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Welcome to Part 3 of Bio 219 Lecturer – David Ray Contact info: Office hours – 1:00-2:00 pm MTW Office location – LSB 5102 Office phone – 293-5102 ext 31454 E-mail – [email protected] Lectures are available online at http://www.as.wvu.edu/~dray go to ‘Teaching’ link How Genes and Genomes Evolve Variation • There is obviously variation among and within taxa. • How does the variation arise in genomes? • Are there patterns to the variation? • How is the variation propagated? • What questions can be addressed using the variation? • What patterns exist in humans with regard to genomic variability? Generating Genetic Variation • Somatic vs. germ line cells – Somatic cells – “body” cells, no long term descendants, live only to help germ cells perform their function. – Germ cells – reproductive cells, give rise to descendants in the next generation of organisms. Generating Genetic Variation • Somatic vs. germ line mutations – Somatic mutations – occur in somatic cells and will only effect those cells and their progeny, cannot not be passed on to subsequent generations of organisms. – Germ mutations – can be passed on to subsequent generations. Generating Genetic Variation • Five types of change contribute to evolution. – Mutation within a gene – Gene duplication – Gene deletion – Exon shuffling – Horizontal transfer – rare in Eukaryotes Generating Genetic Variation • Most changes to a genome are caused by mistakes in the normal process of copying and maintaining genomic DNA. Generating Genetic Variation • Mutations within genes – Point mutations – errors in replication at individual nucleotide sites occur at a rate of about 10-10 in the human genome. – Most point mutations have no effect on the function of the genome – are selectively neutral. Generating Genetic Variation • DNA duplications – Slipped strand mispairing – Unequal crossover during recombination Generating Genetic Variation • Gene duplication allows for the acquisition of new functional genes in the genome Generating Genetic Variation • Gene Duplication: the globin family – A classic example of gene duplication and evolution – Globin molecules are involved in carrying oxygen in multicellular organisms – Ancestral globin gene (present in primitive animals) was duplicated ~500 mya. – Mutations accumulated in both genes to differentiate them - α and β present in all higher vertebrates – Further gene duplications produced alternative forms in mammals and in primates Primates Mammals Generating Genetic Variation • Gene Duplication – Almost every gene in the vertebrate genome exists in multiple copies – Gene duplication allows for new functions to arise without having to start from scratch – Studies suggest the early in vertebrate evolution the entire genome was duplicated at least twice Generating Genetic Variation • Exon Duplication – Duplications are not limited to entire genes – Proteins are often collections of distinct amino acid domains that are encoded by individual exons in a gene – The separation of exons by introns facilitates the duplication of exons and individual gene evolution Generating Genetic Variation • Exon Shuffling – The exons of genes can sometimes be thought of as individual useful units that can be mixed and matched through exon shuffling to generate new, useful combinations Review from last week • Overall theme – There are lots of ways to create genetic variation. Genetic variation is the basis of evolutionary change but the variation must be introduced into the germ line to contribute to evolutionary change. • Two cell lines in multicellular organisms – Somatic – short term genetic repository – Germ line – long term genetic repository • Variation that occurs in the germ line are the only ones that can contribute to evolutionary change • Genetic variation can be accumulated through various events – Mutations in genes – point mutations – DNA duplications – microsatellites (small), unequal crossover (large) – Gene and exon duplications are the major method for generating new gene functions – Exon shuffling can produce new gene functions by creating new combinations of functional exons/protein domains Generating Genetic Variation • Mobile elements contribute to genome evolution in several ways – Exon shuffling – Insertion mutagenesis – Homologous and non-homologous recombination Generating Genetic Variation • What are mobile elements and how do they work? – Fragments of DNA that can copy itself and insert those copies back into the genome – Found in most