Methods used to study mutations

1. Methods used to study mutations Gross chromosomal changes- deletions, insertions, inversions, translocations Cytology- microscopy- karyotype Small mutations Small deletions, insertions and point mutations Recombinant DNA technologies

2. Frameshift mutations A single base-pair deletion or insertion results in a change in the reading frame AUG UUU AGC UUU AGC UUU AGC WT Met Phe Ser Phe Ser Phe Ser Delete C AUG UUU AGU UUA GCU UUA GC Met Phe Ser Leu Ala Leu Insert C AUG UUU AGC CUU UAG CUU UAG C Met Phe Ser Leu STOP

3. Frameshift mutations- Deletion A single base-pair deletion or insertion results in a change in the reading frame AUG UUU AGC UUU AGC UUU AGC Met Phe Ser Phe Ser Phe Ser Delete C AUG UUU AGU UUA GCU UUA GC Met Phe Ser Leu Ala Leu Delete GC AUG UUU AUU UAG CUU UAG C Met Phe Ile Stp Delete AGC AUG UUU UUU AGC UUU AGC Met Phe Phe Ser Phe Ser

4. Frameshift mutations-Insertion A single base-pair deletion or insertion results in a change in the reading frame AUG UUU AGC UUU AGC UUU AGC Met Phe Ser Phe Ser Phe Ser Insert C AUG UUU AGC CUU UAG CUU UAG C Met Phe Ser Leu STOP Insert CC AUG UUU AGC CCU UUA GCU UUA GC Met Phe Ser Pro Leu Ala Leu Insert CCC AUG UUU AGC CCC UUU AGC UUU AGC Met Phe Ser Pro Phe Ser Phe Ser

5. Missense mutations Missense mutations alters ONE codon so that it encodes a different amino acid UUU UUU UGC UUU UUU WT Phe Phe Cys Phe Phe UUU UUU UGG UUU UUU mut Phe Phe Trp Phe Phe

6. Consequences of Missense Mutations Missense mutations alter one of the many amino acids that make a protein Its consequences depend on which amino acid is altered Conservative mutations: K to R Nonconservative mutations: K to E Surface Vs buried Mutations in globular domains Vs un structured tails Silent mutations Mutations in non-coding regions Nonsense mutations

7. Silent Mutations Silent mutations do not alter the amino acid sequence! The Genetic code is degenerate! AUG UUU AGC UUU AGC UUU AGC WT Met Phe Ser Phe Ser Phe Ser AUG UUC AGC UUU AGC UUU AGC Mut Met Phe Ser Phe Ser Phe Ser Mutations that occur in introns are also silent Mutations that occur in non-genic regions are often silent

8. Mutations in non-protein coding regions Mutations in the promoter, splicing junction or ribosome binding site are also mutagenic Reduced expression of mRNA might result in reduced levels of proteins OR Increased expression of mRNA might result in increased levels of protein Mutations in splicing junctions may also be mutagenic improperly spliced mRNA will result in the intron being translated Mutations in tRNA or aminoacyl-tRNA synthase are mutagenic

9. Lactose========>Glucose + Galactose Lactase Human milk is 7% lactose. Lactose is not absorbed through the wall of the digestive tract. In human infants, lactase is secreted in intestine which breaks the lactose into easily absorbed Glucose and Galactose. Production of the lactase enzyme declines in adults. The unabsorbed lactose creates cramps, diarrhea, and nausea. In some humans, lactase continues to be produced throughout adulthood. These individuals are called lactose absorbers (LA). Adult lactose absorption is inherited as an autosomal dominant trait. Lactose persistence and non-persistence reflect inheritence of different alleles of the lactase gene. Lactose intolerance is the result of being homozygous for the recessive lactase (WT) allele Being homozygous or heterozygous for the mutant allele allows lactase expression in adults when normally lactase expression is turned off. Lactose intolerance in humans

