sensitive test to distinguish the relative leakiness of mutations. −, complete loss-of-function, virtually no growth at 25° or 37°; −ts, some growth at 25° but not at 37°; +/−−, +/−, and ++/−, increasing amounts of growth at 37°; +, similar to wild type on pH 8 medium yet acidity mimicking by more stringent criteria such as molybdate hypersensitivity (Caddick et al. 1986). All missense mutations except palC80 permitted significant growth at pH 8 at 25°. The subscript after the allele number refers to the experiment in which the mutation was isolated (see text). The difference in phenotype between the haploid palC181 (His189Gln) mutant and the aneuploid palC100 (His189Gln), “+” vs. “+/−,” respectively, suggests that the mutant phenotype of a new mutation in haploidy might be less pronounced than that in aneuploidy with palC40. fs, frameshift at the indicated codon; + N, where N is a number, indicates the number of frameshifted amino acid residues. Due to the large number of mutations selected, a relatively small number of strains were analyzed in some cases and the possibility cannot be ruled out that the phenotype might be affected by a modifying mutation, although an unexpected phenotype led to analysis in a cross. Naked DNA or DNA from conidiospores from haploid isolates was used as template for PCR amplification using palC-specific primers. Mutations were detected by the direct sequencing of PCR products. The entire coding region was sequenced for all new mutant alleles. Nucleotide numbers refer to the published sequence (Negrete-Urtasun et al. 1999).

  • a The sequenced strain is probably aneuploid and carried palC40 in addition to the new palC mutation.