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Anatomy and Function Anatomy and Function of a Geneof a Gene

Dissection through mutationDissection through mutation

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Outline of Chapter 7Outline of Chapter 7 What mutations areWhat mutations are

How often mutations occurHow often mutations occur What events cause mutationsWhat events cause mutations How mutations affect survival and evolutionHow mutations affect survival and evolution

Mutations and gene structureMutations and gene structure Experiments using mutations demonstrate a gene is a discrete Experiments using mutations demonstrate a gene is a discrete

region of DNAregion of DNA Mutations and gene functionMutations and gene function

Genes encode proteins by directing assembly of amino acidsGenes encode proteins by directing assembly of amino acids How do genotypes correlate with phenotypes?How do genotypes correlate with phenotypes?

Phenotype depends on structure and amount of proteinPhenotype depends on structure and amount of protein Mutations alter genes instructions for producing proteins Mutations alter genes instructions for producing proteins

structure and function, and consequently phenotypestructure and function, and consequently phenotype

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Mutations: Primary tools of genetic Mutations: Primary tools of genetic analysisanalysis

Mutations are heritable changes in base Mutations are heritable changes in base sequences that modify the information sequences that modify the information content of DNAcontent of DNA Forward mutation – changes wild-type to Forward mutation – changes wild-type to

different alleledifferent allele Reverse mutation – causes novel mutation to Reverse mutation – causes novel mutation to

revert back to wild-type (reversion)revert back to wild-type (reversion)

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Classification of mutations by affect Classification of mutations by affect on DNA moleculeon DNA molecule

Substitution – base is replaced by one of the other Substitution – base is replaced by one of the other three basesthree bases

Deletion – block of one or more DNA pairs is lostDeletion – block of one or more DNA pairs is lost Insertion – block of one or more DNA pairs is Insertion – block of one or more DNA pairs is

addedadded Inversion 180Inversion 180oo rotation of piece of DNA rotation of piece of DNA Reciprocal translocation – parts of Reciprocal translocation – parts of

nonhomologous chromosomes change placesnonhomologous chromosomes change places Chromosomal rearrangements – affect many Chromosomal rearrangements – affect many

genes at one timegenes at one time

5Fig. 7.2

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Spontaneous mutations influencing Spontaneous mutations influencing phenotype occur at a very low ratephenotype occur at a very low rate

Mutation rates from wild-type to recessive alleles for five coat color genes in mice

Fig. 7.3 b

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General observations of mutation General observations of mutation ratesrates

Mutations affecting phenotype occur very Mutations affecting phenotype occur very rarelyrarely

Different genes mutate at different ratesDifferent genes mutate at different rates Rate of forward mutation is almost always Rate of forward mutation is almost always

higher than rate of reverse mutationhigher than rate of reverse mutation

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Are mutations spontaneous or Are mutations spontaneous or induced?induced?

Most mutations are spontaneous.Most mutations are spontaneous. Luria and Delbruck experiments - a simple Luria and Delbruck experiments - a simple

way to tell if mutations are spontaneous or way to tell if mutations are spontaneous or if they are induced by a mutagenic agentif they are induced by a mutagenic agent

9Fig. 7.4

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Replica plating verifies preexisting Replica plating verifies preexisting mutationsmutations

Fig. 7.5 a

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Fig. 7.5b

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Interpretation of Luria-Delbruck fluctuation Interpretation of Luria-Delbruck fluctuation experiment and replica platingexperiment and replica plating

Bacterial resistance arises from mutations Bacterial resistance arises from mutations that exist before exposure to bactericidethat exist before exposure to bactericide

After exposure to bactericide, the bactericide After exposure to bactericide, the bactericide becomes a selective agent killing the becomes a selective agent killing the nonresistant cells, allowing only the nonresistant cells, allowing only the preexisting resistant cells to survive.preexisting resistant cells to survive.

