Center for Integrated Animal Genomics Research Experience in Molecular Biotechnology & Genomics Summer 2008 Regina S. Nickel, Toya Lawrence, Becky Weeks, Erik Vollbrecht Program supported by the National Science Foundation Research Experience for Undergraduates DBI Ac-Ds belong to a family of transposons in maize. Transposons are genetic elements that can move throughout the genome. The Ac transposon is an autonomous element that encodes for transposase which gives transposons in the Ac/Ds family the ability to move. In our system a Ds transposon is positioned in a color gene. This knocks out the function of the gene, giving the kernels a yellow color. When Ac is present it can mediate the mobilization of Ds causing it to leave its original location. Figure 1. Mobilization of Ds transposons affect kernel color The overall goal of this project is to determine if a transposon near a gene of interest can be used for local mutagenesis. The purpose of my part of the project is to genotype several lines and determine if a Ds transposon is near a particular gene of interest. These lines will be crossed to initiate remobilization. In the fall, progeny generated from these crosses will be screened for novel insertions in genes of interest Primers were designed for each line using the MacVector program. The sequence flanking the Ds insertion (fDs sequence) was BLASTed against the maize B73 inbred sequence at NCBI to retrieve more sequence surrounding the insertion site. The fDs sequence was then aligned to the NCBI sequence using the ClustalW program. This allowed us to determine the Ds insertion site. Sequences then were BLASTed on ZMGDB to find EST sequences. These sequences are best for primers because they are coding sequences and will have fewer differences between W22 and B73 inbreds. From these sequences, primers were designed 500 bp upstream and downstream from the insertion site using Primer3. PCR was used to test the primers. DNA extractions were performed on each line using the Puregene/Qiagen DNA extraction kit The lines were genotyped using PCR. Ds fDsfDs LC18 JSR01 PvuII Intramolecular Ligation Digest cuts here JSR01 During PCR, the DNA would be amplified from the upstream Ds primer to the downstream gene primer when the transposon is present in the gene of interest. If the transposon is present, a band will appear when the sample is run in an ethidium bromide gel. The size of the band would be around 600bp. Figure 2 IPCR schematic. Picture modified from figure by Justin Schares Figure 3. Orientation of gene specific primers and Ds primers relative to transposon and insertion site. We were able to develop assays for PCR for each gene of interest to test for insertions of Ds. Lines genotypedSamples with insertion B.S R2,3,5 I.S ,2 B.W ,4 I.S ,3,5,6,13,14 I.S ,7,8 I.S ,3,4,6,7 I.S B.S ,3,4,6,7 I.W ,3,8,10 I.S ,7,8,13 I.W B B.S B.S B.W I.S B.S B.S B.S R B.S R B.S R I.S ,2,6,7 5,6 1,2,3 2,3,7,8,9,13 1,2,6 9,11,13 2,3,4,5,6,7,9,14 1,2,6 1 1,5,6,9 2,3,4,6,7 Table 1 Subset of lines genotyped for Ds insertions. Parental Band Mobilized Band Bands Confirming Ds Insertions Figure 4. Example of PCR confirming Ds insertion. In cases where both gene primers failed, new primers were designed from flanking Ds sequences. Since these sequences were derived from the original IPCR, they should be a perfect match to the W22 sequence. In all, 25 lines tested positive for the presence of Ds near the genes of interest Plants with the insertion were tagged and will be outcrossed to initiate remobilization. Progeny will be screened in the fall for novel insertions in genes of interest. When purple kernels are found, flanking sequences are cloned via IPCR (Figure 2) Abstract Introduction Objectives Materials and Methods Ds Color gene Novel insertion site Ds LC18 PvuII Digestion PCR Ligation site PCR products are cloned to isolate the transposed Ds band. Sequence generated from the clones is deposited into a database which can be searched by scientists looking for insertions in particular genes. But what if a Ds element is located close to, but not in a gene of interest? Can it be used for local mutagenesis? In other words, can it be mobilized into the nearby gene? The goal of this project is to determine if a Ds transposon can be used for local mutagenesis. Lines were chosen that possessed Ds insertions near genes of interest. These lines were genotyped using a special PCR assay and tagged for outcrossing to initiate remobilization. We will screen the progeny of these crosses to determine if Ds elements have inserted into the designated genes. If successful, researchers will be able to search Ds Gene-specific primer Gene-specific primer Ds primer Results Conclusions References A special thanks to Erik Vollbrecht for hosting me in his lab and to the members of the Vollbrecht lab for their technical help and advice throughout the project. I would also like to thank Dr. Max Rothschild for his guidance and for giving me the opportunity to participate in the REU Summer Internship program. Last, I would like to thank Linda Wild for working so hard to get me into the REU Summer Internship program. Conrad, L.J. and Brutnell, T.P.(2005). Ac-Immobilized, a stable source of Activator transposase that mediates sporophytic and gametophytic exision of Dissociation elements in Maize. Genetics McCarty, D.R., Settles, A.M., Tan, B.C., Latshaw, S., Porch, T., Robin, K., Bairer, J., Avigne, W., Lai, J. et. al (2005) Steady-state Transposon Mutagenesis in inbred Maize. Plant J. 44, Using the Ac-Ds transposon system in maize as a tool for forward mutagenesis fDs