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Mutations

Mutations. Frameshift mutations. A single base-pair deletion or insertion results in a change in the reading frame. AUG UUU AG C 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 C UU UAG CUU UAG C

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Mutations

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  1. Mutations

  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. Sequencing the whole genomes of a family (2010 Science 328 636). 98 crossovers in maternal genome 57 crossovers in paternal genome Mutation rate is 1x10-8 per position per haploid genome ~70 new mutations in each diploid human genome CpG sites mutate at a rate 11 times higher than other sites Exome sequencing of 2440 individuals (Science 2012 337 40) Each person has ~100 loss of function mutations (~35 nonsense). 20 loss of function mutations are homozygous Common SNP frequencies are the same in all geographic populations Rare SNPs are population specific and their frequencies vary for each geographic population

  4. Frameshift mutation Insertion/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 Delete GC Delete AGC

  5. 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

  6. 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 Insert CC Insert CCC

  7. 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

  8. 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

  9. 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

  10. Silent Mutations Silent mutations do not alter the amino acid sequence! 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

  11. 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

  12. 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

  13. 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

  14. Mutations in splicing of RNA

  15. 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

  16. 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

  17. 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 This allows this tRNA to base pair with a stop codon and ? ---UAC--- ---UAG--- Normal tRNA Mutant tRNA

  18. 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!!

  19. 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.

  20. Spontaneous mutations Replication induced mutations of DNA Usually base substitutions (Most errors are corrected) Meiotic crossing over can induce mutations Small additions and deletions AND Large changes as well Environment induced changes Exposure to physical mutagens - radioactivity or chemicals Depurination (removal of A or G) Repair results in random substitution during replication Deamination (removal of amino group of base) (nitrous acid) Cytosine--uracil--bp adenine--replication-- Oxidation (oxoG) guanine--oxoguanine--bp adenine--replication -- Base analog incorporation during replication BU-T Intercalating agents X-rays- Generation of mutations

  21. Mutation rate There are approximately 1013 cells in the human body Each cell receives 10,000 DNA lesions per day (Lindahl and Barnes 2000). Most pervasive agent is UV. 100,000 lesions per exposed cell per hour (Jackson and Bartek 2009) Ionizing agents are more toxic because they generate double strand breaks (Ward 1988) Chromosome instability (gain or loss of entire segments) is frequent in dividing cells- during manipulation like in vitro fertilization. Occurs even in cleavage stage embryos. 40% of imbalances are entire arm imbalance while 45% are terminal segment imbalance (double strand break and nondysjunction)

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

  23. 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

  24. 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!

  25. Alkaptonuria Degenerative disease. Darkening of connective tissue, arthritis Darkening of urine Garrod characterized the disorder- using Mendels rules- Autosomal recessive. Affected individuals had normal parents and normal offspring. 1908 Garrod termed the defect- inborn error of metabolism Homogentisic acid is secreted in urine of these patients. This is an aromatic compound and so Garrod suggested that it was an intermediate that was accumulating in mutant individuals and was caused by lack of enzyme that splits aromatic rings of amino Acids. 1958 La Du showed that accumulation of homogentistic acid is due to absence of enzyme in liver extracts 1994 Seidman mapped gene to chromosome 3 in human 1996 Gene cloned and mutant identified P230S &V300G 2000 Enzyme principally expressed in liver and kidneys

  26. 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

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