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Gene Order Polymorphism in Yeast
Introduction
We are using Comparative Genomic Hybridization on a Microarray to detect
chromosome segments that have transposed between two divergent strains
of yeast (Saccharomyces cerevisiae). While parental strains will each have a
single copy of a transposed segment, the recombinant haploid progeny can
have either 0, 1 or 2 copies of the same segment, depending on the pattern
of segregation in the tetrad (Fig 1).
Fig 1. A cross between yeast strains carrying a transposition. Two
chromosomes are shown in each parent, differing in the position of
segment B (red). Segregation in meiosis leads to six different tetrads with
equal probability. In one of the tetrads (5), B is duplicated in two spores
and deleted in the other two. Four of the tetrads (1-4) have one duplication,
one deletion, and two expected copy numbers. The remaining tetrad (6)
has no duplications or deletions.
Parents
Tetrads
1
ABC DEF
3
2
ABC DEF
ABC DBEF
4
5
6
ABC DBEF
ABC DBEF
ABC DEF
ABC DBEF
ABC DEF
ABC DEF
ABC DBEF
Cy3
S288C
S90
Array 1 2
21A
Y101
AC DBEF
AC DEF
AC DBEF
AC DEF
AC DBEF
AC DBEF
AC DEF
AC DBEF
AC DEF
AC DEF
AC DBEF
1
1
2
1
1
2
1
1
2
2/3
1
1
2
21B
21C
21D
2
2
0
0
0
4
1/6 1/6
600
400
200
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
0
Fig 6. Map of candidate
transpositions. The 16
chromosomes of yeast are shown.
Each spot is represented to scale by
a blue bar. Within each chromosome,
ORFs on the Watson strand are
shown above and Crick strand below;
intergenic regions are shown in the
middle. The 84 candidate
transpositions are indicated by red
bars. Clusters of candidates can be
seen at nine positions, all on separate
chromosomes. The clump containing
the three spots of greatest interest is
shown in the inset.
3 4 5 6
Fig 3. A DNA Microarray. Each
spot on the array represents a
distinct chromosomal segment in
the yeast genome. The chip used
in these experiments contains
13,380 spots covering the entire 12
Mb yeast genome, including
intergenic regions. The color of
each spot represents the relative
hybridization intensity of Cy3 vs.
Cy5 labeled DNA.
Fig 4. Results of
hybridization against the
reference strain. The ratio of
Cy3 to Cy5 signal seen at
each spot represents the
relative abundance of
reference to sample DNA
complementary to the
reference DNA immobilized
at that spot.
The three spots of greatest interest are IYOL160W, IYOL158C, and NORF60, which all map to
a small region on chromosome 15. For these spots, one tetrad occurred with the pattern that
we would expect to see in 2/3 of tetrads in the presence of a transposition (one duplication, one
deletion, and two within the thresholds).
Normal (1 copy)
1:1 Ratio of Cy3 and
Cy5
Table 1. Normalized ratio values.
Shown are the normalized ratio values
for tetrad 27 and the parents. The
expected spore pattern of one deletion
(green), one duplication (red), and two
parental ratios is consistent in the three
most interesting spots. Two replicates
were performed for each spore in the
tetrad.
Deleted (0 copies)
Low ratio of Cy3:Cy5
Duplicated (2 copies)
High ratio of Cy3:Cy5
Characterization of a Candidate Transposed Region on Chromosome 15
Within
Rearrangement
Outside
Rearrangement
AC DEF
800
Each spot was classified as being below the lower threshold (a deletion), within the thresholds,
or surpassing the upper threshold (a duplication). We developed a computer program to search
for spots that were within the thresholds in the parental samples but that contained duplications
and/or deletions in the tetrads. Because of the apparent overlap in the ratios between putative
duplication, deletion, and equal-copy segments, we were interested in identifying spots for which
any of the tetrad spores exceeded either threshold. Eighty-four spots were identified as
candidate transpositions. Of particular interest were three spots containing at least one spore
below the lower threshold and one above the upper threshold, indicative of a potential
transposition.
We hypothesize an inversion within the candidate transposed segment, with boundaries between the
segments IYOL157C and IYOL155C-1 (Fig.7b). Currently, we are designing long PCR assays to test
our hypothesis. We have designed primers internal to and flanking both the chromosome 15 segment
and the suggested inverted region within the candidate segment. As indicated by the black arrows in
Figure 7b, two primary PCR assays will be used to test the suggested inversion. If our model of the
rearrangement is correct, this experimental design will result in easily-scored amplifications of the
indicated regions in Y101, while no amplification should occur in S90 or S288C.
