TABLE 1.
palC mutations isolated in this work
| Allele | Phenotype pH 8.0 | Nucleotide change(s) | Change in protein | Mutant protein |
|---|---|---|---|---|
| palC114(1) | −ts | T43A,T44G | F15S | F15S |
| palC86(2) | −ts | T44C | F15S | F15S |
| palC103(2) | −ts | T61C | S21P | S21P |
| palC185(1) | − | A193insCGTTCTCTCCGCA | I65fs | 1–64 + 5 |
| palC146(1) | +/−− | T194A,T195A | I65K | I65K |
| palC95(2) | ++/− | T202C | Y68H | Y68H |
| palC158(1) | +/−− | T202A | Y68N | Y68N |
| palC98(2) | −ts | G301A,T302G | V101R | V101R |
| palC111(1) | − | C306T,C307T | Q103stop | 1–102 |
| palC155(1) | −ts | ΔT320–G322 | L107Q,ΔE108 | L107Q,ΔE108 |
| palC93(2) | −ts | T331C | W111R | W111R |
| palC83(2) | −ts | T419A | I140K | I140K |
| palC127(1) | − | T438A | Y146stop | 1–145 |
| palC134(1) | − | C461A | S154stop | 1–153 |
| palC151(1) | − | ΔC461 | S154stop | 1–153 |
| palC97(2) | +/− | T481G,A482T,T483G | Y161V | Y161V |
| palC99(2)a | +/− | T481G | Y161D | Y161D |
| palC91(2) | +/− | A566T | H189L | H189L |
| palC181(1) | + | T567G | H189Q | H189Q |
| palC100(2)a | +/− | T567A | H189Q | H189Q |
| palC149(1) | − | C570G,G571A | S190stop | 1–189 |
| palC178(1) | − | C581G | S194stop | 1–193 |
| palC141(1) | − | ΔA588–A600 | P196fs | 1–196 + 22 |
| palC164(1)a | +/− | T653G,A654G | L218R | L218R |
| palC143(1) | − | T668G | L223stop | 1–222 |
| palC129(1) | − | ΔA701 | D234fs | 1–233 + 51 |
| palC82(2)a | −ts | G703A | D235N | D235N |
| palC81(1) | −ts | G703A,G718A | D235N,A240T | D235N,A240T |
| palC92(2) | +/− | A728C | Q243P | Q243P |
| palC148(1) | − | ΔT747 | D249fs | 1–248 + 36 |
| palC150(1) | − | A748G,G755A,G756A | K250E,W252stop | K250E,1–251 |
| palC85(2) | +/− | G755C | W252S | W252S |
| palC94(2) | +/− | G755C | W252S | W252S |
| palC84(2) | −ts | T813G | C271W | C271W |
| palC88(2) | −ts | A945T | R315S | R315S |
| palC80(1) | − | G961A | G321D | G321D |
| palC144(1) | − | ΔG984 | G328fs | 1–328 + 38 |
| palC116(1) | − | ΔT1093 | S365fs | 1–364 + 2 |
| palC160(1) | − | ΔC1114 | R372fs | 1–371 + 8 |
| palC117(1) | − | A1135T | K379stop | 1–378 |
| palC177(1) | − | G1159T | E387stop | 1–386 |
| palC104(1) | − | T1181G | L394stop | 1–393 |
| palC96(1)a | − | T1181A | L394stop | 1–393 |
| palC89(2) | + | A1193T | D398V | D398V |
| palC112(1) | − | C1204T | R402stop | 1–401 |
| palC153(1) | − | T1336G | L427stop | 1–426 |
| palC179(1) | − | T1336A | L427stop | 1–426 |
| palC159(1) | − | A1337insGA | I428fs | 1–427 + 29 |
| palC90(2) | +/− | C1371T,C1372T | P439F | P439F |
| palC107(1) | +/− | C1371T,C1372T | P439F | P439F |
| palC162(1) | − | ΔG1378–C1380 | ΔR442 | ΔR442 |
| palC113(1) | +/− | G1381A | R442H | R442H |
| palC87(2) | +/−− | T1407A | Y451N | Y451N |
| palC131(1) | +/− | T1419insTT | S455fs | 1–454 + 2 |
| palC102(2) | +/− | T1423G | L456R | L456R |
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).
The sequenced strain is probably aneuploid and carried palC40 in addition to the new palC mutation.