7 05 2005 Lecture 23 Eukaryotic Genes and Genomes IV In the last three lectures we have thought a lot about analyzing a regulatory system in S cerevisiae namely Gal regulation that involved a hand full of genes These studies monitored the increased transcription of Gal genes in the presence of galactose and the absence of glucose we saw that this regulation is achieved by particular proteins or multiprotein complexes that bind to specific sequences in the promoter region upstream from their target genes What if I told you that it is now possible to do the following in S cerevisiae Monitor mRNA expression level for every gene in S cerevisiae in one single experiment Monitor all the binding sites in the S cerevisiae genome for each transcription factor in a single experiment Determine all possible pair wise interactions for every S cerevisiae protein Obviously I wouldn t mention these possibilities if they weren t already happening What I want to do today is to introduce you to the idea of carrying out genetic analyses on a global genome wide scale and hopefully give you some examples that are relevant to what we have already learned along the way So this will be a technology oriented lecture but with some application to what we have already learned Numbers of genes 5 800 per haploid genome about gene regulation in eukaryotes It should also be mentioned that what will be described for S cerevisiae is theoretically possible for 14 000 22 500 any organism whose genome has been completely sequenced and the location of all the genes in that genome have been established So what we will learn today is 19 000 22 500 being or will be applied to higher eukaryotes and mammals Monitor mRNA expression level for every gene in S cerevisiae in one single experiment Global transcriptional profiling Before we consider how it is possible to measure the levels of thousands of mRNA species we will have to step back to consider how the levels of one or two mRNA species can be measured by Northern Blot analysis and I know you must have learned this in 7 01 if not in high school Northern blot analysis is based upon the fact that DNA and RNA molecules that possess complementary base sequences will hybridize together to form a double stranded molecule If the complementarity is perfect the duplex molecule is stable if it is imperfect with base pair mismatches it is relatively less stable This provides the specificity needed to identify perfectly matched DNA RNA duplexes on Northern Blots and DNA DNA duplexes on Southern Blots This specificity is needed to be sure we are measuring the level of one particular transcript and that this is not contaminated with signal from closely related transcripts RNA is isolated from cells size fractionated on a gel All mRNAs separated by size The immobilized mRNA population is probed hybridized with 32P labeled sequences specific for one or two gene products the thousands of mRNAs species form a smear on the gel which is punctuated by the strong ribosomal RNA bands 28S and 18S that do not interfere with the analysis The breakthrough in developing microarrays Northern Blots for analyzing mRNA levels was to reverse the Immobilized mRNA population hybridized logic instead of immobilizing the mRNAs for with labeled DNA probe representing one hybridization with one or two labeled or two genes complementary DNA cDNA probes all possible cDNA probes are immobilized on a solid surface usually glass slides The DNA Microarrays spoting of probes is achieved robotically the Immobilized DNA probes representing all DNA probes are designed to specifically possible genes hybridized with labeled hybridize to only one nucleic acid sequence mRNA population that represents a single mRNA species The thousands of DNA probes are dispensed from 96 well or 384 well plates to an addressable site on the solid surface The mRNA population from each cell type purified and then copied such that the copy is fluorescently labeled This fluorescent population is hybridized to the immobilized probes and the intensity of the fluorescence at each probe spot is proportional to the number of copies of that specific mRNA species in the original mRNA population So let s look at how this would actually work in a real experiment mRNA is isolated from yeast cells in state A e g minus galactose and from yeast cells in state B e g plus galactose and copies of each population is made such that one fluoresces red and the other fluoresces green After mixing these fluorescent molecules are hybridized to the slides containing 5 800 DNA probes each one specific for detecting hybridization of many copies of an individual mRNA species Yeast in state A Yeast in state B What s happening at each spot Isolate mRNA populations AAAAA AAAAA AAAAA AAAAA AAAAA AAAAA AAAAA AAAAA Label copies of mRNA species with RED or GREEN TTTTT TTTTT TTTTT TTTTT TTTTT TTTTT TTTTT TTTTT MIX TTTTT TTTTT TTTTT TTTTT TTTTT TTTTT TTTTT TTTTT Hybridize to the microarray mRNA present much higher in State A than State B mRNA present much higher in State B than State A mRNA present at equal levels in States A and B Hybridization The location and identity of each probe on the microarray slide is known and each probe is specific for a single mRNA The color and intensity of the fluorescence is measured by scanning the slide with lasers and the relative abundance of each mRNA in the cells of State A vs State B can be calculated from the emitted fluorescence i e the relative level of 5 800 mRNAs can be compared between two populations of yeast cells Presenting data for thousands of mRNA transcripts is clearly a challenge You could present endless tables of data but our brains are much more adept at recognizing shapes patterns and colors Northern Blot vs Microarray Colored representations of up and down regulation of transcripts levels is the preferred way to present data Each colored vertical line in the horizontal lane displays the relative expression level of a single mRNA For our purposes here lets look at what genes are up regulated when a glucose grown culture of S cerevisiae is shifted into galactose what genes are upregulated under these conditions Obviously transcripts for Gal1 Gal7 and Gal10 genes will be up regulated as we have discussed in the last couple of lectures But upon looking globally it is now clear that some What transcripts have increased levels other genes are also up regulated when shifted from glucose to galactose In addition Gal2
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