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Mentions: Successful stable transformation of DNA molecules into P. blakesleeanus has been an elusive method , 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
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.
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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