Mutations do not arise in particular genes as Mutations do not arise in particular genes as a direct response to environmental changea direct response to environmental change

Mutations occur randomly at any timeMutations occur randomly at any time

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Chemical and Physical agents cause Chemical and Physical agents cause mutationsmutations

Hydrolysis of a purine Hydrolysis of a purine base, A or G occurs 1000 base, A or G occurs 1000 times an hour in every celltimes an hour in every cell

Deamination removes –NH2 Deamination removes –NH2 group. Can change C to U, group. Can change C to U, inducing a substitution to and inducing a substitution to and A-T base pair after replicationA-T base pair after replication

Fig. 7.6 a,b

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X rays break the X rays break the DNA backboneDNA backbone

UV light produces UV light produces thymine dimersthymine dimers

Fig. 7.6 c, d

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Oxidation from free radicals formed by irradiation damages individual bases

Fig. 7.6 e

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Repair enzymes fix errors created by Repair enzymes fix errors created by mutationmutation

Excision repair Excision repair enzymes enzymes release release damaged damaged regions of regions of DNA. Repair DNA. Repair is then is then completed by completed by DNA DNA polymerase polymerase and DNA ligaseand DNA ligase

Fig. 7.7a

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Mistakes during replication alter Mistakes during replication alter genetic informationgenetic information

Errors during replication are exceedingly Errors during replication are exceedingly rare, less than once in 10rare, less than once in 1099 base pairs base pairs

Proofreading enzymes correct errors made Proofreading enzymes correct errors made during replicationduring replication DNA polymerase has 3’ – 5’ exonuclease DNA polymerase has 3’ – 5’ exonuclease

activity which recognizes mismatched bases and activity which recognizes mismatched bases and excises itexcises it

In bacteria, methyl-directed mismatch repair In bacteria, methyl-directed mismatch repair finds errors on newly synthesized strands and finds errors on newly synthesized strands and corrects themcorrects them

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DNA polymerase proofreadingDNA polymerase proofreading

Fig. 7.8

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Methyl-Methyl-directed directed

mismatch mismatch repairrepair

Fig. 7.9

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Unequal crossing over creates one Unequal crossing over creates one homologous chromosome with a duplication homologous chromosome with a duplication

and the other with a deletionand the other with a deletion

7.10 a

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Transposable elements move around the genome Transposable elements move around the genome and are not susceptible to excision or mismatch and are not susceptible to excision or mismatch

repairrepair

Fig. 7.10 e

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Trinucleotide instability causes Trinucleotide instability causes mutationsmutations

FMR-1 genes in FMR-1 genes in unaffected people unaffected people have fewer than have fewer than 50 CGG repeats. 50 CGG repeats.

Unstable Unstable premutation premutation alleles have alleles have between 50 and between 50 and 200 repeats.200 repeats.

Disease causing Disease causing alleles have > 200 alleles have > 200 CGG repeats.CGG repeats.

Fig. B(1) Genetics and Society

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Trinucleotide repeat in people with Trinucleotide repeat in people with fragile X syndromfragile X syndrom

Fig. A, B(2) Genetics and Society

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Mutagens induce mutationsMutagens induce mutations

Mutagens can be used to increase mutation Mutagens can be used to increase mutation ratesrates

H. J. Muller – first discovered that X rays H. J. Muller – first discovered that X rays increase mutation rate in fruit fliesincrease mutation rate in fruit flies Exposed male Drosophila to large doses of X Exposed male Drosophila to large doses of X

raysrays Mated males to females with balancer X Mated males to females with balancer X

chromosome (dominant Bar eyed mutation and chromosome (dominant Bar eyed mutation and multiple inversions)multiple inversions)

Could assay more than 1000 genes at once on Could assay more than 1000 genes at once on the X chromosomethe X chromosome

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Muller’s experimentMuller’s experiment

Fig. 7.11

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Mutagens increase mutation rate Mutagens increase mutation rate using different mechanismsusing different mechanisms

Fig. 7.12a

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28Fig. 7.12 b

29Fig. 7.12 c

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Consequences of mutationsConsequences of mutations

Germ line mutations – passed on to next Germ line mutations – passed on to next generation and affect the evolution of generation and affect the evolution of speciesspecies

Somatic mutations – affect the survival of Somatic mutations – affect the survival of an individualan individual Cell cycle mutations may lead to cancerCell cycle mutations may lead to cancer

Because of potential harmful affects of Because of potential harmful affects of mutagens to individuals, tests have been mutagens to individuals, tests have been developed to identify carcinogensdeveloped to identify carcinogens

