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Research Experience in Molecular Biotechnology & Genomics Summer 2008 Center for Integrated Animal Genomics Samir M. A’agha1; Dr. Diane Moody Spurlock1; Dawn Elkins1;Hyelee Park1 1Department of Animal Science, Iowa State University, Ames, Iowa 50011 Variation Analyses in a Holstein population in three genes: ATGL, CG1-58 and Perilipin for Lipolysis •No variation was found in the targeted regions of perilipin or beta-adrenergic receptor. Introduction •During early lactation in dairy cows a negative energy balance is created. This negative energy balance occurs when energy used for milk production exceeds the amount of energy consumed. •To balance this deficiency, energy substrates stored in adipose tissue are mobilized. Objective Identify DNA sequence variation (SNP, single nucleotide polymorphisms) for genes that participate in the regulation of lipolysis. Ultimately, associations between alternate alleles of these genes and energy balance traits will be evaluated. •In our study we are looking at four genes, perilipin, beta-adrenergic receptor 1, CGI-58 and ATGL, which are related to the mobilization of energy from adipose tissue. We hypothesize that sequence variation in these genes is associated with energy balance. Materials and Methods • Perilipin that has been phosphorylated acts together with hormone-sensitive lipase to mobilize triacylgycerols from adipose tissue. • ATGL catalyzes the initial step in triglyceride hydrolysis in adipocyte and non-adipocyte lipid droplets. ATGL also has acylglycerol transacylase activity, regulates adiposome size, and may be involved in the degradation of adiposomes. • ATGL plays an important role in energy homeostasis and response to starvation, enhancing hydrolysis of triglycerides and providing free fatty acids to other tissues to be oxidized in situations of energy depletion. • CGI-58 interacts with both perilipin and ATGL, although its role in regulating lipolysis is not well defined. •A SNP resulting in an amino acid change has been reported in the Ensembl database for ATGL. However, our primers failed to amplify this targeted region of ATGL. No other SNP were identified in ATGL. •CGI-58 was not successfully amplified by the primers. Discussion • Perilipin modulates adipocyte lipid metabolism. When unphosphorylated, perilipin coats lipid droplets and prevents the breakdown of triacylglycerols by hormone-sensitive lipase. • Beta-adrenergic receptors are activated when lipolysis is stimulated. Activation of these receptors leads to a cascade of events that result in phosphorylation of hormone sensitive lipase and perilipin. Results Figure 1. Assiting in bleeding a cow via the jugular vein. Figure 2. Extracting serum from blood. •Nine Holstein cows DNA were used in this study. •Blood for DNA was collected via jugular vein in two, 10 ml tubes with EDTA. •Buffy coat was extracted from blood. •DNA was extracted from buffy coat. •Working solutions were prepared for use in PCR. •PCR products were designed. •PCR was used to amplify a region of the genes of interest. •Agarose gel electrophoresis was done to evaluate PCR products. •PCR product was prepared for sequencing. •DNA was sequenced in the DNA Sequencing Facility of Iowa State University. The SNP in ATGL described in the Ensembl database would be of great interest in investigating the function of ATGL in lipolysis. Although the PCR primers used amplified a region of ATGL, the size of the PCR product was smaller than expected and did not include the potential SNP. Further work with additional PCR primers is needed to validate the presence of this SNP in Holstein cattle. Figure 6. SNP not being reached by the amplification of primers during PCR. References Primers ATGL FORWARD 5’- GCA CCC TTC CTT CAA CAT GG- 3’ ATGL REVERSE 5’-ATC CCT GTA GCC CTG TTT GC-3’ Primers for Perilipin and beta andregenic receptor were provided by Dr. Diane Spurlock lab. ATGL primers were design by myself and made by Integrated DNA Technologies. •James G. Granneman, Hsiao-Ping H. Moore, Rachel L. Granneman, Andrew S. Greenberg, Martin S. Obin, and Zhengxian Zhu. 2006. Analysis of Lipolytic Protein Trafficking and Interactions in Adipocytes. •D. Elkins, H. Park and D. Spurlock. Phosphorylation of Perilpin is Associated with Lipolysis in Holstein Cows. Journal of Dairy Science Vol. 91 No. 7, 2008. pg 2919 • Selection of dairy cattle has focused largely on milk production traits for decades. Current trends suggest fitness traits, including health and reproduction, are compromised in favor of production traits. • We are looking for variation genes so that we may select cows to promote the balance of energy mobilization for milk production as well as fitness traits. Acknowledgements Figure 3. ATGL PCR bands in gel. Figure 4. Perilipin PCR bands in gel. Figure 5. Beta andregenic receptor PCR bands in gel. I would like to thank the NSF for funding my research, the REU program and staff, Max Rothschild for giving me the chance to participate in this program. My mentor, Dr. Diane Spurlock, for leading my way and bringing me support in this research. Also Dawn Elkins, graduate student, for helping me day by day on this process, Hyelee Park for always being available when I needed during my experiments. Program supported by the National Science Foundation Research Experience for Undergraduates DBI-0552371