1. Biological Function of RMR2 in Maize: Genetic Study through Fluorescent Tagging of RMR2 Protein : Work In Progress Presented By Han Li Immediate Supervisor: Dr. Jacque Keele & Dr. Anding Luo Lab PI: Dr. Anne Sylvester

2. Outline Introduction Experimental outline and procedures Experimental results Conclusion

3. Central Dogma of Biology DNA (deoxyribonucleic acid)- genetic code Transcription- DNA  mRNA Translation- mRNA  protein This is followed by Mendelian inheritance

4. New Discoveries in genetics Silencing Epigenetic regulation- heritable changes in gene function without a change in DNA sequence Genes can be regulated by: Paramutation – An interaction between alleles of genes that leads to heritable changes in gene expression DNA Methylation Histone acetylation/deacetylation & methylation TGS and PTGS (Transcriptional Genomic Silencing and Post Transcriptional Genomic Silencing, Plants) dsRNA (RNAi) -Chandler, Vicki L. Cells (2007). 128: 641-645 -Griffiths AJF, Miller JH, Suzuki DT, et al. An Introduction to Genetic Analysis. 7 th. 2000. -McGinnis KM et al., (2006) Genetics, 173:1637-1647. B-I*B’ B-I* B-I*/B-I*B-I*/B’ B’ B-I*/B’B’/B’ X

5. Role of RMR Protein RMR2 (required to maintain repression 2) – Gene silencing the production of pigments – Epigenetics studies Transcriptional gene silencing – Regulates RNA polymerase – DNA methylation – Mutation are defective in paramutations RMR1 – Similar to RMR2 Can be reversed by reintroducing the wild type protein McGinnis KM et al., (2006) Genetics, 173:1637-1647.

6. Maize (Corn)- A common biological model used for genetic studies -Biello, David. That Burger You’re Eating Is Mostly Corn. Scientific American. Available from : http://www.scientificamerican.com/article.cfm?id=that- burger-youre-eating-is-mostly-corn --Corn. University of Illinois Extension: Watch your Garden Grow. Available from: http://urbanext.illinois.edu/veggies/corn.cfm - McGinnis KM et al., (2006) Genetics, 173:1637-1647. -ZeaMays. Wikipedia. Available from :http://en.wikipedia.org/wiki/File:ZeaMays.jpg

7. RMR2 in Regulation of Gene Expression in Maize McGinnis KM et al., (2006) Genetics, 173:1637-1647. RMR1 & RMR2 RMR2

8. Aim of the project  To study the role and expression of RMR2 on a molecular level – Fluorescent gene tagging DNA sequence coding for fluorescent protein – Citrine yellow » Produced in the protein product Fluorescent marker Protein product (rmr 2) Tether Gene fragment 1Gene fragment 2 Florescent tag Jackson, David. Sylvester, Anne. Chan, Agnes. Etc. Maize Cell genomics DB (2010). Available from: http://maize.jcvi.org/cellgenomics/

9. Experimental outline Use Multi site Gateway cloning to make the construct – Primer design – Amplification of Rmr2 DNA fragments – BP reaction – Sequencing – LR reaction Introduce the construct into Agrobacterium tumefaciens Transform maize at the Plant Transformation Facility (ISU) – Stable transgenic plant

10. Gateway Cloning Gateway cloning PCR (polymerase chain reaction) Plasmids Special flanking sequences User Manual for MultiSite Gateway Pro (2006) available from: http://www.invitrogen.com/vntigateway

11. Designing primers for Gateway cloning Determining the insertion position – The best position to insert the fluorescent protein is right after the start codon Should incorporate regulatory regions – 3000 bp upstream – 800 bp downstream Primer 3 (http://frodo.wi.mit.edu/) – p1: GGGG ACA AGT TTG TAC AAA AAA GCA GGC Tct agc cac ttg gct gta ctg tg – p4: GGGG AC AAC TTT GTA TAG AAA AGT TGG GTG cat ggt acc ggc ggt ctt gg – p3: GGGG ACA ACT TTG TAT AAT AAA GTT GAG ccg gtc ctc cgc tcc ccg t – p2: GGGG AC CAC TTT GTA CAA GAA AGC TGG GTA ctt gcc ggt gca gta gag tt Steve Rozenand Helen J. Skaletsky. Primer3. (2000) available at http://fokker.wi.mit.edu/primer3/ Promoter Terminator tag

12. p2p3 PCR rmr2 fragments Products are amplified and results were observed on an agarose gel: p1p4~ 3200bp p2p3~3800bp Fig 1Fig 2 p1p4 p2p3 3000 4000 3000 4000 Fluorescent tag p1p4p2p3

