Anatomy and Function of a Gene Dissection through mutation

Outline of Chapter 7 What mutations are Mutations and gene structure How often mutations occur What events cause mutations How mutations affect survival and evolution Mutations and gene structure Experiments using mutations demonstrate a gene is a discrete region of DNA Mutations and gene function Genes encode proteins by directing assembly of amino acids How do genotypes correlate with phenotypes? Phenotype depends on structure and amount of protein Mutations alter genes instructions for producing proteins structure and function, and consequently phenotype

Mutations: Primary tools of genetic analysis Mutations are heritable changes in base sequences that modify the information content of DNA Forward mutation – changes wild-type to different allele Reverse mutation – causes novel mutation to revert back to wild-type (reversion)

Classification of mutations by affect on DNA molecule Substitution – base is replaced by one of the other three bases Deletion – block of one or more DNA pairs is lost Insertion – block of one or more DNA pairs is added Inversion 180o rotation of piece of DNA Reciprocal translocation – parts of nonhomologous chromosomes change places Chromosomal rearrangements – affect many genes at one time

Figure 7.2 Fig. 7.2

Spontaneous mutations influencing phenotype occur at a very low rate Figure 7.3 b Mutation rates from wild-type to recessive alleles for five coat color genes in mice Fig. 7.3 b

General observations of mutation rates Mutations affecting phenotype occur very rarely Different genes mutate at different rates Rate of forward mutation is almost always higher than rate of reverse mutation

Are mutations spontaneous or induced? Most mutations are spontaneous. Luria and Delbruck experiments - a simple way to tell if mutations are spontaneous or if they are induced by a mutagenic agent

Figure 7.4 Fig. 7.4

Replica plating verifies preexisting mutations Figure 7.5 a Fig. 7.5 a

Figure 7.5 b Fig. 7.5b

Interpretation of Luria-Delbruck fluctuation experiment and replica plating Bacterial resistance arises from mutations that exist before exposure to bactericide After exposure to bactericide, the bactericide becomes a selective agent killing the nonresistant cells, allowing only the preexisting resistant cells to survive. Mutations do not arise in particular genes as a direct response to environmental change Mutations occur randomly at any time

Chemical and Physical agents cause mutations Hydrolysis of a purine base, A or G occurs 1000 times an hour in every cell Deamination removes –NH2 group. Can change C to U, inducing a substitution to and A-T base pair after replication Figure 7.6 a,b Fig. 7.6 a,b

X rays break the DNA backbone UV light produces thymine dimers Figure 7.6 c,d Fig. 7.6 c, d

Oxidation from free radicals formed by irradiation damages individual bases Figure 7.6 e Fig. 7.6 e

Repair enzymes fix errors created by mutation Excision repair enzymes release damaged regions of DNA. Repair is then completed by DNA polymerase and DNA ligase Figure 7.7a Fig. 7.7a

Mistakes during replication alter genetic information Errors during replication are exceedingly rare, less than once in 109 base pairs Proofreading enzymes correct errors made during replication DNA polymerase has 3’ – 5’ exonuclease activity which recognizes mismatched bases and excises it In bacteria, methyl-directed mismatch repair finds errors on newly synthesized strands and corrects them

DNA polymerase proofreading Figure 7.8 Fig. 7.8

Methyl-directed mismatch repair Fig. 7.9 Fig. 7.9

Unequal crossing over creates one homologous chromosome with a duplication and the other with a deletion Figure 7.10 a 7.10 a

Transposable elements move around the genome and are not susceptible to excision or mismatch repair Figure 7.10 b Fig. 7.10 e

Trinucleotide instability causes mutations FMR-1 genes in unaffected people have fewer than 50 CGG repeats. Unstable premutation alleles have between 50 and 200 repeats. Disease causing alleles have > 200 CGG repeats. Figure B(1) Genetics and Society Fig. B(1) Genetics and Society

Trinucleotide repeat in people with fragile X syndrom Figure A, B(2) Genetics and Society Fig. A, B(2) Genetics and Society

Mutagens induce mutations Mutagens can be used to increase mutation rates H. J. Muller – first discovered that X rays increase mutation rate in fruit flies Exposed male Drosophila to large doses of X rays Mated males to females with balancer X chromosome (dominant Bar eyed mutation and multiple inversions) Could assay more than 1000 genes at once on the X chromosome

Muller’s experiment Figure 7.11 Fig. 7.11

Mutagens increase mutation rate using different mechanisms Figure 7.12 Fig. 7.12a

Figure 7.12 b

Figure 7.12 b Fig. 7.12 b

Figure 7.12 c Fig. 7.12 c

Consequences of mutations Germ line mutations – passed on to next generation and affect the evolution of species Somatic mutations – affect the survival of an individual Cell cycle mutations may lead to cancer Because of potential harmful affects of mutagens to individuals, tests have been developed to identify carcinogens

The Ames test for carcinogens using his- mutants of Salmonella typhimurium Figure 7.13 Fig. 7.13

What mutations tell us about gene structure Complementation testing tells us whether two mutations are in the same or different genes Benzer’s experiments demonstrate that a gene is a linear sequence of nucleotide pairs that mutate independently and recombine with each other Some regions of chromosomes mutate at a higher rate than others – hot spots

Complementation testing Figure 7.15 a Fig. 7.15 a

Five complementation groups (different genes) for eye color. Figure 7.15 b, c Fig. 7.15 b,c Five complementation groups (different genes) for eye color. Recombination mapping demonstrates distance between genes and alleles.

