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Research Experience in Molecular Biotechnology & Genomics Summer 2007 Center for Integrated Animal Genomics Roles of mop 1 (RdRP) in the Accumulation of Natural Antisense Transcripts in Maize Pamela Reed1, Yi 1The Jia2, Kazuhiro Ohtsu2, and Patrick S.Schnable2 University of New Mexico, Albuquerque, New Mexico, 2 Iowa State University, Ames, Iowa INTRODUCTION Transposon Insertion in mop 1 Gene Results of Gel Electrophoresis Mu-TIR Although most of us learned that DNA is a double stranded helix and that RNA was a single stranded molecule, double stranded RNAs also exist. These double stranded RNAs are comprised of complementary sense and antisense strands. Natural Antisense Transcripts (NATs ) have been discovered in many eukaryotes including plants, animals, and humans (Lapidot et al., 2006). NATs appear to police the organism in both the nucleus as well as the cytoplasm, directing changes anywhere from transcription to translation (Vanhee-Brossollet, 1998). NATs policing activities have yielded some unwelcomed effects, especially in humans; there is strong evidence suggesting that NATs can allow cancer to grow by down regulating tumor suppressors (Lavorgna et al., 2004). What is the origin of natural antisense transcripts? rdrp is an RNA-dependent RNA polymerase 2 gene in Zea mays L.that we hypothesize may be connected to antisense transcription. Finding the starting location of antisense strand production may help researchers to unravel the threads of its tapestry. MATERIALS & METHODS Plant materials and experimental design Kernelsfrom mop1 homozygous or heterozygous Zea mays L. were planted in the growth chambers (Percival Scientific, Perry, IA) at ISU. Each growth chamber provided 15 hours of light at 25 degrees followed by 9hours of darkness at 20 degrees. Tissues were harvested from 14 day old seedlings which were labeled, wrapped in aluminum foil, flash frozen in liquid nitrogen, and stored at -80 until ground into powder. RNA isolation, reverse transcription, and labeling The RNeasy Mini Kit (Qiagen, Valencia, CA) was used to isolate the RNA from the tissue powder following the manufacturer’s protocol. Random primer was used with 80 ug total RNA as template for each reverse transcription reaction. Specific cDNA samples were labeled in the dark with Cy3 and Cy5 fluorescent dyes. Samples were quantified using a Nanodrop spectrophotometer. Microarray hybridization and analysis Three biological replications were performed by hybridizing labeled cDNA samples with our custom microarray. Following hybridization, arrays were scanned and images quantified. Lowess normalization was performed to remove intensity-dependent bias. Mixed model was used for expression pattern analysis. RESULTS & DISCUSSION Plant genotyping Two pairs of primers (rdrf3/rdrr2, rdrf3/Mu-TIR) were used for genotyping to identify the homo- and (Figure 1). Twelve samples were geneotyped as heterozygous and seven sampheter-zygous mop1 plants les were geneotyped as homozygous (Figure 2). ~500bp Rdrf3/rdrr2 rdrf3 rdrr2 671 bp homozygous heterzygous rdrf3/rdrr2 + rdrf3/Mu-TIR + + Figure 1: Diagram of genotyping design for mop 1 plants Rdrf3/Mu-TIR Figure 2: mop1 plants (3766-1) thru (3766-18) And positive control (3766-19) NATs detection via microarray experiment Labeled cDNA samples from above-ground, two-week-old homo- and heter-zygous mop1 seedlings were hybridized to the microarray (Figure 3). Microarray data were normalized and transformed to reduce non-biological variation and facilitate comparison of signal intensities across arrays. Based on the preliminary microarray analysis, there is no evidence that NATs accumulation was significantly different between the homo- and heter-zygous mop1 plants. Thus, the hypothesis that the roles of rdr2 on the NATs accumulation is not supported. One explanation is that the rdr2 has some functional redundancy with some other rdr genes in the maize genome. Hybridization Images Homo Heter Figure 3 Microarray hybridzation images for homo- and heter-zygous samples ACKNOWLEDGEMENTS We thank Pengcheng Lu for microarray image quantification, as well as Cheng-Ting “Eddy” Yeh and Michael Miller for technical assistance. This program was funded by the National Science Foundation. We thank professor Max Rothschild for leading the Research Experience in Molecular Biotechnology and Genomics Program at Iowa State University. Pam thanks professor Mary Anne Nelson of UNM (main campus) for inspiring her to look into summer internships. Pam also thanks professors Claudia Barreto and Celestyn Brozek of UNM-Valencia for writing letters of recommendation. on her behalf. REFERENCES Lapidot M., Pilpel Y. (2006) Genome-wide natural antisense transcription: coupling its regulation to its different regulatory mechanisms.EMBO Reports 7: 1216-1222 Lavorgna G., Dahary D., Lehner B., Sorek R., Sanderson CM, Casari G., (2004) In search of antisense. Trends Biochem Sci 29:88-94 Vanhee-Brossollet C., Vaquero C. (1998) Do natural antisense transcripts make sense in eukaryotes? Gene: An International Journal on Genes and Genomes 211: 1-9 Program supported by the National Science Foundation Research Experience for Undergraduates DBI-0552371