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Mutation and genetic variation Mutations are raw material of evolution. No variation means no evolution and mutations are the ultimate source of variation. Where do new alleles come from? DNA made up of sequence of nucleotides. Each nucleotide includes a sugar, phosphate and one of four possible nitrogenous bases (adenine and guanine [both purines], and thymine and cytosine [both pyrimidines]). 4.1a 4 + 4.1d 4.1b . Where do new alleles come from? The opposite strands of the DNA molecule are complementary because the strands are held together by bonds between the opposing bases and adenine bonds only with thymine and cytosine only with guanine. Thus, knowing the sequence on one strand enables one to construct the sequence on the other strand. 4.2 Where do new alleles come from? Sequence of bases in DNA codes for protein structure as each three base sequence codes for one amino acid in the protein chain. [To refresh yourself on basic DNA structure and protein synthesis see any Introductory Biology textbook] 4.3a Where do new alleles come from? When DNA is synthesized an enzyme called DNA polymerase reads one strand of DNA molecule and constructs a complementary strand. If DNA polymerase makes a mistake and it is not repaired, a mutation has occurred. 4.2 Types of mutations A mistake that changes one base on a DNA molecule is called a point mutation. Two forms: – Transition: one pyrimidine (T or C) substituted for the other pyrimidine or one purine substituted for the other purine (A or G). – Transversion: purine substituted for pyrimidine or vice versa Fig 4.4 Types of mutations Transitions more common than transversions. Perhaps because transitions cause less disruption to the DNA molecule and so are less likely to be noticed by DNA repair molecules. Types of mutations Not all mutations cause a change in amino acid coded for. These are called silent mutations. Mutations that do cause a change in amino acid are called replacement mutations. Types of mutations Another type of mutation occurs when bases are inserted or deleted from the DNA molecule. This causes a change in how the whole DNA strand is read (a frame shift mutation) and produces a nonfunctional protein. Mutation rates Most data on mutations comes from analysis of loss-of-function mutations. Loss-of-function mutations cause gene to produce a non-working protein. Examples of loss-of-function mutations include: insertions and deletions, mutation to a stop codon and insertion of jumping genes. Mutation rates Some mutations cause readily identified phenotypic changes. E.g. Achrondoplastic dwarfism is a dominant disorder. An Achrondoplastic individual’s condition must be the result of a mutation, if his parents do not have the condition. Mutation rates Human estimate is 1.6 mutations/genome/generation. In Drosophila rate is only 0.14 m/g/g, but when corrected for number of cell divisions needed to produce sperm (400 in humans 25 in Drosophila) mutation rates per cell division are very similar. Mutation rates These rates are underestimates as they are based on loss-of-function mutations. Direct estimate of number of mutations of all kinds made for roundworm Caenorhabditis elegans by sequencing mitochondrial DNA. Mutation rates Roundworms can self-fertilize so researchers tracked 74 family lines derived from one female and followed each for 214 generations. At end sequenced 771,672 base pairs of mitochondrial DNA. Found 26 mutations giving rate of 1.6X10-7 mutations per site per generation. Ten mutations were insertion/deletions and 16 substitutions. Mutation rates Applying mutation rates to entire genome gives estimate of approximately 15 mutations/individual/generation. Where do new genes come from? Mutation can produce new alleles, but new genes are also produced and gene duplication appears to be most important source of new genes. Gene duplication Duplication results from unequal crossing over when chromosomes align incorrectly during meiosis. Result is a chromosome with an extra section of DNA that contains duplicated genes 4.7 Gene duplication Extra sections of DNA are duplicates and can accumulate mutations without being selected against because the other copies of the gene produce normal proteins. Gene may completely change over time so gene duplication creates new possibilities for gene function. Globin genes Human globin genes are examples of products of gene duplication. Globin gene family contains two major gene