In the adaptive immune system, V(D)J recombination initiates the production of a diverse antigen receptor repertoire in developing B and T cells. Recombination activating proteins, RAG1 and RAG2 (RAG1/2), catalyze V(D)J recombination by cleaving adjacent to recombination signal sequences (RSSs) that flank antigen receptor gene segments. Previous studies defined the consensus RSS as containing conserved heptamer and nonamer sequences separated by a less conserved 12 or 23 base-pair spacer sequence. However, many RSSs deviate from the consensus sequence. Here, we developed a cell-based, massively parallel assay to evaluate V(D)J recombination activity on thousands of RSSs where the 12-RSS heptamer and adjoining spacer region contained randomized sequences. While the consensus heptamer sequence (CACAGTG) was marginally preferred, V(D)J recombination was highly active on a wide range of non-consensus sequences. Select purine/pyrimidine motifs that may accommodate heptamer unwinding in the RAG1/2 active site were generally preferred. In addition, while different coding flanks and nonamer sequences affected recombination efficiency, the relative dependency on the purine/pyrimidine motifs in the RSS heptamer remained unchanged. Our results suggest RAG1/2 specificity for RSS heptamers is primarily dictated by DNA structural features dependent on purine/pyrimidine pattern, and to a lesser extent, RAG:RSS base-specific interactions.

Natural attenuation of groundwater contamination occurs at some level for all aquifers impacted with organic contaminants. The issues regarding natural attenuation are whether it takes place at a sufficient rate to be protective of human health and the environment. Implementation of a Monitored Natural Attenuation (MNA) remedial alternative for groundwater requires parties responsible for the contamination to demonstrate to regulators and the public that MNA is protective at a given site. Analysis of MNA for remediation of karst aquifers is hampered by a lack of understanding of biodegradation in karst environments. The lack of studies examining biodegradation in karst aquifers may in large part be due to the widespread perception that contaminants are rapidly flushed out of karst aquifers resulting in insufficient residence times for contaminants to biodegrade. In highly developed and well-connected conduit systems, the rate of contaminant migration is perceived to be much faster than the rate of biodegradation. This perception of contaminant transport is largely incorrect. Tracer studies for karst aquifers often indicate that these aquifers are characterized by diverse flow regimes and storage capabilities. Additionally, it is also believed that if bioremediation in bedrock aquifers is dependent upon contact between surface-attached bacteria and contaminants, then bioremediation would be limited by the low surface-area-to-volume ratio (SA/V) of karst aquifers. A quantitative basis, however, for accepting or rejecting the assumption that attached bacteria dominate the biodegradation process in karst conduits has not been shown. The objective of this research was to determine if free-living karst bacteria from contributed as much to toluene OPEN ACCESSWater 2011, 3 1140 biodegradation as attached bacteria. This is an important area of research. Research indicates bacteria are both attached and free-living in karst aquifers and it is unrealistic to think that only the attached bacteria facilitate biodegradation. The groundwater use in all tests was taken from a karst aquifer know to be impacted by BTEX. The resulting first-order rate constants were computed to be 0.014 per hour for the open system and 0.0155 per hour for the packed reactor system. Biodegradation of toluene in flow-through laboratory karst systems of varying SA/V indicated that the observed biodegradation of toluene was attributable to free-living karst bacteria and not limited by low SA/V in karst. This was evidenced by the fact that the systems with five-fold variation in SA/V were shown to have observed pseudo first order reaction rate constants that differed by only 7.0%. If attached bacteria were primarily responsible for biodegradation and limiting, a proportional difference in the observed rates relative to the difference in surface area would be expected.

In the adaptive immune system, V(D)J recombination initiates the production of a diverse antigen receptor repertoire in developing B and T cells. Recombination activating proteins, RAG1 and RAG2 (RAG1/2), catalyze V(D)J recombination by cleaving adjacent to recombination signal sequences (RSSs) that flank antigen receptor gene segments. Previous studies defined the consensus RSS as containing conserved heptamer and nonamer sequences separated by a less conserved 12 or 23 base-pair spacer sequence. However, many RSSs deviate from the consensus sequence. Here, we developed a cell-based, massively parallel V(D)J recombination assay to evaluate RAG1/2 activity on thousands of RSSs. We focused our study on the RSS heptamer and adjoining spacer region, as this region undergoes extensive conformational changes during RAG-mediated DNA cleavage. While the consensus heptamer sequence (CACAGTG) was marginally preferred, RAG1/2 was highly active on a wide range of non-consensus sequences. RAG1/2 generally preferred select purine/pyrimidine motifs that may accommodate heptamer unwinding in the RAG1/2 active site. Our results suggest RAG1/2 specificity for RSS heptamers is primarily dictated by DNA structural features dependent on purine/pyrimidine pattern, and to a lesser extent, RAG:RSS base-specific interactions. Further investigation of RAG1/2 specificity using this new approach will help elucidate the genetic instructions guiding V(D)J recombination.Summary StatementPartially conserved recombination signal sequences (RSSs) govern antigen receptor gene assembly during V(D)J recombination. Here, a massively parallel analysis of randomized RSSs reveals key attributes that allow DNA sequence diversity in the RAG1/2 active site and that contribute to the differential utilization of RSSs in endogenous V(D)J recombination. Overall, these results will assist identification of RAG1/2 off-target sites, which can drive leukemia cell transformation, as well as characterization of bona fide RSSs used to generate antigen receptor diversity.

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