Exploring the Metabolic and Genetic Control of Gene Expression on a Genomic Scale DeRisi, Iyer, and Brown (1997) Science 278,

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. 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  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 factor TUP1 encodes a transcriptional co- repressor that works with Mig1p Compare TUP1 cells to tup1  cells TUP1 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 O 2 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