Gene Expression for Complex Communities
PNNL researchers have examined subtractive hybridization-based methods for studying gene expression, particularly the isolation of genetic information unique to a new environmental condition, in undefined environmental communities.
The total RNA isolated from a soil can be thought of as representing the total genetic and metabolic potential of the soil biome. Within this diverse mixture, some genes (mRNAs) will be associated with contaminant bioattenuation, while many will be involved in unrelated cellular processes. We have used subtractive hybridization-polymerase chain reaction (SH-PCR) strategies to selectively amplify only the sequences unique to contaminant exposure, i.e., SH-PCR can "filter out" RNA sequences common to both samples, and allow collection of gene fragments uniquely expressed in one population relative to another.
Generalized diagram of SH-PCR
Enlarged Detailed View
Generalized diagram of SH-PCR
Enlarged Detailed View
Unique fragments were detected in total RNA from microbial communities derived from hyporrheic, groundwater, and riverwater zones of mesocosm sediments, periphyton exposed to U, as well as in chromium-spiked soils. While this approach is not inclusive of all unique fragments, and often includes ribosomal RNA sequences indicative of microbial species, rather than just genes, it offers a first glimpse inside the "black box" without pre-existing sequence information. We conducted "reciprocal" subtractions for all treatments in order to detect genes that are uniquely lost following the exposures or treatments.
The soil chromium libraries sent for sequencing continue to include gene sequences consistent with the unculturable species Rubrobacter xylanophilus, which is known to grow in arid soils like those used in this experiment. Specifically, we detected a pseudo-uridine synthase gene robustly (i.e., different fragments of this gene were detected in each of two separate subtractions) and a penicillin amidase gene. We resolved past subtraction issues by modifying a commercially available kit for subtractive hybridization of bacteria to work in soils, sediments, and periphyton.
Contact: Vanessa Bailey