Cloning of the Alcaligenes eutrophus alcohol dehydrogenase gene.
AUTOR(ES)
Kuhn, M
RESUMO
Mutants of Alcaligenes eutrophus which are altered with respect to the utilization of 2,3-butanediol and acetoin were isolated after transposon mutagenesis. The suicide vehicle pSUP5011 was used to introduce the drug resistance transposable element Tn5 into A. eutrophus. Kanamycin-resistant transconjugants of the 2,3-butanediol-utilizing parent strains CF10141 and AS141 were screened for mutants impaired in the utilization of 2,3-butanediol or acetoin. Eleven mutants were negative for 2,3-butanediol but positive for acetoin; they were unable to synthesize active fermentative alcohol dehydrogenase protein (class 1). Forty mutants were negative for 2,3-butanediol and for acetoin (class 2). Tn5-mob was also introduced into a Smr derivative of the 2,3-butanediol-nonutilizing parent strain H16. Of about 35,000 transconjugants, 2 were able to grow on 2,3-butanediol. Both mutants synthesized the fermentative alcohol dehydrogenase constitutively (class 3). The Tn5-labeled EcoRI fragments of genomic DNA of four class 1 and two class 3 mutants were cloned from a cosmid library. They were biotinylated and used as probes for the detection of the corresponding wild-type fragments in a lambda L47 and a cosmid gene bank. The gene which encodes the fermentative alcohol dehydrogenase in A. eutrophus was cloned and localized to a 2.5-kilobase (kb) SalI fragment which is located within a 11.5-kb EcoRI-fragment. The gene was heterologously expressed in A. eutrophus JMP222 and in Pseudomonas oxalaticus. The insertion of Tn5-mob in class 3 mutants mapped near the structural gene for alcohol dehydrogenase on the same 2.5-kb SalI fragment.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=210709Documentos Relacionados
- Molecular cloning and DNA sequence of the Arabidopsis thaliana alcohol dehydrogenase gene.
- Alcohol dehydrogenase gene from Alcaligenes eutrophus: subcloning, heterologous expression in Escherichia coli, sequencing, and location of Tn5 insertions.
- Cloning exons of mapping of transcription: characterization of the Drosophila melanogaster alcohol dehydrogenase gene.
- Characterization of alcohol dehydrogenase genes of derepressible wild-type Alcaligenes eutrophus H16 and constitutive mutants.
- Isolation and partial characterization of the Drosophila alcohol dehydrogenase gene.