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24. Functional Genomics and Proteomics.doc

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Biology (Sci)
BIOL 200
Richard Roy

Naveen Sooknanan McGill Fall 2011 Functional Genomics and Proteomics: With the new sequencing technologies available which are capable of sequencing entire genomes in days, even hours, there needs to be technology available to make sense of it all How do these letters (nucleotides) form words (mRNA), paragraphs (proteins) and finally form the whole novel (us) Genomics allows us to do this, by studying the genome as a whole. Genomics has four particular uses: Finding out gene structure Finding out gene function Looking for evolutionary relationships o Such as where divergences take place It is used as a predictive tool for new genes o It gives us the ability to compare a completely new gene against a database to find a gene of similar structure o As example of this is with Rpd3 looking like a histone deacetylase from another organism, allowing us to determine its function Analysis of full genomes has shown us how little we actually know about genes and gene function We have notices that the same basic cellular took kit is shared by almost all organisms o These are the genes that produce materials needed for basic functions such as metabolism, transcription, splicing, etc. Metabolic genes make up a large portion of the total number of genes o Transcription and translation genes also make up a significant number of genes However, the vast majority of genes within the genome are of unknown function o And this is not just in humans, its in even the most basic eukaryotes like yeast The expression of thousands of genes can all be analyzed at once using a technology known as microarray analysis This procedure has been around for 15-25 years, and involves spotting out every gene from a genome onto tiny little wells in a glass chip known as a microarray o These genes can be made from oligonucleotides or cDNA These oligonucleotides can be fluorescently labelled so that their activity can be monitored by a microscope Genes expressed at the around the same time can have similar or complementary functions which are indicative of the cells overall function o I.e. stages of development vs. adulthood These oligonucleotides are designed to that the can only hybridize with one specific cDNA molecule, which allows for specificity 1 Naveen Sooknanan McGill Fall 2011 RNA samples of different origin can be monitored on the same microarray if the two samples are labelled with different fluorescent dyes The microarray experiment can be set up to record results from the same genome subject to different living conditions One sample, left on a plate without serum, cannot divide, meaning they are quiescent The other sample is allowed to grow in the presence of serum and therefore has different gene activity than the quiescent cells mRNAs are isolated from each of these samples (using any methods we have studied earlier) They are then reverse-transcribed into cDNA which are labelled with fluorescent dyes, a different dye for each sample o The process is the same as making a cDNA library These samples are then loaded onto a microarray, each well containing different oligonucleotides which are specific to one cDNA from the sample o Each sample is used to hybridize a full genome microarray independently and the changes are recorded o Therefore, the samples will form hybrids with the oligonucleotides or cDNAs in the wells These samples will show differential hybridizations because of different amounts of mRNA produced by the two types of cells (quiescent vs. actively dividing) Thus, two microarray readouts are produced, which can be superimposed to compare gene activity Take this hybridized microarray: the colors have been assigned so that green means the gene is expressed more in condition A, red means higher expression in condition B, and yellow means unchanged expression Upon completion of the hybridization, a special instrument, a reader, can detect these fluorescent marks emitted by tagged cDNAs Since the oligonucleotides are in excess, they give rough, inaccurate measure for the abundance of mRNA in a sample The more mRNA produced by the cell, the more labelled cDNA will be made, and the higher the level of fluorescence detected by the machine o Each dot on the microarray sample is the activity of a particular gene. You will be able to tell which gene it is because you have recorded which oligonucleotide was placed in this well In this example, green light means this gene was downregulated during cell division, red means it was unregulated, and yellow means it was unchanged This gives an idea of the overall gene expression during cell division These microarray results can be clustered together in order to extract important information: These microarrays are placed so that gene expression can be analyzed over a period of time 2Naveen Sooknanan McGill Fall 2011 o This can be used to figure out which genes are regulated during certain cellular activities For example, This cluster has 5 clusters representing 5 different cell activities o Cluster A represents genes involved in cholesterol biosynthesis o B corresponds to cell cycle genes o C represents immediate cluster genes o D represents genes for angiogenesis o E represents genes for wound healing For all of these clusters, fibroblast samples were grown in the absence of serum, mRNA was collected over a period of time, and a gene activity over time was determined through microarray analysis For example, clusters A and C are downregulated, because the turn green, later in time, while clusters D and E are upregulated later on o Cluster B is downregulated early then is upregulated later If you have a sample of an unknown gene, you can do a microarray analysis and see where it falls in one of these clusters o By doing this, you can assign the gene a function (though it may not be true, but its a fair starting point) This can also be used to monitor the activ
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