10. lactose tolerance There are no mutations in the coding region of the lactase gene. A mutation is observed in the enhancer -13910 bp upstream of the gene in an AP2 consensus sequence. CCCCAGGC • the polymorphism modifies a transcription factor binding site (AP2) • AP2 acts as a repressor but in the mutant it cannot bind and cannot repress the gene- so adults keep producing lactase C/C T/T

11. Mutations in splicing of RNA

12. Nonsense mutations Nonsense mutations alter one codon so that it now encodes for a STOP codon UUU UUU UGC UUU UUU Phe Phe Cys Phe Phe UUU UUU UGA UUU UUU Phe Phe STOP Nonsense mutations insert a stop codon which results in premature termination Truncated polypeptide usually results in loss of function for polypeptide

13. There are NO tRNAs in cells with anti-codons that recognize STOP codons in mRNA What happens if there is a mutation in the anti-codon loop of a specific tRNA Gene that allows a tRNA to recognize a stop codon

14. Trp Trp AUG AUC These are the result of a mutation in the anti-codon loop of a specific tRNA Gene It allows the tRNA to recognize a nonsense codon and base pair with it. Nonsense suppressor mutations! DNA Gene encoding tRNATRP Point mutation occurs in the anticodon loop OF THE tRNA This allows this tRNA to base pair with a stop codon and ? ---UAC---UAG ---UAG---UAG Normal tRNA Mutant tRNA

15. Met Ala Phe Phe Trp AUC AAA Nonsense suppressor --- UUU UUU UAG UUU UUU ----- --- Phe Phe STOP Trp-tRNA has mutation In anticodon This allows it to pair with a stop codon 5’--- UUU UUU UAG UUU UUU -----3’ --- Phe Phe Trp Phe Phe ----> A mutant protein that is larger than normal will be synthesized!!

16. Met Ala Phe Phe Phe Phe Trp Trp AUC AUC AAA AAA AAA ---UAG--- 5’--- UUU UUU UAG UUU UUU -----3’ Nonsense and Nonsense suppressor --- UUU UUU CAG UUU UUU ----- --- Phe Phe Gln Phe Phe --- Nonsense mutation --- UUU UUU UAG UUU UUU ----- --- Phe Phe STOP What will happen if an individual carries both a nonsense mutation in a gene and a nonsense suppressor mutation in the anticodon loop of one of the trp-tRNA genes.

18. Recombinant DNA technology When genes are mutated - proteins are mutated- DISEASE STATES OCCUR Sickle cell Anemia Globin 2 alpha globin chains 2 beta globin chains Mol wt 16100 daltons xfour = 64650 daltons Single point mutation in beta-globin Converts Glu to Val at position 6 Need to know mutation Need to look at genes of individuals Genes lie buried in 6billion base pairs of DNA (46 chromosomes). Molecular analyses necessary Take advantage of enzymes and reactions that naturally occur in bacteria

19. Why all the Hoopla? Why all the excitement over recombinant DNA? It provides a set of techniques that allows us to study biological processes at the level of individual proteins in individuals! It plays an essential role in understanding the genetic basis of cancer in humans Recently found that mutations in a single gene called p53 are the most common Genetic lesion in cancers. More than 50% of cancers contain a mutation in p53 Cells with mutant p53 Chromosomes fragment Abnormal number of chromosomes Abnormal cell proliferation!

20. To understand the complete biological role of p53 protein and its mutant phenotype we need to study the gene at multiple levels: Genetics- mutant gene- mutant phenotype Now what? Genetics will relate specific mutation to specific phenotype It usually provides No Information about how the protein generates the phenotype For p53 We would like to know The nucleotide sequence of the gene and the mutation that leads to cancer When and in which cells the gene is normally expressed (in which cells is it transcribed) At the protein level--Amino acid sequence Three-dimensional structure Interactions with other proteins Cellular information Is the location in the cell affected How does it influence the behavior of the cell during division Organism phenotype p53