Isolation and expression of the acetate-inducible isocitrate lyase gene (acu-3) from Neurospora crassa: evidence for a second constitutive isozyme.
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.
Department of Microbiology, University of Reading, UK.
L D
LD
Gainey
K
K
Kölble
I F
IF
Connerton
D000085:Acetates / Q000494:pharmacology*; D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics; D004586:Electrophoresis; D004790:Enzyme Induction; D007522:Isocitrate Lyase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D009492:Neurospora crassa / Q000201:enzymology*; D012150:Polymorphism, Restriction Fragment Length; D012331:RNA, Fungal / Q000235:genetics; D015183:Restriction Mapping
D000085:Acetates; D004271:DNA, Fungal; D007527:Isoenzymes; D012331:RNA, Fungal; D007522:Isocitrate Lyase