Download Exploring the Metabolic and Genetic Control of Gene

Exploring the Metabolic
and Genetic Control of
Gene Expression on a
Genomic Scale
DeRisi, Iyer, and Brown (1997)
Science 278, 680-686
Introduction to Yeast
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Free-living fungus; generally single-celled
Eukaryotic; possesses a nucleus and other
intracellular organelles
Grows by budding; size of bud reflects
progression through the cell cycle
Can exist either as a haploid (1N) or diploid
(2N); haploids can be mated to form new
diploids; diploids can be sporulated to make
new haploids
First eukaryotic genome to be completely
sequenced (1996)
Yeast Genetic Nomenclature
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All named genes have a three-letter
abbreviation followed by a number
Wild type genes are denoted as italicized
capital letters (i.e. ACT1)
Mutant alleles (which are usually recessive)
are denoted by small-case letters, followed
by an allele number (i.e. ura3-52); dominant
alleles are denoted by capital letters
Frank deletions are usually followed by the
D symbol (i.e. his3D)
More Yeast Genetic
Nomenclature
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Not all genes in the genome have been named
Such loci are therefore referred to by their chromosome location
 The first space is always a Y
 The second space is a letter referring to the chromosome number
(A = chromosome I, B = II, etc.)
 The third space is either R or L, and refers to whether the locus is
to the Right or Left of the centromere as the chromosome is
conventionally drawn (long arm is Left arm)
 A three digit number follows; this is the locus number on that arm
 The last space is either a W or a C; referring to either the Watson
or Crick strand (top or bottom respectively as conventionally
drawn)
Questions
1. Have all the genes required for a particular
process (in this case, global carbon utilization
pathways) been identified?
2. How do such genes change in expression over
time?
3. Are the genes involved in a particular process
coordinately regulated?
DeRisi, Iyer, and Brown
Experiment: Diauxic shift
Metabolism of glucose v. ethanol
Inoculate culture; soon thereafter
isolate cells and make RNA, from this
create cDNA labeled with Cy3-dUTP
At ~9h post-inoculation and every 2h
thereafter, isolate cells and make
RNA, from these create cDNA labeled
with Cy5-dUTP
Diauxic Shift:
Experiment Parameters
Figure 5
Figure 1
One microarray, this one compares the first two
time points, after initial inoculation (green), then
9.5 h later (red)
Results: As time proceeds,
more and more differences are
observed
By the last time point:
710 genes induced at least 2x
(183 genes at least 4x)
1030 genes repressed at least
2x (203 genes at least 4x)
>870 of these previously
unknown to be associated with
this process
Figure 2: Repeated views of box outlined in Figure 1a
Changes in metabolism as
diauxic shift proceeds;
red genes are the ones
turned on;
green ones are the ones
turned off
Figure 3
Group behavior: Genes that work coordinately are
regulated coordinately
Figure 4
Genes whose expression increases markedly,
but only in the last time point; most have a
glucose-repressible carbon source response
element (CSRE) in their promoters
Figure 5
Ribosomal protein genes, all decline
b/c of loss of Rap1 mRNA. Rap1
encodes a transcription factor
required for synthesis of these genes
Figure 5
Other uses of microarrays
explored here
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Determine effects of loss of a
common transcription factor
TUP1 encodes a transcriptional corepressor that works with Mig1p
 Compare TUP1 cells to tup1D cells
 TUP1 cDNA = green; tup1D cDNA =
red;
 red spots therefore genes suppressed
by Tup1p
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Many (10%) of the same
genes induced by diauxic
shift are also induced by the
absence of Tup1p;
suggesting Tup1p is
important for diauxic shift
Figure 2 (bottom center panel)
Logic of the Yeast Metabolic
Cycle: Temporal
Compartmentalization of
Cellular Processes
Tu et al., (2005) Science 310, 1152
Oxygen consumption varies in a periodic manner:
What genes are expressed in a periodic manner?
Figure 1
Correlation of gene expression with O2 consumption
Figure 2
Mitochondrial large
ribosomal subunit protein
Fatty acyl CoA oxidase
Adenine deaminase
Figure 2
Expression of Genes
Note that energy and metabolism protein genes are more
likely to be periodic than others
Table 1
Genes can be arranged in
superclusters
Figure 3D