A twin-reporter vector for simultaneous analysis of expression signals of divergently transcribed, contiguous genes in filamentous fungi.
To analyze the promoter region(s) of divergently transcribed fungal genes, a twin-reporter vector was constructed. This vector contains two divergently oriented reported genes, encoding Escherichia coli beta-glucuronidase (uidA) and E. coli beta-galactosidase (lacZ). Terminator regions of the Aspergillus nidulans nitrate and nitrite reductase-encoding genes, niaD and niiA, respectively, have been cloned 3' to the reporter genes to ensure proficient transcription termination of the reporter genes. The reporter genes have been separated by a unique NotI restriction site, which can be used for the insertion of expression signals. A mutant argB selection marker has been introduced in order to obtain A. nidulans transformants with a single copy of the vector integrated at the argB locus. The use of the vector was demonstrated by insertion of the A. nidulans niaD-niiA intergenic region and analysis of A. nidulans transformants obtained with this construct. Control of expression of both reporter genes was found to be in accordance with previously published data on control of nitrate assimilation [Cove, Biol. Rev. 54 (1979) 291-327].
TNO Medical Biological Laboratory, Rijswijk, The Netherlands.
P J
PJ
Punt
P A
PA
Greaves
A
A
Kuyvenhoven
J C
JC
van Deutekom
J R
JR
Kinghorn
P H
PH
Pouwels
C A
CA
van den Hondel
D001233:Aspergillus nidulans / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D005800:Genes, Fungal; D014644:Genetic Variation; D005822:Genetic Vectors; D005966:Glucuronidase / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000096:biosynthesis / Q000235:genetics*; D009572:Nitrite Reductases / Q000096:biosynthesis / Q000235:genetics*; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics*
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D009565:Nitrate Reductases; D009572:Nitrite Reductases; D050901:Nitrate Reductase; D001616:beta-Galactosidase; D005966:Glucuronidase