Lecture 19 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 genome DNA content of a complete haploid set of chromosomes DNA content of a gamete sperm or egg Species Chromosomes cM DNA content haploid Mb year sequence completed genes haploid genes have introns E coli 1 N A 5 1997 4 200 no S cerevisiae 16 4000 12 1997 5 800 rarely C elegans 6 300 100 1998 19 000 nearly all D melanogaster 4 280 180 2000 14 000 nearly all M musculus 20 1700 3000 2002 draft 2005 finished 22 500 nearly all H sapiens 23 3300 3000 2001 draft 2003 finished 22 500 nearly all Note cM centi Morgan 1 recombination Mb megabase 1 million base pairs of DNA Kb kilobase 1 thousand base pairs of DNA 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 noncoding 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 sequences that do not encode for gene protein these exons introns intervening sequences 1 2 3 are called introns chromosome ds DNA transcription primary transcript ss RNA 1 2 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 3 addition of 5 cap 3 polyadenylation splicing out of introns mRNA ssRNA MeG cap 1 2 AUG protein amino acids 3 translation 1 2 AAAAA stop 3 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 Saccharomyces cerevisiae YFL046W RGD2 YFL040W FET5 TUB2 RP041 YFL030W HAC1 YFL034W STE2 0 50 SEC53 YFL044C YFL042C ACT1 YPT1 MOB2 RPL22B RIM15 CAK1 CAF16 GYP8 BST1 EPL1 Drosophila melanogaster CG3131 syt 0 CG15400 50 CG16987 CG2964 CG3123 Human 0 GATA1 HDAC6 LOC139168 PCSK1N Figure by MIT OCW 50 Gene Regulation in Yeast In the next few lectures we will consider how eukaryotic genes and genomes can be 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 Characterizing function and regulation of S cerevisiae genes We are going 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 Mini Tn7 Tn7TR lacZ URA3 In E coli Tn7TR tet Reporter of transcription Selection in yeast URA3 tet Tn7TR Tn7TR In yeast Required for transposition lacZ Selection in Required for E coli transposition Tn7TR lacZ UR A3 tet Tn7TR Tn7TR lacZ URA3 tet Tn7TR Yeast genomic DNA E coli The mini Tn7 is introduced into a population of E coli that harbor a plasmid library of the S cerevisiae Tn7 genome i e each E coli cell is home Tn7 donor to a plasmid that contains a different Yeast genomic segment of the S cerevisiae genome plasmid library Random yeast insertion library 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 the plasmid or E coli chromosome are selected as Tetracycline resistant colonies Plasmid DNA is purified from these transformants and retransformed into tetracycline sensitive E coli the resulting tetracycline resistant bacteria harbor only plasmids that have an integrated mini Tn7 transposon Plasmid is isolated from these cells and the yeast genomic fragments are isolated by digestion with an appropriate restriction enzyme So now we have a library of yeast genomic fragments each of which has the transposon inserted these genomic fragments can be transformed into S cerevisiae cells that are ura3 Each Ura transformant colony will have recombined a Tn7 transposon containing genomic DNA into its genome This essentially gives us a library of yeast with transposons randomly integrated into it genome Fusion protein URA3 AUG N Gene X encoded amino acids C Mini Tn7 encoded amino acids LacZ encoded amino acids top Tn7TR mRNA lacZ ns tet Tn7TR ti o rip sc n a Tr lacZ URA3 URA3 rt t sta ta r on on s i t p i i t r sc sla an an
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