clusters (alpha and beta) that code for the protein subunits of hemoglobin. Globin genes Hemoglobin (the oxygen-carrying molecule in red corpuscles) consists of an iron-binding heme group and four surrounding protein chains (two coded for by genes in the Alpha cluster and two in the Beta cluster). Globin genes Ancestral globin gene duplicated and diverged into alpha and beta ancestral genes about 450-500 mya. Later transposed to different chromosomes and followed by further subsequent duplications and mutations. From Campbell and Reese Biology 7th ed. Globin genes Lengths and positions of exons and introns in the globin genes are very similar. Very unlikely such similarities could be due to chance. Exons (blue), introns (white), number in box is number of nucleotides. 4.9 Globin genes Different genes in alpha and beta families are expressed at different times in development. For example, in very young human fetus, zeta (from alpha cluster) and epsilon (from beta cluster) chains are present initially then replaced. Similarly G-gamma and A-gamma chains present in older fetuses are replaced by beta chains after birth. 4.8 Gestation (weeks) Post-birth(weeks) Fetal hemoglobin has a higher affinity for oxygen than adult hemoglobin. Enhances oxygen transfer from mother to offspring. Chromosomal alterations Two major forms important in evolution: inversions and polyploidy. Inversions A chromosome inversion occurs when a section of chromosome is broken at both ends, detaches, and flips. Inversion alters the ordering of genes along the chromosome. 4.10 Inversions Inversion affects linkage (linkage is the likelihood that genes on a chromosome are inherited together i.e., not split up during meiosis). Inverted sections cannot align properly with another chromosome during meiosis and crossingover within inversion produces non-functional gametes. Genes contained within inversion are inherited as a set of genes also called a “supergene” Inversions Inversions are common in Drosophila (fruit flies) Frequency of inversions shows clinal pattern and increases with latitude. Inversions are believed to contain combinations of genes that work well in particular climatic conditions. Inversions Drosophila introduced to Washington and Chile from Old World. Spread along both coasts and within a few years had developed similar clines in number of inversions to those found in native range. 4.11 Polyploidy Polyploidy is the duplication of entire sets of chromosomes. A polyploid organism has more than two sets of chromosomes. E.g. A diploid (2n chromosomes) organism can become tetraploid (4n), [where n refers to one set of chromosomes]. Polyploidy Polyploidy is common in plants, rare in animals. Half of all angiosperms (flowering plants) and almost all ferns are polyploid. Polyploidy Polyploid animal taxa include earthworms and flatworms, which can self-fertilize, and some other groups including insects that can reproduce asexually (parthenogenesis). Polyploidy Polyploidy can occur if an individual produces diploid gametes and selffertilizes generating tetraploid offspring. If an offspring later self fertilizes or crosses with its parent, a population of tetraploids may develop. FIG 4.12 Polyploidy If a sterile plant undergoes polyploidy and self-fertilization a new species can develop essentially immediately. Polyploidy Cross-fertilization of different species, followed by polyploidy, was responsible for the development of many crop plants e.g. wheat. Initial cross-fertilization produces sterile offspring, because chromosomes cannot pair up during meiosis. Polyploidy Triticum monococcum (AA) X wild Triticum (BB) cross produced sterile hybrid with 14 chromosmomes (AB; 1-7A and 1-7B). {capitalized letters symbolize species source of chromosomes, number denotes individual chromosome e.g. 1A, 3B} Polyploidy of first sterile hybrid produced Emmer Wheat T. turgidum (AABB) which has 28 chromosomes. Emmer Wheat isn’t sterile. It has two copies of each chromosome (e.g. two 1A chromosomes, two 3B chromosomes, etc.). Polyploidy Further cross between Emmer Wheat and T. tauschii which has a total of 14 chromosomes (DD) produced a sterile hybrid with 21 chromosomes (ABD). Further polyploid error in meiosis produced T. aestivum Bread Wheat with 42 chromosomes (AABBDD). Those chromosomes are derived from 3 ancestral species. Example of inter-species cross-fertilization and meiotic errors leading to production of a fertile hybrid Polyploidy Genes in polyploid chromosome sets are free to develop new functions through natural selection and evolution. Polyploidy is a key source of genetic variation. What is mutation rate to polyploidy in plants? Flowering plants produce diploid gametes at frequency of 0.00465 per generation Thus tetraploid offspring produced at rate of approx 0.00465 X 0.00465 or 2.16 X 10-5 per generation (approx. 