The regulation of biochemical pathways is controlled at a variety of levels, from transcriptional timing to protein localization. Here we study the corticosteroid synthesis pathway in order to understand a transition from a biochemical pathway controlled by allosteric regulation to a phenotypically equivalent pathway controlled by differential expression of paralogs and enzyme specificity.The corticosteroids aldosterone and cortisol are produced in different layers of the adrenal cortex – a phenomenon known as zonation. Most mammals have a single enzyme, CYP11B, which produces aldosterone in the exterior layer of the adrenal cortex and cortisol in the interior layers. In mammals with a single‐CYP11B, the specific production of aldosterone and cortisol in different tissues is thought to be accomplished by allosteric regulation by another enzyme, CYP11A, which is only present in the interior layer of the cortex. It is thought that the presence of CYP11A changes the function of CYP11B so that it preferentially produces cortisol instead of aldosterone.However, throughout mammalian evolution CYP11B has duplicated independently in several lineages, giving rise to two CYP11B paralogs. These species still experience zonation and produce aldosterone and cortisol in different layers of the adrenal cortex. It has been demonstrated that this zonation in species with two CYP11B enzymes is accomplished by differential expression of the paralogs in the different tissues along with the evolution of enzymatic preference for their respective products. However, it is unknown whether the interaction between CYP11B and CYP11A has been lost and whether the zonation of hormones produced is regulated solely by the two differential expression and enzymatic activity of the CYP11B enzymes.Here we used Forster Resonance Energy Transfer (FRET) High Performance Liquid Chromatography (HPLC) to determine whether CYP11B and CYP11A physically interact and whether or not that interaction would contribute to the allosteric regulation of this pathway. Our preliminary analyses demonstrate a physical interaction between CYP11B and CYP11A in a single‐CYP11B species. Further experiments are aimed to test the interaction of duplicated CYP11Bs with CYP11A. We will monitor in vivo enzymatic assays by HPLC to determine what hormones are produced in the presence of different enzyme combinations. These results will shed light on the various ways metabolic pathways are regulated.Support or Funding InformationSD BRIN P20GM103443SD EPSCOR IIA‐1355423This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Speciation is the source of diversity on the planet, but the genomic mechanisms that lead to the reproductive isolating barriers between populations that cause speciation are not well understood. Here we are studying the genomic differentiation between two species of milkweed that grow in the Western (Asclepias speciosa) and Eastern United States (A. syrica). In addition to different geographic locations, these species have diverged in a number of important phenotypes, including: production of secondary metabolites, flower morphology, and drought resistance. In the central US, these species have come into secondary contact and sometimes form hybrids in the wild. We are interested in quantifying the amount of gene flow between these species and identifying genomic locations that may be indicative of local adaptation within populations and reproductive isolation between species.To compare these species of milkweed, we isolated DNA from A. speciousa, A. syrica, and individuals that displays intermediate phenotypes from the hybridization zone. We extracted and isolated genomic DNA and sequenced their genomes with Oxford Nanopore technology. With the aligned genomes, we will be able to compare polymorphisms in these species and begin to quantify the amount of gene flow between them. We will also identify polymorphisms in candidate genes and pathways that may have led to ecological specialization between these species.Support or Funding InformationSouth Dakota EPSCoR IIA‐1355423South Dakota BRIN P20GM 103443This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cellular metabolism is a complex set of interwoven interactions. That complexity challenges the prediction of how metabolism responds to change. To try to increase our ability to predict how single‐gene changes result in global metabolic shifts, we studied how changing expression in the indolic glucosinolate chemical defense pathway in Arabidopsis thaliana influences production in the related aliphatic glucosinolate pathway.Aliphatic glucosinolates are synthesized in a pathway parallel to the indolic pathway and share a number of enzymes (SUR1, GGP1). Research on the aliphatic pathway has shown aliphaticspecific and shared gene knockdowns have no control over the indolic pathway. However, by knocking down activity of each step in the indolic pathway and measuring the production of all glucosinolates via High Performance Liquid Chromatography (HPLC), our results suggest crosstalk between these pathways.Preliminary results reveal that when the gene GTSF9 of the indolic pathway is knocked down, the production of aliphatic glucosinolates is reduced, but the production of indolic glucosinolates is not affected. These results are puzzling given that GSTF9 is not thought to be shared between the pathways like the enzymes SUR1 and GGP1. Indeed, previous research suggests GSTF9 is predicted to act in the aliphatic pathway exclusively. To further study this pathway cross‐talk, we will extract and sequence RNA from single‐gene knockouts of the genes in the indolic pathway to track how changes in gene expression of individual enzymes change global patterns of expression. These results will allow us to begin to understand the genetic mechanisms that mediate cross‐talk between these pathways.Support or Funding InformationSD BRIN P20GM103443 SD EPSCOR IIA‐1355423This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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