Fall 2006 7 03 1 7 03 2006 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 Numbers of genes per haploid genome Gene Density bp per gene E coli E coli 4 200 22 500 4 200 22 500 5 Mbs sequenced in 1997 3000 Mbs sequenced in 2005 1 2 Kb per gene 115 5 Kb per gene S cerevisiae 5 800 12 Mbs sequenced in 1997 22 500 S cerevisiae 5 800 3000 Mbs sequenced in 2005 1 9 Kb per gene 22 500 121 5 Kb per gene D melanogaster D melanogaster 14 000 22 500 14 000 22 500 131 Mbs sequenced in 2000 3000 Mbs sequenced in 2003 9 5 Kb per gene 127 9 Kb per gene Mb megabase 1 million base pairs Kb kilobase 1 thousand base pairs 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 Fall 2006 7 03 2 that 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 EXONS intron 1 intron 2 intron 3 genomic DNA intron 4 genomic DNA 5 transcription transcription AAA Primary Transcript start 3 4 5 Alternative splicing AAA nuclear pore AAA mRNA ssRNA AAA translation 2 AAA nuclear pore stop 1 RNA processing export 5 cap 3 polyadenylation splicing out of introns AAA cap AAA Protein amino acids 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 Fall 2006 7 03 3 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 In E coli Mini Tn7 Tn7TR lacZ URA3 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 genome i e each E coli cell is home Tn7 to a plasmid that contains a different Tn7 donor segment of the S cerevisiae genome Yeast genomic plasmid library such that the whole geneome is Random yeast insertion library 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 Fall 2006 7 03 4 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 URA3 mRNA AUG Fusion protein N Gene X encoded amino acids C Mini Tn7 encoded amino acids LacZ encoded amino acids p s to Tn7TR lacZ i on tet Tn7TR s an Tr pt cri lacZ URA3 URA3 t ta r rt n s n sta o i t o p i i t r sc sla an an Tr
View Full Document
Unlocking...