| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2003: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2002: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
Histidine kinase, Hik33, functions as both low-temperature sensor and osmosensor in Synechocystis sp. PCC 6803. To elucidate the molecular mechanisms of stress perception by Hik33, the study was focused on the identification of stress-sensing domains by domain-swap experiments.
(1)Making domain-swap constructs
Domain swapping was designed to use Hik31 that is not involved in the perception of cold and osmotic stress. According to the tight relation between stress perception and physical state of membranes, transmembrane and/or periplasmic regions of Hik33 were swapped with corresponding regions of Hik31. Then, to produce plasmids for genome-integration, resultant chimeric genes sandwiched between 5' and 3' franking regions of the hik33 gene:
(2)Production of a deletion mutant of an ORF of the hik33 gene
To avoid the possibility of the homologous recombination between the coding regions of Hik33 between genome and plasmid that sometimes disrupt the ORF, a deletion mutant of the whole ORF of the hik33 gene was made. Although Synechocystis contains over 10 copies of the genome per cell, segregation of deletion mutation was completed for all of genomes.
(3)Introduction of the chimeric genes into genomes of the hik33 deletion mutant
Homologous recombination at 5' and 3' franking regions of the hik33gene was successfully operated for all of domain-swap constructs.
(4) Complementation test of domain-swapped genes
To identify the regions of stress sensing in Hik33, checking of the responsibility of domain-swap mutants obtained was scheduled. As an initial test, wild-type hik33 gene was introduced into the deletion mutant by homologous recombination and analyzed stress-inducible genes by DNA microarray experiments. Unfortunately, deletion of the hik33 gene was not complemented completely by the wild-type hik33 gene. Thus, experimental systems should be developed as soon as possible.
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