GENE-EXPRESSION PER GENE DOSE, A SPECIFIC MEASURE OF GENE-EXPRESSION IN AQUATIC MICROORGANISMS

A method for the measurement of specific levels of gene expression that combines target mRNA and target DNA quantitation has been developed. The use of target gene dose as a normalizing factor for mRNA provides an alternative to 16S or 23S rRNA, which are unsuitable for use in the environment because of their presence in nontarget organisms. Both target mRNA and DNA are recovered from replicate samples and detected by using antisense and sense single-stranded RNA gene probes. For efficient mRNA recovery, the use of Millipore Durapore filters and multiple extractions was necessary. Quantitation was performed by radiometric detection by using a beta-scanner and comparison of the sample signal against target mRNA and DNA standard curves. This method enabled the measurement of expression of the catechol-2,3-dioxygenase gene (xylE) contained on the thermoregulated plasmid pLV1013 in a marine Vibrio strain in culture and in the environment. In studies of the relationship between mRNA levels and enzyme activities, the appearance of enzyme activity lagged behind xylE mRNA synthesis by an hour after temperature induction. This suggests that mRNA analysis is well suited for determining rapid regulation of microbial gene expression at the transcriptional level in water column microbial populations.