1. Gene Order Polymorphism in Yeast Dina Faddah Vision Lab Meeting- February 18, 2005

2. Background Differences in the order of genes on chromosomes among individuals within a population may influence expression at those affected loci We do not know how frequently such variations in gene order occur among individuals in a population We do not know the degree to which such differences in chromosomal location affect gene expression at those transposed loci

3. Outline I.Detecting Transposition-Using DNA Microarrays and SpotProb II.Characterization of a Candidate Transposed Region using PCR assays III.Joe & Eric  looking at the expression data and utilizing genome mismatch scanning to predict the genomic locations of transposed segments in Y101

4. Detecting Transpositions Using Comparative Genomic Hybridization on a Microarray (CGHM) to detect genomic segments that have transposed between two stains of yeast (Saccharomyces cerevisiae) S288C- sequenced reference strain S90- thought to be very similar to S288C Y101- known to lack 4 open reading frames (ORFs) which are present in S288C Transposed: Equal copy number, but at non-syntenic positions

5. Parents 1/6 2/3 Tetrads ABC DEF AC DEF AC DBEF ABC DBEF ABC DEF ABC DBEF AC DEF AC DBEF ABC DEF ABC DBEF AC DBEF AC DEF ABC DBEF ABC DEF AC DBEF AC DEF ABC DBEF AC DEF ABC DEF AC DBEF 12543 6 Parental 402222 Deletion 021111 Duplication 021111 x ABC DEF AC DBEF Segregation of a Transposed Segment

6. Methods 1.Genomic DNA is extracted from each parent (S90 and Y101) and from the four spores in a tetrad (7, 21, 27, 36) 2.The reference DNA is labeled with one fluorescent dye, Cy3, and the sample DNA is labeled with another fluorescent dye, Cy5 3.The reference is mixed with the sample and then hybridized onto an array S288CY101S90 7B7C7D7A Arrays Cy5 5 6 4 3 2 1 Cy3

7. DNA Microarrays 14,097 spots on the array cover the entire yeast genome, including intergenic regions Duplicated (2 copies) High ratio of Cy3:Cy5 Deleted (0 copies) Low ratio of Cy3:Cy5 Normal (1 copy) 1:1 Ratio of Cy3 and Cy5

8. We have ratio values for each ORF and intergenic in each of the four spores for each tetrad (21 & 27) Now what? Questions?

9. Analysis of ratios The normalized hybridization log ratios are used in analysis The spot intensities were normalized for each sample separately by transforming the logarithm of the ratio to a standard normal distribution (subtracting the mean and dividing by the std dev) By inspection of the tails of the distribution, specific upper and lower threshold were chosen common to all samples Examined each spot in each spore of each tetrad Threshold -2.9-- 2.0

10. SpotProb Each spot was classified as being: –Below the lower threshold (Deletion) –Within the thresholds –Surpassing upper threshold (Duplication) SpotProb searches for spots with the following criteria: I.Within the thresholds in the parent samples II.In a given tetrad, it is duplicated in one spore, deleted in one spore, and present in equal copy number in the other two spores III.Or, in a given tetrad, there is a duplication or a deletion in one spore, but not both 84 spots were identified as candidate transpositions (I & II) and (I & III) 3 spots fit criteria (I & III) TOTAL: 57 candidate transposed segments

11. Map of Candidate Transpositions

12. Refresh We’ve identified 57 putative transposed segments between S90 and Y101 using DNA microarrays and SpotProb. Lets look closer at the region on Chromosome 15 in which the three spots show a 1:1:2 pattern Questions?

13. PCR Assays Each open reading frame (ORF) and intergenic within the area of interest was amplified in the two parents (S90 and Y101) and all four spores in a tetrad Analysis of the PCR band pattern among the parental strains and two tetrads allowed us to characterize segments within, outside, or on the boundaries of the transposed region

14. Expected PCR Band Patterns -++-++--+-+ Endpoints, or primer mismatch +++++++++++ Outside Rearrangement +++-+++-+++ Within Rearrangement 27D27C27B27A21D21C21B21AS90Y101S288C

15. Within Rearrangement +++-+++-+++ Within Rearrangement 27D27C27B27A21D21C21B21AS90Y101S288C -Spots termed as ‘Within Rearrangement’ are also termed ‘transpositions’ - Remember, for a transposed segment, within a tetrad, 1 spore will be missing the segment, 1 spore will have two copies of the segment, and 2 spores will each have a single segment Deleted Spore Y101 Copy S90 Copy Duplicated S90 Copy

16. Endpoints, or primer mismatch -++-++--+-+ 27D27C27B27A21D21C21B21AS90Y101S288C Transposed location in Y101 Pre-Transposed location in Y101 Deleted Spore Y101 Copy S90 Copy Duplicated S90 Copy

17. Characterization of Chromosome 15 Region Contains 5 genes Spans ~15kB of genomic DNA **

18. Refresh We have identified 57 putative transposed segments using DNA microarrays and SpotProb We have used PCR assays to delineate the extent of the Chromosome 15 region

19. Current Work Hybridization of additional tetrads (21, 36, 7) to allow for better predictions of where these transposed segments are located in Y101 2/3 of the tetrads should show the 1:1:2 pattern for a transposed segment We are using these transpositions as markers to map Y101

20. Future Work Contour-clamped homogeneous electric field (CHEF) analysis will be used to determine the exact chromosomal location of the transposed segment in Y101 We would also like to examine a.How transposition of the five genes affects their gene expression b.What the frequency of this rearrangement is among a larger sample of natural yeast strains c.Whether there are any clues as to the transposition mechanism in the sequences in and around the transposed segment

21. Conclusion Developing a new procedure to systematically identify transpositions Using CGHM in a novel way. Now we are no longer limited to preselected or adjacent regions We can make a genome-wide map of transposed segments Hopefully, our procedure will be applied to more complex eukaryotic genomes in the future