13. Gateway Cloning User Manual for MultiSite Gateway Pro (2006) available from: http://www.invitrogen.com/vntigateway

14. Gel purification and BP reaction Bands were cut out and gel purified. 1 µL of each product was loaded on a gel BP reaction was conducted to introduce the DNA fragments into the pDONOR vector. Kanamycin resistance The vectors are then introduced into the kanamycin sensitive bacteria (E. coli) through transformation. Heat shock Fig 3 4000 3000 P2P3P1P4

15. Bacteria were plated on kanamycin plates and were grown at 37 ◦ C overnight. Colonies are then subjected to colony PCR – Amplify the DNA sequences in the plasmid – Plasmid specific primers Colony PCR Fig 3 Fig 4 Samples 1-12 p1p4 Samples 13-23 p1p4 Control “-”

16. Colony PCR with Fresh Primers Fig 5Fig 6 p2p3 p1p4 Control “+” 3000 4000 5000 p2p3 p1p4 5000 3000 4000

17. Restriction Digest with HaeIII and HindIII Hae IIIHind III Davis, M. Wayne. APE. Version 2.0.44. 12 from: http://biologylabs.utah. edu/jorgensen/wayne d/ape/ P1P4 P2P3 3000 4000 400 1000

18. Work Progress and Conclusion  All steps prior to the BP reaction were successful  Bacteria have the plasmid  Survived on the kanamycin plates  Indicated that plasmid is present  Most of the bands are smaller than expected  Restriction digest confirmed that fragments of Rmr2 were inserted  Possible reasons:  Recombination alteration  Bacteria excreting plasmids out  Dimers  Maize sequence repeats Future Trouble Shooting Use a different bacteria vector Utilize only PCR reactions New primers Avoid Maize genome issues: common repeats, GC rich (different physical structure)

19. Acknowledgment Special thanks to: Dr. Anne Sylvester Dr. Jacque Keele Dr. Anding Luo Rest of Sylvester’s Lab EPSCoR NSF

20. Questions?

21. Work Cited Biello, David. That Burger You’re Eating Is Mostly Corn[internet]. Scientific American. [12 November 2008; 29 March 2012]. Available from : http://www.scientificamerican.com/article.cfm?id=that-burger-youre-eating-is-mostly-corn Chandler, Vicki L. Paramutation: From Maize to Mice. Cells [internet]. [2007; cited 12 April 2012]. 128: 641-645. Available from: ftp://ftpmips.gsf.de/plasmar/epigenetics/Cell%20review%20Issue/Paramutation%20- %20From%20Maize%20to%20Mice.pdf Commonly Used Promoters [internet]. African Biosafety Network Expertise. [2010; cited 29 March 2012] available from :http://www.nepadbiosafety.net/for-regulators/resources/subjects/biotechnology/commonly-used-promoters Corn [internet]. University of Illinois Extension: Watch your Garden Grow. [2012: 10 April 2012]. Available from: http://urbanext.illinois.edu/veggies/corn.cfm Corn and Sorgnum Pictures/corn ears [internet]. Texas A&M. [1 March 2002; cited 29 March 2012]. Available from: http://soilcrop.tamu.edu/photogallery/cornsorghum+/pages/corn%20ears.htm Davis, M. Wayne. APE [internet download]. Version 2.0.44. 12 April 2012. Available from: http://biologylabs.utah.edu/jorgensen/wayned/ape/ Jackson, David. Sylvester, Anne. Chan, Agnes. Etc. Characterizing Sub-cellular Compartments in Maize Using Fluorescent Protein Tagging Lines. Maize Cell genomics DB. [18 December 2010; cited 20 April 2012]. Available from: http://maize.jcvi.org/cellgenomics/ McGinnis KM, Springer C, Lin Y, Carey CC, Chandler V (2006) Transcriptionally silenced transgenes in maize are activated by three mutations defective in paramutation. Genetics, 173:1637-47. Griffiths AJF, Miller JH, Suzuki DT, et al. An Introduction to Genetic Analysis. 7 th. New York: W. H. Freeman; 2000. Steve Rozenand Helen J. Skaletsky (2000) Primer3 on the WWW for general users and for biologist programmers.In: Krawetz S, Misener S (eds)Bioinformatics Methods and Protocols: Methods in Molecular Biology.Humana Press, Totowa, NJ, pp 365-386 Source code available at http://fokker.wi.mit.edu/primer3/ User Manual for MultiSite Gateway Pro (Invitrogen 2006) Using gateway technology to simultaneously clone multiple DNA fragments. Available from: http://www.invitrogen.com/vntigateway ZeaMays [Internet]. Wikipedia. [2012; cited 29 March 2012]. Available from :http://en.wikipedia.org/wiki/File:ZeaMays.jpg