Fig 7a. Classification of ORF and intergenic amplicons corresponding to spots on the
microarray. Shown below are the classifications of each ORF and intergenic spacer in the candidate
region, based on the PCR band pattern among the parental strains and two tetrads. Coordinates are in
S288C. Asterisks indicate two of the spots initially observed.
Fig 7b. Hypothesized Inversion in Y101. Shown below is the hypothesized model for an inversion in
Y101. Primers are displayed for the two primary PCR assays for validation of the inverted segment.
IYOLCDELTA2 is a putative primer mismatch.
Table 2. Expected PCR Band Patterns. Analysis of the PCR band pattern
among the parental strains (S90 and Y101) and two tetrads (21 and 27) were
used to classify spots as inside, outside, or on the boundaries of the
rearrangement. A (+) indicates a PCR sizeable amplicon, and a (-) indicates
a PCR non-amplicon.
ABC DEF
1000
Fig 5. Histogram of log
ratio values. Shown are
values for one spore of
tetrad 27. Lower and upper
thresholds are shown by
arrows.
log(ratio)
S288C Y101 S90 21A 21B
ABC DBEF
1200
Cy5
We developed Polymerase Chain Reaction (PCR) assays to delineate the
extent of the chromosome 15 region. Each open reading frame (ORF) and
intergenic spacer within the area of interest was amplified. Analysis of the
PCR band pattern (Table 2) among the parental strains (S90 and Y101) and
two tetrads revealed segments within, outside, and on the boundaries of the
transposed region (Fig. 7a/b).
AC
ABC
DBEF
DEF
x
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 standard
deviation). By inspection of the tails of the combined distribution, specific lower and upper
threshold were chosen common to all the samples (Fig 5).
Fig 2. Labeling of DNA. The genomic DNA, including the parents and the
tetrads, was mixed with the reference DNA, S288C, and subsequently
hybridized onto an array.
Differences in the chromosomal position of genes among individuals may affect
the transcriptional regulation of those genes and thus contribute to phenotypic
variation. However, we do not know how frequently such variations in gene
location occur among individuals within populations. Additionally, we do not
know the degree to which such differences in chromosomal location affect gene
expression at the transposed loci. We are studying this issue using Comparative
Genomic Hybridization on a Microarray (CGHM) to detect genomic segments
that have transposed between two divergent strains of yeast. CGHM allows us
to determine whether a gene is duplicated, deleted, or present in the same copy
number between two genomes. To date, two tetrads from a cross between
strains Y101 and S90 have been analyzed. While S90 is thought to be very
similar to the sequenced strain, S288C, Y101 is known to lack seven open
reading frames (ORFs) which are present in S288C. Out of eighty-four
candidate transposed segments, the three spots of greatest interest mapped to a
single region on chromosome 15 (in S288C). Polymerase Chain Reaction
(PCR) assays were used to characterize the region and identify probable
endpoints. Currently, we are testing a hypothesized inversion within the
candidate region. We aim to use the identified gene order polymorphisms to
study the effects of such polymorphisms on gene expression.
ABC
DEF
Genomic DNA was extracted from the parents of the cross (S90 and Y101) and from
the four spores in two tetrads (numbers 21 and 27). DNA was also extracted from a
reference strain. We used strain S288C as the reference, since it is the sequenced
strain of yeast that was used to design the microarray. We labeled the reference DNA
with one fluorescent dye (Cy3) and the sample DNA with another (Cy5).
-5.1
Abstract
Identification of Candidate Transposed Segments
frequency
Dina Faddah, Jason Lieb, and Todd Vision
Department of Biology
University of North Carolina at Chapel Hill
[email protected]
Endpoints, or
primer
mismatch
21C 21D 27A 27B
27C 27D
+
+
+
-
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
-
-
+
+
-
+
+
-
With the use of both CGHM and PCR data, we were able to identify probable
endpoints of the rearrangement within the segments IYOL161C-0 and
IYOL155C-0.
a.
*
*
b.
Duplication
Deletion
Parental
Discussion
In conclusion, we have identified eighty-four candidate transposed segments present in equal copy number but located at different
genomic positions in S90 and Y101. We identified a particularly striking segment located on chromosome 15 in the reference strain
S288C. This region contains five genes, spans approximately 15 kB of genomic DNA, and is hypothesized to contain an inversion. In
the future, contour-clamped homogeneous electric field (CHEF) analysis will be used to determine the exact chromosomal location of
the transposed segment in Y101. Also, we would 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, and (c) whether there are any clues as
to the transposition mechanism in the sequences in and around the transposed segments.
Acknowledgements
Dina thanks Ostranda Williams, Dr. Sean Hanlon,
Dr. Michael Buck , Dr. Cheol Koo Lee, and Dr. Eric
Ganko for their support, encouragement, and
knowledge. This work is supported by NSF DBI0227314 to TJV and the Smallwood Foundation.