Transcript 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
• 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
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
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
Segregation of a Transposed Segment
ABC
DEF
Parents
Tetrads
AC
x
DBEF
5
6
ABC DBEF
ABC DBEF
ABC DEF
ABC DEF
ABC DEF
ABC DBEF
ABC DEF
AC DBEF
AC DEF
AC DBEF
AC DEF
AC DBEF
AC DEF
AC DBEF
AC DEF
AC DEF
AC DBEF
1
2
3
ABC DEF
ABC DEF
ABC DBEF
ABC DBEF
ABC DBEF
AC DEF
AC DBEF
4
1
1
1
1
2
0
Duplication
1
1
1
1
2
0
Deletion
2
2
2
2
0
4
Parental
1/6
1/6
2/3
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
Cy5
Cy3
S288C
S90
1
Arrays
Y101
7A
2
3
4
5
6
7B
7C
7D
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
We have ratio values for each
ORF and intergenic in each of
the four spores for each
tetrad (21 & 27)
Now what?
Questions?
Analysis of ratios
• The normalized hybridization log
ratios are used in analysis
1200
1000
frequency
• 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)
800
600
400
200
• Examined each spot in each spore
of each tetrad
• Threshold
-2.9-- 2.0
log(ratio)
3.7
3.1
2.4
1.8
1.2
0.6
-0.1
-0.7
-1.3
-1.9
-2.6
-3.2
-3.8
-4.4
• By inspection of the tails of the
distribution , specific upper and
lower threshold were chosen
common to all samples
-5.1
0
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
Map of Candidate Transpositions
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?
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
Expected PCR Band Patterns
S288C Y101 S90 21A 21B 21C 21D 27A 27B 27C 27D
Within
Rearrangement
+
+ +
+ +
+
Outside
Rearrangement
+
+ + + + + + + + +
+
Endpoints, or
primer
mismatch
+
-
-
+
-
-
+ + +
-
+ +
-
-
+ +
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
Deleted
Spore
S90 Copy
Y101 Copy
Duplicated
S90 Copy
Deleted
Spore
Y101 Copy
Endpoints, or primer mismatch
S288C Y101 S90 21A 21B 21C 21D 27A 27B 27C 27D
Endpoints, or
primer
mismatch
+
-
+
-
-
+ +
-
+ +
-
Duplicated
Deleted
Spore
Pre-Transposed
location in Y101
Transposed
location in Y101
S90 Copy
Y101 Copy
Duplicated
S90 Copy
Deleted
Spore
Y101 Copy
Characterization of Chromosome 15 Region
*
*
•Contains 5 genes
•Spans ~15kB of genomic DNA
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
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
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
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