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MIT 7 03 - EUKARYOTIC GENES AND GENOMES I

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7.03, 2005, Lecture 20 EUKARYOTIC GENES AND GENOMES I For the last several lectures we have been looking at how one can manipulate prokaryotic genomes and how prokaryotic genes are regulated. In the next several lectures we will be considering eukaryotic genes and genomes, and considering how model eukaryotic organisms are used to study eukaryotic gene function. During the course of the next six lectures we will think about genes and genomes of some commonly used model organisms, the yeast Saccharomyces cerevisiae and the mouse Mus musculus. But first let’s look how the genes and genomes of these organisms compare to E. coli at one extreme, and humans at the other. Gene Density – bp per gene5,8001.9 Kb per gene14,0009.5 Kb per gene~22,500115.5 Kb per gene~22,500121.5 Kb per gene~22,500127.9 Kb per geneKb = kilobase = 1 thousand base pairs4,2001.2 Kb per geneE. coliS. cerevisiaeD. melanogasterGene Density – bp per gene5,8001.9 Kb per gene14,0009.5 Kb per gene~22,500115.5 Kb per gene~22,500121.5 Kb per gene~22,500127.9 Kb per geneKb = kilobase = 1 thousand base pairs4,2001.2 Kb per geneE. coliS. cerevisiaeD. melanogasterNumbers of genes per haploid genome5,80012 Mbs, sequenced in 199714,000131 Mbs, sequenced in 2000~22,500~3000 Mbssequenced in 2005~22,500~3000 Mbssequenced in 2005~22,500~3000 Mbssequenced in 2003Mb = megabase = 1 million base pairs4,2005 Mbs, sequenced in 1997E. coliS. cerevisiaeD. melanogasterNumbers of genes per haploid genome5,80012 Mbs, sequenced in 199714,000131 Mbs, sequenced in 2000~22,500~3000 Mbssequenced in 2005~22,500~3000 Mbssequenced in 2005~22,500~3000 Mbssequenced in 2003Mb = megabase = 1 million base pairs4,2005 Mbs, sequenced in 1997E. coliS. cerevisiaeD. melanogaster Let’s think about the number of genes in an organism and the size of the organism’s genome. The average protein is about 300 amino acids long, requiring 300 triplet codons, or roughly 1Kb of DNA. Thus it makes sense that to encode 4,200 genes E. coli requires a genome of 5 million base pairs. However, the human genome encodes about 22,500 proteins, and this should require a genome of lets say 25 million base pairs. Instead, humans have a genome that is ~ 3000 million base pairs, or ~ 3,000 Mb, i.e., ~ 3 billion base pairs. In other words, there is about 100-fold more DNA in the human genome than is required for encoding 22,500 proteins. What is it all doing? Some of it constitutes promoters upstream of each gene, some is structural DNA around centromeres and telomeres (the end of chromosomes, some is simply intergenic regions (non-coding regions between genes) but much of it is present as introns. What does it mean “Genes Have Introns”. This represents one of the fundamental organizational differences between prokaryotic and eukaryotic genes. Eukaryotic genes turn out to be interrupted with long DNA sequencesthat do not encode for protein…these “intervening sequences” are called introns. The DNA segments that are ultimately expressed as protein, i.e., the DNA sequence that contains triplet codon information, are called exons. The intronic sequences are removed from the primary transcript by splicing. transcriptiongenomic DNAAAA…Primary TranscriptmRNA (ssRNA)Protein (amino acids)5’ cap, 3’polyadenylation, splicing out of intronstranslationstart123 5412 3 45stopintron intron intron intronEXONStranscriptiongenomic DNAAAA…Primary TranscriptmRNA (ssRNA)Protein (amino acids)5’ cap, 3’polyadenylation, splicing out of intronstranslationstart123 5412 3 45stoptranscriptiongenomic DNAgenomic DNAAAA…AAA…Primary TranscriptmRNA (ssRNA)Protein (amino acids)5’ cap, 3’polyadenylation, splicing out of intronstranslationstart123 5412 3 45stopintron intron intron intronEXONSgenomic DNAtranscriptionRNA processingexportnuclear porenuclear poreAAA… AAA…Alternative splicing AAA…capAAA…AAA…AAA…genomic DNAtranscriptionRNA processingexportnuclear porenuclear poreAAA…AAA… AAA…AAA…Alternative splicingAAA…AAA…capAAA…AAA…AAA…AAA…AAA…AAA… A major consequence of this arrangement is the potential for alternative splicing to produce different proteins species from the same gene and primary transcript. This gives the potential for tremendous amplification of the complexity of mammals (and other eukaryotes) through many more thousands of possible proteins. Note that lower eukaryotes such as the yeast S. cerevisiae only have ~ 5% of their genes interrupted by introns, but for multicellular organisms, like humans, >90% of all genes are interrupted by anywhere between 2 and 60 introns, but most genes have between 5 and 12 introns. If we look at a “typical” 50 Kb region of the genome of yeast, flies and humans we immediately see how differently their genes are constructed. (Black represents exons, gray represents introns.Gene Regulation in Yeast the next few lectures we will consider how eukaryotic genes and genomes can haracterizing function and regulation of S. cerevisiae genes: We are e mini-Tn7 is introduced into a he plasmid or E. coli chromosome are Inbe manipulated and studied, and we will begin with an example of examining how genes are regulated in S. cerevisiae. First, let’s figure out how to use some neat genetics to identify some regulated genes, and in the next lecture we will figure out how one can use genetics to dissect the mechanism of that regulation. Cgoing to combine a few neat genetic tools that you learned about in Prof. Kaiser’s lectures for this, namely a library of yeast genomic fragments cloned into a bacterial plasmid, a modified transposon (mini-Tn7), and the lacZ gene embedded within the transposon. In this experiment the lacZ gene is going to be used as a reporter for transcriptional activity of yeast genes. Thpopulation of E.coli that harbor a plasmid library of the S. cerevisiae genome; i.e., each E. coli cell is home to a plasmid that contains a different segment of the S. cerevisiae genome, such that the whole geneome is represented many times over in this population of E. coli. The mini-Tn7 is allowed to transpose by integrating into either the plasmid DNA or the bacterial DNA; the original DNA that carries the mini-Tn7 can not replicate, but cells that have integrated the mini-Tn7 into tselected as Tetracycline resistant colonies. Plasmid DNA is purified from these transformants and retransformed into tetracycline sensitive E. coli; the resulting tetracycline


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MIT 7 03 - EUKARYOTIC GENES AND GENOMES I

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