Cloning, Analysis and Inactivation of thendhKGene Encoding a Subunit of NADH Quinone Oxidoreductase fromAnabaenaPCC 7120
| Autor: | David A. Day, James Whelan, G. D. Price, Crispin A. Howitt |
|---|---|
| Rok vydání: | 1996 |
| Předmět: |
Operon
Immunoblotting Molecular Sequence Data Cyanobacteria Polymerase Chain Reaction Biochemistry NADP Transhydrogenases Animals Coding region Amino Acid Sequence Cloning Molecular Quinone Reductases Gene Conserved Sequence DNA Primers Base Sequence biology Anabaena Cell Membrane Synechocystis Nucleic acid sequence NADH dehydrogenase biology.organism_classification Recombinant Proteins Molecular Weight Chloroplast Blotting Southern Mutagenesis Insertional Genes Bacterial biology.protein bacteria Cattle |
| Zdroj: | European Journal of Biochemistry. 240:173-180 |
| ISSN: | 1432-1033 0014-2956 |
| Popis: | The function of the type-1 pyridine nucleotide dehydrogenase (NDH-1) in the cyanobacterium Anabaena PCC 7120 was investigated. Immunological analysis with antibodies raised against NdhK from Synechocystis PCC 6803, a subunit of NDH-1, showed that NdhK in Anabaena PCC 7120 is only present on the plasma membrane, which confirms the results of previous studies [Howitt, C. A., Smith, G. D. & Day, D. A. (1993) Biochim. Biophys. Acta 1141, 313–320]. Southern analysis with probes from the operon encoding ndhC-K-J from Synechocystis PCC 6803 showed that this operon is also conserved in Anabaena PCC 7120. Part of the operon was amplified using PCR with degenerate primers designed against two sequences encoding regions of NdhC and NdhJ that are conserved between cyanobacteria and chloroplasts. The nucleotide sequence of ndhK encodes a protein of 245 amino acids with a predicted molecular mass of 27.5 kDa. The coding regions of ndhC and ndhK overlap by 7 bp, as found in the chloroplasts of liverwort, maize, and rice. This is markedly different from the case in Synechocystis PCC 6803 where a 71-bp non-coding, intergenic spacer region lies between ndhC and ndhK. The ndhK clone was interrupted by the insertion of a kanamycin-resistance gene and used to transform Anabaena PCC 7120. 20 unsegregated transformants were produced, all of which died during attempts to segregate them. This indicates that under the selection conditions used, ndhK is an essential gene in Anabaena PCC 7120. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|