exploring the metabolic and genetic control of gene expression on a genomic scale

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

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

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 symbol (i.e. his3)

More Yeast Genetic Nomenclature

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)

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?

Questions

DeRisi, Iyer, and Brown Experiment: Diauxic shift

Metabolism of glucose v. ethanolInoculate 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

One microarray, this one compares the first two time points, after initial inoculation (green), then 9.5 h later (red)

Figure 2: Repeated views of box outlined in Figure 1a

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 3

Changes in metabolism as diauxic shift proceeds; red genes are the ones turned on; green ones are the ones turned off

Figure 4

Group behavior: Genes that work coordinately are regulated coordinately

Figure 5

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

Other uses of microarrays explored here

Determine effects of loss of a common transcription factorTUP1 encodes a transcriptional co-

repressor that works with Mig1pCompare TUP1 cells to tup1 cellsTUP1 cDNA = green; tup1 cDNA =

red; red spots therefore genes suppressed

by Tup1p

Figure 2 (bottom center panel)

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

Logic of the Yeast Metabolic Cycle: Temporal Compartmentalization of Cellular Processes

Tu et al., (2005) Science 310, 1152

Figure 1

Oxygen consumption varies in a periodic manner: What genes are expressed in a periodic manner?

Figure 2

Correlation of gene expression with O2 consumption

Figure 2

Mitochondrial large ribosomal subunit protein

Fatty acyl CoA oxidase

Adenine deaminase

Expression of Genes

Table 1

Note that energy and metabolism protein genes are more likely to be periodic than others

Genes can be arranged in superclusters

Figure 3D