ABSTRACT
Analysis of the promoter region of the acetate-induced isocitrate lyase gene (acu-3) of Neurospora crassa was undertaken. A series of deletions in the 5' non-transcribed region were constructed and the effects of these mutations on the enzyme levels following growth on sucrose and transfer to acetate were measured. Sequences within the region -603 to -271 relative to the transcription start site appear essential for transcription. The region -950 to -1278 is required for sucrose repression, which is consistent with previous protein/DNA gel retardation results of protein extracts from N. crassa cultured on sucrose. Protein extracts from acetate-induced mycelia identify alternative promoter regions apparently involved in acetate-induced gene transcription.
Subject(s)
Isocitrate Lyase/genetics , Neurospora crassa/enzymology , Neurospora crassa/genetics , Promoter Regions, Genetic , Acetates/pharmacology , Base Sequence , Enzyme Induction , Isocitrate Lyase/biosynthesis , Molecular Sequence Data , Mutagenesis , Restriction Mapping , Sequence Deletion , Sucrose/pharmacology , Transcription, GeneticABSTRACT
The nucleotide sequences of the genes encoding the acetate-inducible glyoxylate cycle enzyme isocitrate lyase from the ascomycete fungi Aspergillus nidulans (acuD) and Neurospora crassa (acu-3) are presented. The respective A. nidulans and N. crassa genes are interrupted at identical positions by two introns and encode proteins of 538 and 543 amino acids, which have 75% identity. The predicted protein sequences do not demonstrate the C-terminal tripeptide S-K-L that has been implicated in peroxisomal targeting and found in the glyoxysomally located enzyme malate synthase from the same species. However, the protein sequences do exhibit a partial repeat which, in common with malate synthase, is located in regions that are absent from, or non-homologous with, the E. coli enzyme, which is not compartmentalized.
Subject(s)
Aspergillus nidulans/genetics , Glyoxylates/metabolism , Isocitrate Lyase/genetics , Microbodies/enzymology , Neurospora crassa/genetics , Amino Acid Sequence , Aspergillus nidulans/enzymology , Base Sequence , DNA, Fungal/genetics , Genes, Fungal , Isocitrate Lyase/chemistry , Malate Synthase/chemistry , Malate Synthase/genetics , Molecular Sequence Data , Neurospora crassa/enzymology , Repetitive Sequences, Nucleic AcidABSTRACT
Heterologous hybridisation of the Aspergillus nidulans structural gene for isocitrate lyase (acuD) to a lambda genomic library of Neurospora crassa identified a recombinant phage containing the hybridising sequence on an internal 9 kb EcoRI fragment. A restriction fragment length polymorphism (RFLP) enabled the fragment to be assigned to linkage group V (LG V), the location of the acetate-inducible isocitrate lyase, acu-3 of Neurospora. Functional ectopic complementation by co-transformation of an am-, acu- double mutant using independent plasmid clones, carrying the entire 9 kb EcoRI fragment (pICLG1) and the selectable marker am+ (NADP-glutamate dehydrogenase), demonstrated that the clone contains the entire acetate-inducible transcription unit. However, Northern analysis revealed two species of mRNA, only one of which was inducible on acetate. Native polyacrylamide gel electrophoresis separated two iso-enzymic activities, again only one of which was acetate-inducible and deficient in acu-3- mutants. Further hybridisation of the acu-3 gene probe to an electrophoretic karyotype of Neurospora crassa identified sequences in an additional linkage group as well as in LG V, as anticipated. The isozymes are therefore sequence-related.
