lecture19 - Lecture 19 EUKARYOTIC GENES AND GENOMES I For...

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Unformatted text preview: 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) DNA content/ haploid (Mb) 5 12 100 180 3000 3000 year sequence completed 1997 1997 1998 2000 2002 draft 2005 finished? 2001 draft 2003 finished genes/ haploid 4,200 5,800 19,000 14,000 22,500? 22,500? genes have introns? no rarely nearly all nearly all nearly all nearly all Species E. coli S. cerevisiae C. elegans D. melanogaster M. musculus H. sapiens Chromosomes cM 1 16 6 4 20 23 Note: N/A 4000 300 280 1700 3300 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 3 addition of 5' cap 3' polyadenylation splicing out of introns mRNA (ssRNA) MeG cap 1 AUG 2 translation 2 3 3 stop AAAAA 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. protein (amino acids) 1 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 0 YFL040W FET5 TUB2 RP041 YFL034W YFL030W HAC1 STE2 50 SEC53 YFL044C YFL042C ACT1 YPT1 MOB2 RPL22B RIM15 CAK1 CAF16 GYP8 BST1 EPL1 Drosophila melanogaster 0 syt CG3131 CG15400 50 CG16987 CG2964 CG3123 Human 0 GATA1 HDAC6 LOC139168 50 PCSK1N Figure by MIT OCW. 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 In E. coli Tn7TR lacZ URA3 tet Tn7TR Tn7TR lacZ URA3 tet Tn7TR Tn7TR lacZ UR A3 tet Tn7TR In yeast Tn7TR lacZ URA3 tet Tn7TR Required for transposition Reporter of transcription Selection in yeast Selection in Required for E. coli transposition E. coli Yeast genomic DNA 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. Note that the lacZ gene in the transposon does not carry its own transcription or a translation start site, but if the transposon inserts in the correct orientation downstream of a yeast gene promoter, and in the correct triplet codon reading frame, the lacZ gene comes under the control of that promoter and when transcription is activated from that promoter a LacZ-fusion protein is expressed, and most LacZ-fusion proteins display robust -galactosidase activity. Promoter of gene X t ta r rt n s n sta ti o tio rip sc sla an an Tr Tr Tn7TR lacZ Promoter of gene X Tn7TR lacZ URA3 tet Tn7TR One in two insertions will be in the incorrect orientation and will not produce a LacZ-fusion protein Only one in three correct orientation insertions can produce a LacZ-fusion proten At most, only one in six insertions produce a functional LacZ-fusion proten ti o rip sc an Tr URA3 ns top mRNA Fusion protein AUG NMini-Tn7 encoded amino acids -C LacZ encoded amino acids Gene X encoded amino acids Yeast cells expressing -galactosidase activity can easily be detected by growth in the presence of 5-bromo-4-chloro3-indolyl-beta-D-galactopyranoside, better known as X-gal. LacZ cleaves Xgal to release a chemical moiety that has a brilliant blue color...and so the colonies turn bright blue! Fusion Protein has -galactosidase activity There are at least two useful things to come out of such a collection of yeast strains: (1) Any transposon that integrated into a gene will essentially disrupt that gene and is likely to cause a null mutation. (2) For transposons that integrate into a yeast gene such that the lacZ gene is in frame with the genes coding region, the level of -galactosidase activity in these cells therefore becomes a reporter for the transcription of that gene. Tn7TR lacZ URA3 tet Tn7TR Here are just two examples of how such a library can be used: (1) to identify genes that protect cells against a DNA damaging agent that causes cancer; lets take the example of one of the many many compounds found in tobacco smoke; and (2) to identify genes whose transcription is up-regulated in response to being exposed to this tobacco smoke chemical. The chemical we'll use as an example is 4-(Methylnitrosoamino)-1-(3-pyridyl)-1butanone (NNK). The yeast random insertion library is first plated out so that individual cells give rise to a colony; these colonies are then replicated onto test plates. To screen the library for genes that protect against the cell killing that can be induced by NNK the colonies are replica plated onto agar medium that either does or does not contain a high dose of NNK. To screen the library for genes that are transcriptionally regulated in the presence of this nasty carcinogenic compound, the colonies are replica plated onto agar medium containing either X-gal alone or X-gal plus a low dose of NNK. Random library of Tn7lacZ insertion mutants Phenotypic screen for NNK sensitivity Random library of Tn7lacZ insertion mutants screen for NNK-regulated genes Minus NNK Plus NNK high dose NNK sensitive strain + X-Gal X-Gal + NNK low dose Interesting colonies can be retrieved from the master plate for further study and for identification (and subsequent cloning) of the gene responsible for the interesting phenotype. Once we have identified a gene that is transcriptionally up or down regulated in response to an environmental change, how can we use genetics to figure out how regulation is achieved. This is the topic of the next lecture. ...
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