These tools will no longer be maintained as of December 31, 2024. Archived website can be found here. PubMed4Hh GitHub repository can be found here. Contact NLM Customer Service if you have questions.


Pubmed for Handhelds

PUBMED FOR HANDHELDS

Search MEDLINE/PubMed

  • Title: Molecular cloning of genes encoding branched-chain keto acid dehydrogenase of Pseudomonas putida.
    Author: Sykes PJ, Burns G, Menard J, Hatter K, Sokatch JR.
    Journal: J Bacteriol; 1987 Apr; 169(4):1619-25. PubMed ID: 3549697.
    Abstract:
    We cloned the structural genes for the individual subunits of the branched-chain keto acid dehydrogenase multienzyme complex on a 7.8-kilobase EcoRI-SstI restriction fragment of Pseudomonas putida chromosomal DNA by cloning into the broad-host-range vector pKT230. A direct selection system for growth on valine-isoleucine agar was achieved by complementation of P. putida branched-chain keto acid dehydrogenase mutants. The recombinant plasmid, pSS1-1, increased expression of branched-chain keto acid dehydrogenase up to five times in wild-type P. putida. The complex was expressed constitutively in P. putida(pSS1-1) but was inducible in Escherichia coli HB101(pSS1-1) by high valine. E. coli minicells transformed with pSS1-1 produced three polypeptides which did not match the four polypeptides of the purified complex. To resolve this problem, we inserted P. putida DNA from pSS1-1 into pUC18 and pUC19. The pUC-derived plasmids were used as DNA templates in an E. coli transcription-translation system. Four polypeptides were produced from the pUC18-derived plasmid which had the correct molecular weights, showing that the structural genes had been cloned. Since only weak bands were produced with the pUC19-derived plasmid, the direction of transcription was established. The locations and order of all the structural genes of branched-chain keto acid dehydrogenase were located by restriction enzyme mapping.
    [Abstract] [Full Text] [Related] [New Search]