2 in every 100,000 offspring per generation are tetraploid). This is comparable to rate of point mutations in individual genes. Measuring genetic variation in populations Classical view: little genetic variation expected because one allele at a locus expected to be superior, favored by natural selection and thus fixed in the population. Now recognized that there is substantial genetic variation in natural populations. Identifying genotypes Gel electrophoresis used to determine genotypes of individuals. DNA isolated, amplified and cut using restriction enzymes. Different patterns on gel can be distinguished. Also direct sequencing can be used to identify different alleles. Calculating allele frequencies Recall Δ32 (delta32) variant of CCR5 gene (CCR5 used as coreceptor by HIV to enter white blood cells). Δ32 allele offers protection against HIV. How can its frequency in a population be estimated? Calculating allele frequencies Simplest way is to count frequencies, but can also be estimated from the frequencies of heterozygotes and homozygotes as we will see when we study Hardy-Weinberg equilibrium shortly. How much genetic diversity exists in a typical population? Electrophoretic studies have shown that most populations have substantial genetic variation. In general, 33-50% of enzyme loci are polymorphic and average individual is heterozygous at 4-15% of loci. Frequency distribution of enzyme heterozygosity in plants. Heterozygosity is the mean percentage of loci hetoerozygous per individual or the mean percentage of individuals heterozygous per locus. 4.16 Why are populations genetically diverse? Two major hypotheses. Selectionist: Diversity maintained by selection. Selection favors rare individuals, heterozygotes or different alleles at different times and places. Neutral: Most alleles at polymorphic loci functionally and selectively equivalent. Neutralist vs Selectionist controversy Both neutralist and selectionist schools recognize the importance of selection. I.e. that many alleles are fixed as a result of selection. The disagreement is about the proportion of molecular variation that is due to the effects of selection. Neutralist vs Selectionist controversy Neutralist argument is that most of the diversity seen in DNA at the molecular level is functionally neutral (i.e. does not have an effect on fitness). In most cases, neutralists argue that a change in a codon leads to an amino acid change that has no effect on the functioning of the protein produced. Neutralist vs Selectionist controversy Through the process of genetic drift (chance) different alleles may become fixed in different populations. Selectionist would argue that most variation is subject to selection. Neutralist vs Selectionist controversy One way to evaluate the situation in nature is to look at synonymous and non-synonymous changes in nucleotides. Synonymous changes are ones where the codon is changed but the amino acid produced is not [i.e. a silent mutation] . This will be invisible to selection and so must be . In non-synonymous changes, the amino acid is changed and this change may result in a phenotypic change and thus be selected for or against. McDonald-Kreitman test You would expect that if selection acts on most nucleotides that the ratio Dn/Pn of fixed to polymorphic nucleotides for non-synonymous codons would be much higher than the same ratio Ds/Ps for synonymous codons (not subject to selection). This is because fixation implies a particular nucleotide is better than the alternatives at that site. Fixed means that only one nucleotide occurs at a site in a species, but there are differences between species. Polymorphic means multiple nucleotides occur at a site in a species. Neutralist vs Selectionist controversy Using an extension of the McDonaldKreitman test it is possible to estimate the proportion of nucleotide substitutions fixed by natural selection. It is estimated that 45% of the amino acid differences between two species of Drosophila (D. simulans and D. yakuba) have been fixed by selection and 35% of AA differences in primates have been fixed by selection (Barton et al. Evolution 2007).