Melissa Ann Metzler and Sarah Todd
Melissa Ann Metzler
Melissa Ann Metzler was an active researcher during her four years at Bellarmine, but gained that experience by working in four different research laboratories. During her first summer in college she worked in Dr. Pete Mirabito’s lab at the University of Kentucky. This lab studied the genes involved in regulating mitosis in fungi. After that she worked in the lab of Dr. Nick Delamere in the Department of Medicine at the University of Louisville. There Melissa studied the effect of a drug on the cell signaling process in, of all things, the kidney cells of the opossum! This research resulted in the publication of a refereed journal article entitled “Ouabain induces cell proliferation through calcium-dependent phosphorylation of Akt (protein kinase B) in opossum kidney proximal tubule cells” in the American Journal of Physiology and Cell Physiology in December, 2006. After working with opossum kidneys, Melissa worked on campus at Bellarmine’s Department of Biology in the lab of Dr. Steven Wilt. There she studied the underlying causes of retinal detachment (a kind often seen in diabetics). Later, Ms. Metzler conducted research with Dr. Joann Lau and Dr. Dave Robinson at Bellarmine. Her project involved inserting the gene from glowing fireflies (luciferase) into plants with the objective of developing glowing plants. The abstract below is from a poster she co-authored and presented at the Botany and Plant Biology Joint Congress in Chicago, IL in 2007. Ms. Metzler has also presented her research at the Kentucky Academy of Sciences (twice).
Glowing Results: A Laboratory Exercise for Demonstrating Plant Transformation
Melissa A. Metzler, Whitney S. Theis, Ted T. Miles, Dave L. Robinson and Joann M. Lau
Department of Biology, Bellarmine University, Louisville, KY, USA, 40205
The objective of this experiment was to develop an Agrobacterium-mediated transformation laboratory exercise for undergraduate students. The firefly luciferase gene driven by the constitutively-expressed Cauliflower Mosaic Virus (CaMV) promoter was transformed into Arabidopsis, thus allowing the plant to become bioluminescent. The plasmid construct, pLuk07 (GenBank Accession U84006), was obtained from the Arabidopsis Biological Resource Center (Ohio State University). This construct contains an intron which prevents expression in Agrobacterium thus allowing for early detection following transformation. After electroporation into A. tumefaciens, and plating on YM (Yeast Extract/Mannitol) medium containing ampicillin, wild-type Arabidopsis plants were transformed twice using the floral-dip method. After seeds matured, they were collected, sterilized, and screened on MS (Murashige and Skoog) Basal Medium containing ampicillin. Seedlings were grown to the cotyledonary stage and transplanted into soil. A subset of these seedlings were assessed for bioluminescence by soaking in either 1, 5, or 10 mM luciferin for 10 hours and transferred to x-ray film for 16 hours. Untransformed wildtype Arabidopsis seedlings served as a negative control. The intensity of illumination was greatest in transgenic seedlings incubated in 10 mM luciferin, whereas untransformed seedlings produced no detectable luminescence. Mature transformed plants (T0) were screened for transgene insertion by PCR (using luciferase-gene primers) and gel electrophoresis. This project is an effective means of demonstrating to students important molecular techniques like restriction mapping of a plasmid, floral-dip transformation using Agrobacterium, antibiotic-screening, autoradiography, DNA extraction, PCR, gel electrophoresis, and reporter analysis using a constitutively-expressed promoter. It can be accomplished in a single 4-month semester and is appropriate for courses in Plant Biology, Plant Molecular Biology, Biotechnology, or Senior Research.
Sarah Louise Todd
Sarah Louise Todd worked in the lab of Dr. Mary Huff beginning in her freshman year. Her research was on the effect of two known carcinogens (cadmium chloride and sodium arsenate) on the growth of lung cancer cells. She found that these two chemicals induce proliferation in female cancer lines, but not in male lines. Since these two chemicals have similar characteristics to the female hormone estrogen, Ms. Todd also did research to show that they have the same effect on inducing transcription of estrogen-response genes. She has presented her research at the Kentucky Academy of Sciences (for 3 years) and at the Butler University Undergraduate Research Conference for 3 years. Ms. Sarah Todd is currently in medical school at the University of Louisville.
Role of environmental estrogens in the etiology of human lung adenocarcinoma cells
Sarah L. Todd1, Allison S. Bleser1, Susan M. Dougherty2, Carolyn M. Klinge2 and Mary O. Huff1
1Department of Biology, Bellarmine University, Louisville, KY 40205 USA and
2Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, Louisville, KY 40202
Of the 65 carcinogens in cigarette smoke, cadmium chloride and sodium arsenate are considered environmental estrogens because they mimic the effect of estrogen in living systems. Since recent studies support a role for estrogen in the etiology of lung cancer, we wanted to determine if these environmental estrogens could stimulate cellular proliferation in a similar manner. Treatment of a female derived adenocarcinoma cell line H1793 for 4 days with nanomolar concentrations of cadmium chloride or sodium arsenate induced cellular proliferation similarly to estrogen. Furthermore, inhibition of the estrogen signaling pathway using an estrogen receptor antagonist ICI 180,780 partially reduced the observed proliferation induced by treatment with these environmental estrogens supporting the involvement of the estrogen signaling pathway. To determine if these environmental estrogens can directly activate transcription of estrogen responsive genes, a reporter gene was transfected into the same h1793 cell line, which was then treated for 30 hr with estrogen, cadmium chloride or sodium arsenate. The results suggest that like estrogen, cadmium chloride and sodium arsenate activate transcription. To determine the gene responses induced by these environmental estrogens, RNA is being isolated after treatment and the expression of a group of estrogen responsive genes will be analyzed.