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The Ames test The Ames test for carcinogens for carcinogens

using hisusing his-- mutants of mutants of Salmonella Salmonella

typhimuriumtyphimurium

Fig. 7.13

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What mutations tell us about gene What mutations tell us about gene structurestructure

Complementation testing tells us whether Complementation testing tells us whether two mutations are in the same or different two mutations are in the same or different genesgenes

Benzer’s experiments demonstrate that a Benzer’s experiments demonstrate that a gene is a linear sequence of nucleotide pairs gene is a linear sequence of nucleotide pairs that mutate independently and recombine that mutate independently and recombine with each otherwith each other

Some regions of chromosomes mutate at a Some regions of chromosomes mutate at a higher rate than others – hot spotshigher rate than others – hot spots

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Complementation testingComplementation testing

Fig. 7.15 a

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Five complementation groups (different genes) for eye color.Recombination mapping demonstrates distance between genes and alleles.

Fig. 7.15 b,c

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A gene is a linear sequence of A gene is a linear sequence of nucleotide pairsnucleotide pairs

Seymour Benzer mid 1950s – 1960sSeymour Benzer mid 1950s – 1960s If a gene is a linear set of nucleotides, If a gene is a linear set of nucleotides,

recombination between homologous recombination between homologous chromosomes carrying different mutations chromosomes carrying different mutations within the same gene should generate wild-type within the same gene should generate wild-type

T4 phage as an experimental systemT4 phage as an experimental system Can examine a large number of progeny to detect Can examine a large number of progeny to detect

rare mutation eventsrare mutation events Could allow only recombinant phage to proliferate Could allow only recombinant phage to proliferate

while parental phages diedwhile parental phages died

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Benzer’s experimental procedureBenzer’s experimental procedure

Generated 1612 spontaneous point mutations and Generated 1612 spontaneous point mutations and some deletionssome deletions

Mapped location of deletions relative to one Mapped location of deletions relative to one another using recombinationanother using recombination

Found approximate location of individual point Found approximate location of individual point mutations by deletion mappingmutations by deletion mapping

Then performed recombination tests between all Then performed recombination tests between all point mutations known to lie in the same small point mutations known to lie in the same small region of the chromosomeregion of the chromosome

Result – fine structure map of the Result – fine structure map of the rIIrII gene locus gene locus

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How recombination within a gene How recombination within a gene could generate wild-typecould generate wild-type

Fig. 7.16

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Working with T4 phageWorking with T4 phage

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Phenotpyic properties of T4 phagePhenotpyic properties of T4 phage

Fig. 7.17 b

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Complementation test to for Complementation test to for mutations in different genesmutations in different genes

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Detecting recombination between Detecting recombination between two mutations in the same genetwo mutations in the same gene

Fig. 7.17 d

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Deletions for rapid mapping of point Deletions for rapid mapping of point mutations to a region of the chromosomemutations to a region of the chromosome

Fig. 7.18 a

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Recombination Recombination mapping to identify mapping to identify the location of each the location of each

point mutation point mutation within a small within a small

regionregion

Fig. 7.18 b

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Fine structure map of Fine structure map of rIIrII gene gene regionregion

Fig. 7.18 c

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What mutations tell us about gene What mutations tell us about gene functionfunction

One gene, one enzyme hypothesis: a gene contains One gene, one enzyme hypothesis: a gene contains the information for producing a specific enzymethe information for producing a specific enzyme Beadle and Tatum use Beadle and Tatum use auxotrophicauxotrophic and and prototrophicprototrophic

strains of strains of NeurosporaNeurospora to test hypothesis to test hypothesis Genes specify the identity and order of amino Genes specify the identity and order of amino

acids in a polypeptide chainacids in a polypeptide chain The sequence of amino acids in a protein The sequence of amino acids in a protein

determines its three-dimensional shape and determines its three-dimensional shape and functionfunction

Some proteins contain more than one polypeptide Some proteins contain more than one polypeptide coded for by different genescoded for by different genes

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Beadle and Tatum – One gene, one Beadle and Tatum – One gene, one enzymeenzyme