A gene is a linear sequence of nucleotide pairs Seymour Benzer mid 1950s – 1960s If a gene is a linear set of nucleotides, recombination between homologous chromosomes carrying different mutations within the same gene should generate wild-type T4 phage as an experimental system Can examine a large number of progeny to detect rare mutation events Could allow only recombinant phage to proliferate while parental phages died

Benzer’s experimental procedure Generated 1612 spontaneous point mutations and some deletions Mapped location of deletions relative to one another using recombination Found approximate location of individual point mutations by deletion mapping Then performed recombination tests between all point mutations known to lie in the same small region of the chromosome Result – fine structure map of the rII gene locus

How recombination within a gene could generate wild-type Figure 7.16 Fig. 7.16

Working with T4 phage Figure 7.17 a

Phenotpyic properties of T4 phage Figure 7.17 b Fig. 7.17 b

Complementation test to for mutations in different genes Figure 7.17 c

Detecting recombination between two mutations in the same gene Figure 7.17 d Fig. 7.17 d

Deletions for rapid mapping of point mutations to a region of the chromosome Figure 7.18 a Fig. 7.18 a

Recombination mapping to identify the location of each point mutation within a small region Figure 7.18 b Fig. 7.18 b

Fine structure map of rII gene region Figure 7.18 c Fig. 7.18 c

What mutations tell us about gene function One gene, one enzyme hypothesis: a gene contains the information for producing a specific enzyme Beadle and Tatum use auxotrophic and prototrophic strains of Neurospora to test hypothesis Genes specify the identity and order of amino acids in a polypeptide chain The sequence of amino acids in a protein determines its three-dimensional shape and function Some proteins contain more than one polypeptide coded for by different genes

Beadle and Tatum – One gene, one enzyme 1940s – isolated mutagen-induced mutants that disrupted synthesis of arginine, an amino acid required for Neurospora growth Auxotroph – needs supplement to grow on minimal media Prototroph – wild-type that needs no supplement; can synthesize all required growth factors Recombination analysis located mutations in four distinct regions of genome Complementation tests showed each of four regions correlated with different complementation group (each was a different gene)

Figure 7.20 a Fig. 7.20 a

Figure 7.20 b Fig. 7.20 b

Interpretation of Beadle and Tatum experiments Each gene controls the synthesis of an enzyme involved in catalyzing the conversion of an intermediate into arginine

Genes specify the identity and order of amino acids in a polypeptide chain Proteins are linear polymers of amino acids linked by peptide bonds 20 different amino acids are building blocks of proteins NH2-CHR-COOH – carboxylic acid is acidic, amino group is basic R is the side chain that distinguishes each amino acid Figure 7.21 a Fig. 7.21 a

R is the side group that distinguishes each amino acid Fig. 7.21 b Figure 7.21 b

Figure 7.21 b

Figure 7.21 b Fig. 7.21 b

N terminus of a protein contains a free amino group C terminus of protein contains a free carboxylic acid group Figure 7.21 c Fig. 7.21 c

Genes specify the amino acid sequence of a polypeptide – example, sickle cell anemia Mutant b chain of hemoglobin form aggregates that cause red blood cells to sickle Figure 7.22 a Fig. 7.22 a

Sequence of amino acids determine a proteins primary, secondary, and tertiary structure Figure 7.23 Fig. 7.23

Some proteins are multimeric, containing subunits composed of more than one polypeptide Figure 7.24 Fig. 7.24

How do genotypes and phenotypes correlate? Alteration of amino acid composition of a protein Alteration of the amount of normal protein produced Changes in different amino acids at different positions have different effects Proteins have active sites and sites involved in shape or structure

Dominance relations between alleles depend on the relation between protein function and phenotype Alleles that produce nonfunctional proteins are usually recessive Null mutations – prevent synthesis of protein or promote synthesis of protein incapable of carrying out any function Hypomorphic mutations – produce much less of a protein or a protein with weak but detectable function; usually detectable only in homozygotes Incomplete dominance – phenotype varies in proportion to amount of protein Hypermorphic mutations – produces more protein or same amount of a more effective protein Dominant negative – produces a subunit of a protein that blocks the activity of other subunits Neomorphic mutations – generate a novel phenotype; example is ectopic expression where protein is produced outside of its normal place or time