Lecture #8Date _________ n Chapter 19~ The Organization and Control of Eukaryotic Genomes.
Genome organization Eukaryotic genomes are complex and DNA amounts and organization vary widely...
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Transcript of Genome organization Eukaryotic genomes are complex and DNA amounts and organization vary widely...
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Genome organization
Eukaryotic genomes are complex and DNA amounts and organization vary
widely between species.
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Genome OrganizationG
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C value paradox:
The amount of DNA in the haploid cell of an organism is not related to its evolutionary complexity or number of genes.
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Highly Repeated Sequences
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There are different classes of eukaryotic DNA based on sequence complexity.
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105 106 107 108 109 1010 1011 1012
basepairs
Amount of DNA in a Genome Does Not Correlate with
Complexity
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How many genes do humans have?
Original estimate was between 50,000 to 100,000 genes
We now think humans have ~ 20,000 genes
How does this compare to other organisms?
Mice have ~30,000 genes
Pufferfish have ~35,000
Nematodes (C. elegans), have ~19,000
Yeast (S. cerevisiae) has ~6,000
The microbe responsible for tuberculosis has ~4,000
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Single Copy Sequences
Exome
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Even the Amount of DNA a Gene Spans Differs Among Species
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Problems?• Some gene products are RNA (tRNA,
rRNA, others) instead of protein
• Some nucleic acid sequences that do not encode gene products (noncoding regions) are necessary for production of the gene product (protein or RNA).
• Eukaryotic genes are complex!
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Gene Identification• Open reading frames• Sequence conservation
– Database searches– Synteny
• Sequence features– CpG islands
• Evidence for transcription– ESTs, microarrays
• Gene inactivation– Transformation, RNAi
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Unique genes
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Noncoding regions
• Regulatory regions– RNA polymerase binding site
– Transcription factor binding sites
• Introns
• Polyadenylation [poly(A)] sites
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Splice Sites
• Eukaryotes only • Removal of internal parts of the newly
transcribed RNA.• Takes place in the cell nucleus• Splice sites difficult to predict
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One gene, many proteins via alternative splicing , 3’ cleavage and polyadenlyation
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Exon Shuffling
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Trans-Splicing in Higher Eukaryotes
Gingeras, Nature (2009) 461, 206-211
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Non-contiguous Transcription Generates An Enormous Number of Possible
Transcripts
Gingeras, Nature (2009) 461, 206-211
Six 2-exon co-linear combinations from four exons
325 combinations of 3-exons, non-colinear
• Reassortment of exons coding for ncRNA or protein domains could dramatically increase number of functional products beyond the number of ‘genes’
• Trans-splicing exists in higher eukaryotes as well as in lower ones like Trypanosomes
Blue: only co-linearRed: all combinations
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Why genome size isn’t the only concern (size doesn’t matter?)
• More sophisticated regulation of expression?
• Proteome vastly larger than genome?
– Alternate splicing
– RNA editing
• Postranslational modifications?
• Cellular location?
• Moonlighting
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Gene families
• E.g. globins, actin, myosin• Clustered or dispersed• Pseudogenes
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Pseudogenes
• Nonfunctional copies of genes
• Formed by duplication of ancestral gene, or reverse transcription (and integration)
• Not expressed due to mutations that produce a stop codon (nonsense or frameshift) or prevent mRNA processing, or due to lack of regulatory sequences
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Duplicated genes• Encode closely related (homologous) proteins• Formed by duplication of an ancestral gene
followed by mutation
Five functional genes and two pseudogenes
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Paralogs vs Orthologs
• Different members of the globin gene family are paralogs, having evolved one from another through gene duplication. Paralogs are separated by a gene duplication event.
• Each specific gene family member (e.g. a specific gene in human) is an ortholog of the same family member in another species (e.g. mouse). Both evolved from an ancestral globin gene. Orthologs are separated by a speciation event.
• It is not always easy to distinguish true orthologs from paralogs , especially in polyploid organisms!
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Protein - coding sequences less than 1.5% of the genome in humans!
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Noncoding RNAs (ncRNA)
• Do not have translated ORFs• Small• Not polyadenylated
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Functions of Known lncRNAs
Ponting et al, Cell (2009) 136, 629-641
• Transcriptional interference -lncRNA transcription turns off transcription of nearby gene
• Initiation of chromatin remodeling - lncRNA transcription turns on transcription of nearby gene
• Promoter inactivation - lncRNA binds to TFIIB and to promoter DNA
• Activation of an accessory protein - lncRNA binds to allosteric effector protein TLS and inhibits histone acetyltransferase, decreasing transcription
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Functions of Known lncRNAs
• Activation of transcription factors - binding of lncRNA to Dlx2 activates Dlx5/6 activity
• Oligomerization of an accessory protein - lncRNA induces heat shock factor trimerization
• Epigenetic silencing of gene clusters -Xist RNA inactivates X chromosome
• Epigenetic repression of genes in trans -HOTAIR binds PRC2, leading to methylation and silencing of several genes in HOXD locus
• Transport of transcription factors -lnRNA NRON keeps NFAT out of nucleus
Ponting et al, Cell (2009) 136, 629-641
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• ~97-98% of the transcriptional output of the human genome is ncRNA– Introns– Transfer RNAs (tRNA)
• ~ 500 tRNA genes in human genome– Ribosomal RNAs
• Tandem arrays on several chromosomes• 150-200 copies of 28S – 5.8S – 18S
cluster• 200-300 copies of 5S cluster
ncRNA
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• The level of transcription from human chromosomes 21 and 22 is an order of magnitude higher than can be accounted for by known or predicted exons
• Almost half of all transcripts from well-constructed mouse cDNA libraries are ncRNAs (identified because they do not code for an open reading frame of larger than 100 codons)
Genome Organization - ncRNA
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Repeat sequences – 50% or more of the genome
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Repetitive DNA
• Moderately repeated DNA– Tandemly repeated rRNA, tRNA and histone genes
(gene products needed in high amounts)– Large duplicated gene families– Mobile DNA
• Simple-sequence DNA– Tandemly repeated short sequences– Found in centromeres and telomeres (and others)– Used in DNA fingerprinting to identify individuals
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Segmental duplications
• Found especially around centromeres and telomeres
• Often come from nonhomologous chromosomes
• Many can come from the same source• Tend to be large (10 to 50 kb)• Unique to humans?
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Repetitive DNA - Segmental duplications
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Mobile DNA
• Moves within genomes• Most of the moderately repeated DNA
sequences found throughout higher eukaryotic genomes– L1 LINE is ~5% of human DNA (~50,000
copies)– Alu is ~5% of human DNA (>500,000 copies)
• Some encode enzymes that catalyze movement
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Repetitive DNA – Highly repetitive satellite DNA
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