1940s – isolated mutagen-induced mutants that 1940s – isolated mutagen-induced mutants that disrupted synthesis of arginine, an amino acid disrupted synthesis of arginine, an amino acid required for required for NeurosporaNeurospora growth growth AuxotrophAuxotroph – needs supplement to grow on minimal – needs supplement to grow on minimal

mediamedia PrototrophPrototroph – wild-type that needs no supplement; can – wild-type that needs no supplement; can

synthesize all required growth factorssynthesize all required growth factors Recombination analysis located mutations in four Recombination analysis located mutations in four

distinct regions of genomedistinct regions of genome Complementation tests showed each of four Complementation tests showed each of four

regions correlated with different complementation regions correlated with different complementation group (each was a different gene)group (each was a different gene)

47Fig. 7.20 a

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Fig. 7.20 b

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Interpretation of Beadle and Tatum Interpretation of Beadle and Tatum experimentsexperiments

Each gene controls the synthesis of an Each gene controls the synthesis of an enzyme involved in catalyzing the enzyme involved in catalyzing the conversion of an intermediate into arginineconversion of an intermediate into arginine

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Genes specify the identity and order of Genes specify the identity and order of amino acids in a polypeptide chainamino acids in a polypeptide chain

Proteins are linear polymers of amino acids linked Proteins are linear polymers of amino acids linked by peptide bonds by peptide bonds 20 different amino acids are building blocks of proteins20 different amino acids are building blocks of proteins NHNH22-CHR-COOH – carboxylic acid is acidic, amino -CHR-COOH – carboxylic acid is acidic, amino

group is basicgroup is basic R is the side chain that distinguishes each amino acidR is the side chain that distinguishes each amino acid

Fig. 7.21 a

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R is the side group that distinguishes each R is the side group that distinguishes each amino acidamino acid

Fig. 7.21 b

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53Fig. 7.21 b

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N terminus of a protein contains a free amino groupN terminus of a protein contains a free amino groupC terminus of protein contains a free carboxylic acid groupC terminus of protein contains a free carboxylic acid group

Fig. 7.21 c

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Genes specify the amino acid sequence of a Genes specify the amino acid sequence of a polypeptide – example, sickle cell anemiapolypeptide – example, sickle cell anemia

Mutant chain of hemoglobin form aggregates that cause red blood cells to sickle

Fig. 7.22 a

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Sequence of amino acids determine a proteins Sequence of amino acids determine a proteins primary, secondary, and tertiary structureprimary, secondary, and tertiary structure

Fig. 7.23

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Some proteins are multimeric, containing subunits Some proteins are multimeric, containing subunits composed of more than one polypeptidecomposed of more than one polypeptide

Fig. 7.24

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How do genotypes and phenotypes How do genotypes and phenotypes correlate?correlate?

Alteration of amino acid composition of a Alteration of amino acid composition of a proteinprotein

Alteration of the amount of normal protein Alteration of the amount of normal protein producedproduced

Changes in different amino acids at Changes in different amino acids at different positions have different effectsdifferent positions have different effects Proteins have active sites and sites involved in Proteins have active sites and sites involved in

shape or structureshape or structure

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Dominance relations between alleles depend on the Dominance relations between alleles depend on the relation between protein function and phenotyperelation between protein function and phenotype

Alleles that produce nonfunctional proteins are usually recessiveAlleles that produce nonfunctional proteins are usually recessive Null mutationsNull mutations – prevent synthesis of protein or promote synthesis of – prevent synthesis of protein or promote synthesis of

protein incapable of carrying out any functionprotein incapable of carrying out any function Hypomorphic mutationsHypomorphic mutations – produce much less of a protein or a protein – produce much less of a protein or a protein

with weak but detectable function; usually detectable only in with weak but detectable function; usually detectable only in homozygoteshomozygotes

Incomplete dominance – phenotype varies in proportion to Incomplete dominance – phenotype varies in proportion to amount of proteinamount of protein Hypermorphic mutationsHypermorphic mutations – produces more protein or same amount of a – produces more protein or same amount of a

more effective proteinmore effective protein Dominant negativeDominant negative – produces a subunit of a protein that blocks the – produces a subunit of a protein that blocks the

activity of other subunitsactivity of other subunits Neomorphic mutationsNeomorphic mutations – generate a novel phenotype; example is ectopic – generate a novel phenotype; example is ectopic

expression where protein is produced outside of its normal place or timeexpression where protein is produced outside of its normal place or time