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. Kb = kilobase = 1 thousand base-pairs of DNA= DNA content of a gamete (sperm or egg)genome = DNA content of a complete haploid set of chromosomesH. sapiensM. musculusD. melanogasterC. elegansS. cerevisiaeE. coligenes/ haploidyear sequence completedDNA content/ haploid (Mb)cMChromosomesSpecies116642023N/A40003002801700330051210018030003000199719971998200019,00014,00022,500?22,500?genes have introns?norarelynearly allnearly allnearly allnearly allMb = megabase = 1 million base-pairs of DNANote: cM = centi Morgan = 1% recombination2002 draft2001 draft2005 finished?2003 finished4,2005,800Kb = kilobase = 1 thousand base-pairs of DNA= DNA content of a gamete (sperm or egg)genome = DNA content of a complete haploid set of chromosomesH. sapiensM. musculusD. melanogasterC. elegansS. cerevisiaeE. coligenes/ haploidyear sequence completedDNA content/ haploid (Mb)cMChromosomesSpecies116642023N/A40003002801700330051210018030003000199719971998200019,00014,00022,500?22,500?genes have introns?norarelynearly allnearly allnearly allnearly allMb = megabase = 1 million base-pairs of DNANote: cM = centi Morgan = 1% recombination2002 draft2001 draft2005 finished?2003 finished4,2005,800Kb = kilobase = 1 thousand base-pairs of DNA= DNA content of a gamete (sperm or egg)genome = DNA content of a complete haploid set of chromosomesH. sapiensM. musculusD. melanogasterC. elegansS. cerevisiaeE. coligenes/ haploidyear sequence completedDNA content/ haploid (Mb)cMChromosomesSpecies116642023N/A40003002801700330051210018030003000199719971998200019,00014,00022,500?22,500?genes have introns?norarelynearly allnearly allnearly allnearly allMb = megabase = 1 million base-pairs of DNANote: cM = centi Morgan = 1% recombination2002 draft2001 draft2005 finished?2003 finished4,2005,800 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 centromeresand 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 sequences that do not encode for protein…these “intervening sequences” are called introns. chromosome (ds DNA)1 2 3geneexons intronstranscription1 2 3primary transcript (ss RNA) mRNA (ssRNA) translation1 2 3protein (amino acids) 1 2 3MeGcap AAAAA addition of 5’ cap3’ polyadenylationsplicing out of intronsAUGstopchromosome (ds DNA)1 2 3genegeneexonsexons intronsintronstranscriptiontranscription1 2 31 2 3primary transcript (ss RNA) mRNA (ssRNA) translationtranslation1 2 31 2 3protein (amino acids) 1 2 3MeGcap AAAAA addition of 5’ cap3’ polyadenylationsplicing out of introns1 2 3MeGcap AAAAA addition of 5’ cap3’ polyadenylationsplicing out of intronsAUGAUGstopstop 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. 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. Drosophila melanogastersytCG2964 CG3123CG16987CG15400CG31310 50HumanGATA1 HDAC6 LOC139168PCSK1N0 50Saccharomyces cerevisiaeRGD2SEC53 ACT1FET5 TUB2 RP041 YFL034W HAC1 STE2YFL046WYFL044C YPT1MOB2RPL22BCAK1 BST1 EPL1RIM15CAF16YFL042C0 50GYP8YFL040W YFL030WFigure 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. Tn7TR lacZ URA3 t etTn7TRReporter of transcriptionSelection in yeastSelection in E. coliRequired for transpositionRequired for transpositionMini-Tn7Tn7TR lacZ URA3 t etTn7TRTn7TR lacZ URA3 t etTn7TRReporter of transcriptionSelection in yeastSelection
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