eukaryotic genomes – Humans – Alu (SINE); Ta, PreTa (LINEs); SVA; plus several families that are no longer active Generating Genetic Variation: Normal SINE mobilization Reverse transcription and insertion Pol III transcription 1. Usually a single ‘master’ copy 2. Pol III transcription to an RNA intermediate 3. Target primed reverse transcription (TPRT) – enzymatic machinery provided by LINEs Generating Genetic Variation • Mobile elements contribute to genome evolution in several ways – Exon shuffling Generating Genetic Variation: Exon shuffling via SINE mobilization exon 1 SINE exon 2 intron DNA copy of transcript SINE exon 2 SINE transcription can extend past the normal stop signal Reverse transcription creates DNA copies of both the SINE and exon 2 Reinsertion occurs elsewhere in the genome Generating Genetic Variation • Mobile elements contribute to genome evolution in several ways – Exon shuffling – Insertion mutagenesis • The insertion of mobile elements can disrupt gene structure and function Generating Genetic Variation ALU INSERTIONS AND DISEASE LOCUS BRCA2 Mlvi-2 DISTRIBUTION de novo de novo (somatic?) SUBFAMILY Y Ya5 de novo Familial Ya5 Yb8 about 50% Ya5 Familial Y Familial one Japanese family Ya5 Yb8 familial Ya4 C1 inhibitor ACE de novo about 50% Y Ya5 Factor IX 2 x FGFR2 GK a grandparent De novo ? Ya5 Ya5 NF1 APC PROGINS Btk IL2RG Cholinesterase CaR Sx DISEASE Breast cancer Associated with leukemia Neurofibromatosis Hereditary desmoid disease Linked with ovarian carcinoma X-linked agammaglobulinaemia XSCID Cholinesterase deficiency Hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism Complement deficiency Linked with protection from heart disease Hemophilia Apert’s Syndrome Glycerol kinase deficiency REFERENCE Miki et al, 1996 Economou-Pachnis and Tsichlis, 1985 Wallace et al, 1991 Halling et al, 1997 Rowe et al, 1995 Lester et al, 1997 Lester et al, 1997 Muratani et al, 1991 Janicic et al, 1995 Stoppa Lyonnet et al, 1990 Cambien et al, 1992 Vidaud et al, 1993 Oldridge et al, 1997 McCabe et al, (personal comm.) Generating Genetic Variation • Gene expression alteration via a Pelement mobilization in Drosophila Generating Genetic Variation • Mobile elements contribute to genome evolution in several ways – Exon shuffling – Insertion mutagenesis • The insertion of mobile elements can disrupt gene structure and function – Homologous and non homologous recombination • 10,000 – 1,000,000 + nearly identical DNA fragments scattered throughout the genome Generating Genetic Variation Unequal crossover due to non-homologous recombination ALU/ALU RECOMBINATION AND GERM-LINE DISEASE LOCUS 8 x LDLR 5 x -globin 5 x C1 inhibitor C3 HPRT DMD ADA Ins. Rec. Antithrombin XY Lysyl hydroxylase DISTRIBUTION DISEASE REFERENCE Kindreds Hypercholesterolemia Lehrman et al, 1985, 1987 Yamakawa et al, 1989 Rudiger et al, 1991 Chae et al, 1997 Kindreds Nicholls et al, 1987 -thalassaemia Flint et al, 1996 Harteveld et al, 1997 Ko et al, 1997 Kindreds Angioneurotic adema Stoppa-Lyonnet et al, 1990 Ariga et al, 1990 Kindred C3 deficiency Botto et al, 1992 Individual Lesch-Nyhan Marcus et al, 1993 syndrome Kindred Duchenne’s muscular Hu et al, 1991 dystrophy Individual ADA deficiency-SCID Markert et al, 1988 Individual Insulin-independent Shimada et al, 1990 diabetes Individual Thrombophilia Olds et al, 1993 Individual XX male Rouyer et al, 1987 Kindreds Ehlers-Danlos Pousi et al, 1994 syndrome ALU/ALU RECOMBINATION AND CANCER LOCUS 10 x ALL-1 DISTRIBUTION Somatic MECHANISM Alu-Alu recomb Dup. intron 1-6 DISEASE Acute myelogenous leukemia 7x BCR/Abl Somatic X-Alu recomb. CML All-1/AF9 Somatic Alu-Alu translocation 2x BRCA1 Somatic & A kindred 2x MLH1 2 kindreds Alu-Alu recomb (del exon 17; del. Promoter) Alu-Alu recomb. (del exon 16) (exons 13-16) Acute myelogenous leukemia Breast cancer TRE RB EWS Somatic Interchromosomal Alu-Alu recomb Common Alu-Alu recomb. (799 bp del.) Subset of Africans Alu-Alu recomb. (del 2 kb) REFERENCE Strout et al, 1998 So et al, 1997; Schichman et al, 1994 Jeffs et al, 1998 Chen et al, 1989 de Klein et al, 1986 Super et al, 1997 Ewing's sarcoma Puget et al, 1997 Swensen et al, 1997 Nystrom-Lahti et al, 1995 Mauillon et al, 1996 Onno et al, 1992 Association with glioma Protective against Ewing Sarcoma? Rothberg et al, 1997 Zucman-Rossi et al, 1997 HNPCC Generating Genetic Variation • Gene transfer can move genes between entire genomes – Horizontal gene transfer – Main problem with the development of drug resistant strains of bacteria Generating Genetic Variation • Bacterial