Transcript Document

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