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Gene Order Polymorphism in Yeast

Gene Order Polymorphism in Yeast. Dina Faddah Vision Lab Meeting- February 18, 2005. Background. Differences in the order of genes on chromosomes among individuals within a population may influence expression at those affected loci

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Gene Order Polymorphism in Yeast

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  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 • Detecting Transposition-Using DNA Microarrays and SpotProb • Characterization of a Candidate Transposed Region using PCR assays • 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. ABC DEF AC DBEF Segregation of a Transposed Segment Parents x 6 1 2 3 4 5 ABC DEF ABC DBEF ABC DBEF ABC DEF ABC DEF Tetrads ABC DBEF ABC DBEF ABC DEF ABC DEF ABC DBEF ABC DEF ABC DBEF AC DBEF AC DEF AC DBEF AC DEF AC DBEF AC DEF AC DEF AC DBEF AC DEF AC DEF AC DBEF AC DBEF 1 1 1 1 2 0 Duplication 1 1 1 1 2 0 Deletion 2 2 2 2 0 4 Parental 2/3 1/6 1/6

  6. 7A 7B 7C 7D Methods • Genomic DNA is extracted from each parent (S90 and Y101) and from the four spores in a tetrad (7, 21, 27, 36) • The reference DNA is labeled with one fluorescent dye, Cy3, and the sample DNA is labeled with another fluorescent dye, Cy5 • The reference is mixed with the sample and then hybridized onto an array Cy5 Cy3 S288C S90 Y101 1 2 3 Arrays 4 5 6

  7. DNA Microarrays • 14,097 spots on the array cover the entire yeast genome, including intergenic regions Normal (1 copy) 1:1 Ratio of Cy3 and Cy5 Deleted (0 copies) Low ratio of Cy3:Cy5 Duplicated (2 copies) High ratio of Cy3: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: • Within the thresholds in the parent samples • 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 • 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 S288C Y101 S90 21A 21B 21C 21D 27A 27B 27C 27D Within Rearrangement + + + - + + + - + + + Outside Rearrangement + + + + + + + + + + + Endpoints, or primer mismatch + - + - - + + - + + -

  15. Within Rearrangement -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 S288C Y101 S90 21A 21B 21C 21D 27A 27B 27C 27D Within Rearrangement + + + - + + + - + + + Duplicated Duplicated S90 Copy Deleted Spore Deleted Spore S90 Copy Y101 Copy Y101 Copy

  16. Endpoints, or primer mismatch S288C Y101 S90 21A 21B 21C 21D 27A 27B 27C 27D Endpoints, or primer mismatch + - + - - + + - + + - Duplicated Duplicated S90 Copy Deleted Spore Deleted Spore S90 Copy Y101 Copy Pre-Transposed location in Y101 Y101 Copy Transposed location in Y101

  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 • How transposition of the five genes affects their gene expression • What the frequency of this rearrangement is among a larger sample of natural yeast strains • 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

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