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The leuA point mutation segregates with leucine auxotrophy.(A) Phenotypic analysis of A721, UBC21 and a subset of 12 of the 104 progeny from the A721 x UBC21 cross. Spores were plated onto YNB media containing HCl at a concentration of 5 mM, supplemented with leucine (+leu), adenine (+ade), or both (+leu +ade) to test for leuA and purC mutations. Growth on neither the YNB+leu nor the YNB+ade medium indicates that the strain is mutant for both the leuA and purC phenotypes, while growth on both indicates that the strain is WT. Growth on the YNB+ade medium with no growth on the YNB+leu medium indicates that the strain is an adenine auxotroph, mutant for the purC phenotype but WT for the leuA phenotype. (B) PCR of the leuA gene with primers ALID0469 and ALID0474, and digestion with HpyCH4IV (the G-C point mutation of A721 alters the ACGT site recognized by the HpyCH4IV restriction enzyme). (C and D) PCR of markers on two independent chromosomes confirm independent segregation. (C) PCR from the chromosome containing pyrG with primers ALID0251-ALID0252 and digestion with EcoRV. (D) PCR from the chromosome containing sex with ALID0397-ALID0398 and digestion with XhoI. DNA fragments were resolved on 1% agarose gels. The size markers between samples from strains A721 and UBC21 are the 1 kb + ladder (Invitrogen).
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pone-0011605-g003: The leuA point mutation segregates with leucine auxotrophy.(A) Phenotypic analysis of A721, UBC21 and a subset of 12 of the 104 progeny from the A721 x UBC21 cross. Spores were plated onto YNB media containing HCl at a concentration of 5 mM, supplemented with leucine (+leu), adenine (+ade), or both (+leu +ade) to test for leuA and purC mutations. Growth on neither the YNB+leu nor the YNB+ade medium indicates that the strain is mutant for both the leuA and purC phenotypes, while growth on both indicates that the strain is WT. Growth on the YNB+ade medium with no growth on the YNB+leu medium indicates that the strain is an adenine auxotroph, mutant for the purC phenotype but WT for the leuA phenotype. (B) PCR of the leuA gene with primers ALID0469 and ALID0474, and digestion with HpyCH4IV (the G-C point mutation of A721 alters the ACGT site recognized by the HpyCH4IV restriction enzyme). (C and D) PCR of markers on two independent chromosomes confirm independent segregation. (C) PCR from the chromosome containing pyrG with primers ALID0251-ALID0252 and digestion with EcoRV. (D) PCR from the chromosome containing sex with ALID0397-ALID0398 and digestion with XhoI. DNA fragments were resolved on 1% agarose gels. The size markers between samples from strains A721 and UBC21 are the 1 kb + ladder (Invitrogen).

Mentions: Successful stable transformation of DNA molecules into P. blakesleeanus has been an elusive method [38], and so Mendelian genetic analysis was used to confirm that the bp change identified in strain A721 confers the leucine auxotrophy. Strain A721 was crossed with a wild type strain of opposite sex type (UBC21), and 104 progeny isolated and scored for their ability to grow in the absence of leucine and adenine. Genomic DNA was extracted from these strains, and the putative leuA gene amplified with primers ALID0469 and ALID0474. The G-C bp difference between strains A721 and UBC21 alters the ACGT site recognized by the HpyCH4IV restriction enzyme, enabling a PCR-RFLP assessment of the two alleles at this locus (Figure 3). Out of the A721 x UBC21 progeny, 43 were assessed as wild type and all 43 bore the wild type leuA allele, while 61 were assessed as leucine auxotrophs and carried the leuA mutant allele.

Two Origins for the Gene Encoding α-Isopropylmalate Synthase in Fungi

Larson EM, Idnurm A - PLoS ONE (2010)

Bottom Line: The pathway is a proposed target for antimicrobial therapy.To confirm the function of the gene, Phycomyces leuA was used to complement the auxotrophic phenotype exhibited by mutation of the leu3+ gene of the ascomycete fungus Schizosaccharomyces pombe.The identification of leuA in Phycomyces adds to the growing body of evidence that some primary metabolic pathways or parts of them have arisen multiple times during the evolution of fungi, probably through horizontal gene transfer events.

Affiliation: Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, Missouri, United States of America.

ABSTRACT

Background: The biosynthesis of leucine is a biochemical pathway common to prokaryotes, plants and fungi, but absent from humans and animals. The pathway is a proposed target for antimicrobial therapy.

Methodology/principal findings: Here we identified the leuA gene encoding alpha-isopropylmalate synthase in the zygomycete fungus Phycomyces blakesleeanus using a genetic mapping approach with crosses between wild type and leucine auxotrophic strains. To confirm the function of the gene, Phycomyces leuA was used to complement the auxotrophic phenotype exhibited by mutation of the leu3+ gene of the ascomycete fungus Schizosaccharomyces pombe. Phylogenetic analysis revealed that the leuA gene in Phycomyces, other zygomycetes, and the chytrids is more closely related to homologs in plants and photosynthetic bacteria than ascomycetes or basidiomycetes, and suggests that the Dikarya have acquired the gene more recently.

Conclusions/significance: The identification of leuA in Phycomyces adds to the growing body of evidence that some primary metabolic pathways or parts of them have arisen multiple times during the evolution of fungi, probably through horizontal gene transfer events.

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