Case Study

One test of a mutagenesis protocol is the rediscovery of previously described mutant alleles. On screening the first 800 Basta-resistant strains, a mutant (KOMO 303) showed a phase of entrainment that was opposite that of wildtype in the 16-h temperature cycle protocol (22 to 27°C). In a light-dark cycle using 3 ^mol of photons/m2/s, it completely failed to entrain, and in constant darkness, it was arrhythmic (see Fig. 5). These phenotypes resemble the null frequency mutant, frq9, and also the white collar (wc) mutants. The wc mutants are blind for light-induced mycelial carotenoids, in addition to having clock defects (11,12).

1. We determined that the mutation was not likely in the wc genes by a simple light-induced carotenoid assay using mycelial pads, showing near-normal levels. The frq locus was investigated for insertion of the Basta-resistance cassette with Southern analysis, revealing insertion in the open reading frame, in approximately the same location as the frq9 mutation is found (13). Furthermore, the Southern analysis showed a clear double insertion (i.e., two copies) of the drug resistance marker at this site.

2. One of the first insertions, KOMO 58, rescued as described in Subheading 3.5., was further characterized by amplifying the insertion site from the genomic mutant DNA. Primers were designed using the genomic DNA sequence predicted to flank the insertion. A positive result (a robust fragment of an appropriate molecular weight) would indicate that the drug-resistance marker is inserted with no large deletions or translocations. A negative result (no fragment) would indicate that genomic DNA was lost in the recombination leading to insertion, or that a translocation occurred. These possibilities can be finally determined with

Fig. 4. (opposite page) Flow diagram of insertion rescue protocol. (A) The area surrounding the insertion is mapped by restriction digestion and Southern blotting. (B) The genomic DNA is digested and the desired fragment is enriched by size selection. (C) The pool of fragments is ligated into pSK-II. (D) The appropriate ligation product is amplified with nested polymerase chain reactions, and gel-purified. (E) The fragment is sequenced. RE, restriction endonuclease site; Xbal and HindIII, restriction enzymes; T3 and T7, standard RNA polymerase sites used for primer binding; bar2.1 and bar2.2, primers specific for the Basta-resistance gene, used in this protocol.

DD, K0M03Q3

TC, KOMO303

LC, K0M03Q3

Fig. 5. A frequency mutant was generated by insertional mutagenesis. Race tubes and double plots for a frq mutant generated in this study are shown here. The mutant is compared with the bd strain in constant darkness, a 16-h temperature cycle (TC), and a 12-h light cycle (LC). For each condition a control and a mutant race tube is shown, as well as a sample double plot. The gray areas in the plots indicate the cold (TC) or the dark phase (LC). Note the phase of entrainment of KOMO303 in the temperature cycle is approximately opposite that of bd. Furthermore conidiation is not stimulated by (or driven by) the onset of the cold phase. KOMO303 completely fails to entrain in light cycles. These phenotypes are consistent with those for the frq9 strain (8). KOMO303 was determined to be a frq mutant by Southern blotting (data not shown).

Southern blot or by rescuing the other end of the insertion. In either of the latter cases, the mutant is much less interesting, as it is almost impossible to determine where the mutant phenotype derives from. In the case of KOMO 58, the fragment generated in the PCR reaction was larger than would have been predicted. Sequence analysis showed that there was a duplication in the inserted plasmid (an extra copy of the ampicillin resistance gene was present), whereas the genomic DNA was intact except for a few nucleotides at the junctions.

3. To finally determine that the insertion is acting through a specific gene, the gene should be knocked out (13) and the null mutant strain then assayed for clock characteristics. If the gene is essential, an overexpression construct can be engineered using the inducible qa-2 promoter (14) and transformed ectopically. After growing in the precence of an inducer, functional tests can be carried out in its absence.

Fig. 5. A frequency mutant was generated by insertional mutagenesis. Race tubes and double plots for a frq mutant generated in this study are shown here. The mutant is compared with the bd strain in constant darkness, a 16-h temperature cycle (TC), and a 12-h light cycle (LC). For each condition a control and a mutant race tube is shown, as well as a sample double plot. The gray areas in the plots indicate the cold (TC) or the dark phase (LC). Note the phase of entrainment of KOMO303 in the temperature cycle is approximately opposite that of bd. Furthermore conidiation is not stimulated by (or driven by) the onset of the cold phase. KOMO303 completely fails to entrain in light cycles. These phenotypes are consistent with those for the frq9 strain (8). KOMO303 was determined to be a frq mutant by Southern blotting (data not shown).

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