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Jarod Hughes and George Inglis

Variation in Alcohol Dehydrogenase a cross P. leucopus Populations Jarod Hughes and George Inglis. Jarod Hughes and George Inglis. Introduction. Alcohol dehydrogenase (ADH) encompasses classes of enzymes that break down ethanol and other –OH compounds

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Jarod Hughes and George Inglis

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  1. Variation in Alcohol Dehydrogenase across P. leucopusPopulationsJarod Hughes and George Inglis Jarod Hughes and George Inglis

  2. Introduction • Alcohol dehydrogenase (ADH) encompasses classes of enzymes that break down ethanol and other –OH compounds • Six different classes (ADH I – ADH VI), each highly variable • Multiple alleles for each enzyme • Looking at Dr. Nelson’s data of all the P. leucopusspecimens, we noticed the variety of different ADH-I alleles (A, B, C, or D)

  3. Goal • study differences in expression of the alleles across various populations • To start off, chose specimens from the list that were homozygous for alleles and from different localities

  4. Initial Specimen Collection Hard to find D alleles, so had to resort to using heterozygotes

  5. Lab Work / Methods • Needed to verify DNA was viable before testing • Ran DNA on gels to check for bands. • Ran 8 samples on the first gel: • 20872 • 20826 • 20769 • 19979 • 19961 • 19936 • 19927 • 19949 • Only one didn’t have good DNA (19936), couldn’t use

  6. Lab Work / Methods • With good DNA, could start to use primers to isolate specific sequences of DNA and use PCR • Looked at GenBank data to find potential primer sequences for exons 1 and 6 • Studied M. musculusADH-IA sequences • Ultimately we got primers for exons 3-5 and 6-7, since Dr. Nelson made extras • Started running gels with them

  7. Gel 1 (exons 6-7) • DNA Ladder • 20826 • 19927 • Control

  8. Gel 2 (exons 2-5) (from right to left) • DNA ladder • 20826 • 20769 • 19979 • Negative control • 19927 • 19949

  9. Gel 3 (exons 2-5 / 6-7) (from right to left) • DNA Ladder • 20872 (exons 2-5) • 19979 (exons 2-5) • 20872 (exons 6-7) • 20869 (exons 6-7) • 19979 (exons 6-7) • 19949 (exons 6-7) • Negative Control (exons 6-7)

  10. Overall Results -19936 didn’t have viable DNA -29961 had too little DNA for later tests

  11. Looking Back… • Always used TD 60, standard settings • Didn’t get DNA for all of the specimens, but could have done so if tried PCR at different settings • Didn’t get a chance to do DNA sequencing, so couldn’t look at actual sequence variation between them

  12. Ideally... • Would have gotten sequence data for all of the DNA we managed to isolate with the primers • See just how different P. leucopus from different areas were (i.e. how different the alleles were, or if there was intra-allele variation based on location) • Make a tree with MEGA • From there, test more samples of each allele from each locality, see if the differences hold constant on a larger scale

  13. For Further Research • Get live samples of mice from studied localities, perform tests to observe breakdown (dehydrogenation) of ethanol • Do real-time PCR to determine any differences in how much ADH is expressed • Analyze other classes of ADH, since we were just focusing on ADH-I • Examine environmental differences between localities

  14. Conclusions • From an evolutionary viewpoint, could be the start of description of new, divergent species • More experimentation needs to be done, but the primers work (maybe, might not result in exceptionally clean DNA sequences) • Could better the general understanding of how ADH works • Need more time and DNA to make real progress

  15. Works Cited • Höög, Jon-Olov, Jesper J. Hedberg, PatrikStrömberg, and Stefan Svensson. "Mammalian Alcohol Dehydrogenase." Biomedical Science (2001): 71-76. PubMed. NIH, 2001. Web. 3 Sept. 2011. <http://www.ncbi.nlm.nih.gov/pubmed/11173978>. • Nelson, Kimberyln, Robert J. Baker, and Rodney L. Honeycutt. "Mitochondrial DNA and Protein Differentiation between Hybridizing Cytotypes of the White-Footed Mouse, PeromyscusLeucopus." Evolution 41.4 (1987): 864-72. Print. • Stangl, Frederick B. "Aspects of a Contact Zone Between Two Chromosomal Races of PeromyscusLeucopus (RodenteCrictidae)." Mammology 67.3 (1986): 465-73. Print.

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