conjugation Reconstructing Life’s Tree • Evolutionary theory predicts that organisms that are derived from a common ancestor will share genetic signatures • Organisms that shared an ancestor more recently will be more similar than those that shared a more distant common ancestor • Similarity can include sequence composition, genome organization, presence/absence of mobile elements, presence/absence of gene families, etc. 09_15_Phylogen.trees.jpg 09_16_Ancestral.gene.jpg 09_22_genetic.info.jpg 09_17_Human_chimp.jpg Chromosome 1 Review from last time • Overall themes: Genetic variation can be introduced due to the activities and presence of mobile elements (MEs); Genetic information can be introduced into organisms through horizontal transfer. • MEs are fragments of DNA that can make copies of themselves and insert those copies back into the genome – MEs can lead to variation through exon shuffling, insertion mutagenisis, and recombination – Many human diseases are the result of MEs • Horizontal transfer can introduce genetic variation into bacteria via the process of conjugation • Introduction of concepts for discussion of “Reconstructing life’s tree” – All sorts of variation provide information on the relationships among organisms – Homology – derived from the same ancestral source – Phylogeny – a reconstruction of relationships based on observations Reconstructing Life’s Tree • Basic terms – Homologous – derived from a common ancestral source – Phylogeny – a reconstruction of relationships based on observed patterns Reconstructing Life’s Tree • Homologous genes can be recognized over large amounts of evolutionary time Reconstructing Life’s Tree • Homologous genes can be recognized over large amounts of evolutionary time • Why? – Selectively advantageous genes and sequences tend to be conserved (preserved) – Selectively disadvantageous genes and sequences are tend not to be passed on to offspring Reconstructing Life’s Tree • Most DNA of most genomes is non-coding – Changes to much of this DNA are selectively neutral – cause no harm or good to the genome – Different portions of the genome will therefore diverge at different rates depending on their function The neutral regions tend to change in a clock-like fashion – We can estimate divergence times for certain groups 09_19_human_mouse1.jpg Reconstructing Life’s Tree • Most DNA of most genomes is non-coding – Changes to much of this DNA are selectively neutral – cause no harm or good to the genome – Different portions of the genome will therefore diverge at different rates depending on their function • The neutral regions tend to change in a clocklike fashion – We can estimate divergence times for certain groups Reconstructing Life’s Tree • The accumulation of changes can be quantified by several logical methods – Parsimony – the best hypothesis is the one requiring the fewest steps (i.e. Occam’s razor) – Distance – count the number of differences between things, the ones with the fewest numbers of differences are most closely related – Sequence based models – take into account what we know about the ways sequences change over time Reconstructing Life’s Tree: An example using distance • These slides and the sequence files used to produce them are available as a supplement on the class website: • DNA sequence from six taxa Sumatran orang Bornean orang gorilla bonobo chimp common chimp human Reconstructing Life’s Tree: An example using parsimony ATGGCT CAGGCT AAGACG CAGGCT AAGACT A-C T-G G-A G-A A-C T-A 6 steps Reconstructing Life’s Tree: An example using parsimony ATGGCT CAGGCT AAGACG AAGACT CAGGCT T-G G-A G-A T-A A-C 5 steps Reconstructing Life’s Tree: An example using parsimony ATGGCT AAGACT AAGACG CAGGCT CAGGCT T-G G-A T-A A-C 4 steps Reconstructing Life’s Tree • The accumulation of changes can be quantified by several logical methods • The accumulation of mobile elements provides a nearly perfect record of evolutionary relationships Phylogenetic Inference Using SINEs Phylogenetic Inference Using SINEs Species A Species B Species C Species D Resolution of the Human:Chimp:Gorilla Trichotomy (H,C)G (H,G)C (C,G)H (H,C,G) Phylogenetic Analysis PCR of 133 Alu loci 117 Ye5 13 Yc1 1 Yi6 1 Yd3 1 undefined subfamily PNAS (2003) 22:12787-91 Alu Elements and Hominid Phylogeny PNAS (2003) 22:12787-91 Review from last time • The variation that is present in genomes allows us to make determinations about the relationships among living things • Different parts of the genome accumulate variation at different rates depending on their function (or