ISOLATION AND EXPRESSION OF THE ACETATE-INDUCIBLE ISOCITRATE LYASE GENE (ACU-3) FROM NEUROSPORA-CRASSA - EVIDENCE FOR A 2ND CONSTITUTIVE ISOZYME

被引:15
作者
GAINEY, LDS
KOLBLE, K
CONNERTON, IF
机构
[1] UNIV READING, DEPT MICROBIOL, LONDON RD, READING RG1 5AQ, BERKS, ENGLAND
[2] UNIV OXFORD, DEPT BIOCHEM, MRC, IMMUNOCHEM UNIT, OXFORD OX1 3QU, ENGLAND
来源
MOLECULAR AND GENERAL GENETICS | 1991年 / 229卷 / 02期
关键词
NEUROSPORA-CRASSA; ACETATE; ISOCITRATE LYASE; ISOZYMES; PULSED FIELD GEL ELECTROPHORESIS;
D O I
10.1007/BF00272163
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:253 / 260
页数:8
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