An integrated systems approach for understanding cellular responses to gamma radiation.. Halobacterium salinarum NRC-1

Cellular response to stress entails complex mRNA and protein abundance changes which translate into physiological adjustments for maintaining homeostasis as well as to repair and minimize damage to cellular components. We have characterized the response of the halophilic archaeon Halobacterium salinarum NRC-1 to 60Co ionizing gamma radiation in an effort to understand correlation between genetic information processing and physiological changes. The physiological response model we have constructed is based on integrated analysis of temporal changes in global mRNA and protein abundance along with protein-DNA interactions and evolutionarily conserved functional associations. This systems view reveals cooperation amongst several cellular processes including DNA repair, increased protein turnover, apparent shifts in metabolism to favor nucleotide biosynthesis and an overall effort to repair oxidative damage. Further, we demonstrate the importance of time dimension while correlating mRNA and protein levels and suggest that steady state comparisons may be misleading while assessing dynamics of genetic information processing across transcription and translation. Keywords: Time course after gamma irradiation Overall design: The wild type strain of Halobacterium salinarium strain NRC-1 was used for the gamma radiation experiments. Culturing of all strains was done in a liquid Complete Medium (CM; at 42 ºC with shaking at 220 rpm. Cell pellets from two 180mL cultures (control and experimental) of Halobacterium NRC-1 (OD600nm= 0.4) were resuspended in 1/20 volume in a basal salt solution (CM without peptone) and exposed to 2500 Gy of gamma-ray at 22oC using a 26,000-curie (9.6E14 Bq) 60Co gamma source at Univ. of Maryland College Park Gamma Test Facility at a dose rate of 62.01 Gy/min. Irradiated and control cultures were resuspended in the original volume of CM, split into 20mL aliquots in baffled flasks and incubated at 42oC and 220rpm shaking. Time course samples were placed on ice, pelleted (5000 x g, 4oC, 5 min) and flash-frozen in a dry ice/ethanol bath after decanting the supernatant. RNA extractions were performed using the Stratagene Absolute RNA kit and RNA quality checked with the Agilent Bioanalyzer and with PCR. Microarrays were fabricated at the Institute for Systems Biology Microarray Facility. The arrays contain 4 spots per unique 70-mer oligonucleotides for each of 2400 non-redundant genes in Halobacterium NRC-1. Bias in dye incorporation was accounted for by reversing the labeling dyes (dye-flip). Raw data was processed and converted into log10 ratios with lambda values determined by a maximum likelihood method.