lack thereof) • The presence of different rates allows for different questions to be addressed depending on the level of divergence • Several methods are available to analyze variation for phylogenetic signal – Parsimony, distance, sequence based models • Patterns of mobile element insertion can be used to infer relationships among taxa Reconstructing Life’s Tree • Much of the “junk” DNA is dispensible – The Fugu (Takifugu rubripes) genome is almost completely devoid of unnecessary sequences – Exon number and organization is similar to mammals – Compared to other vertebrates • Intron size (not number) is reduced • Intergenic regions are reduced in size • No mobile elements 09_21_Fugu.introns.jpg Reconstructing Life’s Tree • Using all of the available information, we can reconstruct relationships between organisms back to the earliest forms of life Our Own Genome • The human genome is large and complex – 23 pairs of chromosomes – ~3.2 x 109 (3.2 billion) nucleotide pairs – Human genome composition 09_26_noncoding.jpg 09_25_Chromosome22.jpg Our Own Genome • Nuclear genome –3300 Mb –23 (XX) or 24 (XY) linear chromosomes –30-35,000 genes –1 gene/40kb –Introns –3% coding –Repetitive DNA sequences (45%) Our Own Genome • The human genome is large and complex – 23 pairs of chromosomes – ~3.2 x 109 (3.2 billion) nucleotide pairs – Human genome composition – The human genome project was one of the largest undertakings in human history Our Own Genome • Progress in human genome sequencing – Hierarchical vs. whole genome shotgun (WGS) sequencing – Repetitive DNA represents a significant problem for WGS sequencing in particular 10_09_Shotgun.sequenc.jpg 08_03.jpg Our Own Genome • Progress in human genome sequencing – Hierarchical vs. whole genome shotgun (WGS) sequencing – Repetitive DNA represents a significant problem for WGS sequencing in particular 10_10_Repetit.sequence.jpg Our Own Genome • Progress in human genome sequencing – Hierarchical vs whole genome shotgun sequencing – Repetitive DNA represents a significant problem for WGS sequencing in particular –Landmark papers in Nature and Science (2001) • Venter et al Science 16 February 2001; 291: 13041351 • Lander et al Nature 409 (6822): 860-921 Our Own Genome • A typical highthroughput genomics facility Our Own Genome • Exploring and exploiting the genome sequences • BLAST/BLAT and other tools – BLAST - Basic local alignment search tool • Input a sequence and find matches to human or other organisms – publication information – DNA and protein sequence (if applicable) Our Own Genome • Exploring and exploiting the genome sequences • BLAST/BLAT and other tools – BLAT – BLAST-like alignment tool • A “genome browser” • Genomes available : – human, chimp, rhesus monkey, dog, cow, mouse, opossum, rat, chicken, Xenopus, Zebrafish, Tetraodon, Fugu, nematode (x3), Drosophila (x10), Apis (x3), Saccharomyces (yeast), SARS • example: chr6:121,387,504-121,720,836 Our Own Genome • BLAT can be used to make direct comparisons between our genome and others. Query sequence - Callithrix Human ortholog Our Own Genome • Comparisons with other genomes inform us about our own –Important genes and regulatory sequences can easily be identified if they are conserved between genomes Our Own Genome • Human variation –~0.1% difference in nucleotide sequence between any two individual humans –Translates to about 3 million differences in the genome –Most of these differences are Single Nucleotide Polymorphisms (SNPs) –We can use these differences to investigate human variation, population structure and evolution Our Own Genome • Human evolution –Coalescence analyses (mtDNA and Y chromosome) –Mutiregional vs. Out of Africa • Predictions of the Multiregional Hypothesis – Equal diversity in human subpopulations – No obvious root to the human tree • Predictions of the Out of Africa Hypothesis – Higher diversity in African subpopulations – Root of the human tree in Africa Population Relationships Based on 100 Autosomal Alu Elements Africa Asia Europe S. India Our Own Genome • Human evolution –Higher diversity in African subpopulations • Insulin minisatellite Table 12.6 in text • 22 divergent lineages exist in the human population • All are found in Africa. Only 3 are found outside of Africa. Our Own Genome • Interpreting the information generated by the human genome project –The complexity of genome function makes interpretation difficult –Ex. What are the regulatory sequences? –Ex. Exons can be spliced together in different ways in different tissues 09_30_alt.splice.RNA.jpg