Transcription profiling of E. coli groESL(-) cells expressing human Hsp60(wt)/Hsp10 or Hsp60-(p.Val98Ile)/Hsp10 operons

ABSTRACT: To identify regulatory responses that could give clues as to the cause(s) of the growth arrest observed in E. coli cells expressing mutant Hsp60-(p.Val98Ile) and Hsp10 versus cells expressing wild type Hsp60 and Hsp10, we performed microarray analyses. We reasoned that the decreased chaperonin activity of the Hsp60-(p.Val98Ile) protein would greatly impair or abolish folding of E. coli proteins that depend on chaperonin assistance. The resulting decreased levels of various activities may cause compensatory up-regulation or deregulation of genes. The transcriptomes of cells shifted to expression of the arabinose-inducible chaperonin operon with either the Hsp60-(p.Val98Ile) mutant or the wild type Hsp60 from the second plasmid were compared. RNA samples from three independent cultures expressing the mutant or two independent cultures expressing wild type Hsp60 were pooled, respectively. Experiment Overall Design: B178 (groELS)-deleted cells double-transformed with two plasmids harboring an IPTG-inducible operon with wild type human Hsp60 and Hsp10 and a second plasmid harboring an arabinose inducible operon with either Hsp60-(p.Val98Ile) and Hsp10 (mutant cells) or wild type Hsp60 and Hsp10 (wild type cells) were grown in dYT medium (21) supplemented with kanamycin (10 mg/L), ampicillin (100 mg/L) and IPTG (0.1mM) at 30°C in a shaking water bath. At OD536 â?? 0.1 bacteria were harvested by centrifugation at ambient temperature, resuspended in fresh medium, and 0.2% arabinose was added. Growth was continued at 30°C. After 10 hours 3ml samples were withdrawn and RNA was isolated. Experiment Overall Design: Two independent transformant clones of the wild type cells and three of the mutant cells were grown in this way. The RNA preps from the 2 wild type and the three mutant cells, respectively, were pooled, cDNA was synthesized, labelled and subjected to Affymetrix arrays.

ORGANISM(S): Escherichia coli

SUBMITTER: Peter Bross 

PROVIDER: E-GEOD-10974 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress