Isolation and characterization of the Aspergillus niger pyruvate kinase gene

The Aspergillus niger gene encoding pyruvate kinase was cloned by heterologous hybridization using a fragment from the corresponding yeast gene as a probe. The primary structure of the gene, including 5' and 3' flanking sequences, was determined. The structural part of the A. niger pkiA ge...

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Bibliographic Details
Published in:Current genetics 1992-07, Vol.22 (1), p.21-27
Main Authors: Graaff, L. de (Agricultural Univ., Wageningen (Netherlands). Dept. of Genetics, Section of Molecular Genetics), Broeck, H. van den, Visser, J
Format: Article
Language:English
Subjects:
Amino Acid Sequence
ANALISIS MICROBIOLOGICO
ANALYSE MICROBIOLOGIQUE
ASPERGILLUS NIGER
Aspergillus niger - enzymology
Base Sequence
Biological and medical sciences
DNA, Fungal
Electrophoresis, Polyacrylamide Gel
EXPRESION GENICA
EXPRESSION DES GENES
Fundamental and applied biological sciences. Psychology
Fungal transformation
GENE
GENE EXPRESSION
Gene structure
GENES
Genes. Genome
GENETIC TRANSFORMATION
Laboratorium voor Erfelijkheidsleer
Laboratory of Genetics
MICROBIOLOGICAL ANALYSIS
Molecular and cellular biology
Molecular genetics
Molecular Sequence Data
Nucleic Acid Hybridization
nucleotide sequence
PIRUVATO QUINASA
pkiA gene
prediction
PRI BIOS Applied Genomics & Proteomics
Primary structure
PYRUVATE KINASE
Pyruvate Kinase - genetics
Pyruvate Kinase - isolation & purification
Restriction Mapping
Sequence Homology, Nucleic Acid
TRANSFORMACION GENETICA
TRANSFORMATION GENETIQUE
Transformation, Genetic
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Summary:The Aspergillus niger gene encoding pyruvate kinase was cloned by heterologous hybridization using a fragment from the corresponding yeast gene as a probe. The primary structure of the gene, including 5' and 3' flanking sequences, was determined. The structural part of the A. niger pkiA gene is 2054 bp long and is interrupted by seven putative introns. Splicing of the intron sequences results in an open reading frame of 1578 bp, encoding a protein of 526 amino-acid residues and a molecular weight of 58,130 Da. Extensive homology is found with pyruvate kinase from A. nidulans; only 33 amino acids are different between both proteins. Transformation experiments using the pyrA gene as a selection marker and the subcloned pkiA gene as a co-transforming marker led to increased levels of pyruvate kinase. Analysis of the transformants showed that in none of the transformants integration had occurred at the pkiA locus. Predominantly co-integration of the pyrA- and the pkiA-containing plasmids was found in the cases examined.
ISSN:0172-8083
1432-0983
DOI:10.1007/BF00351737