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

This means that most traits are multifactorial. Most mutations are harmful in their effects; only rarely are mutations beneficial. A gene with one wild-type allele is monomorphic; a gene with two or more wild-type alleles is polymorphic. The vast majority of traits are determined by alleles of more than one gene. This means that most traits are multifactorial. A Heterogeneous Trait is One That May be caused by mutations in more than one gene. Human deafness is an example of a heterogeneous trait: mutations in any of at least 50 genes lead to deafness. (How can one tell if two deaf individuals carry mutations in the same gene or mutations in different genes?) An important class of mutations are conditional mutations. In these mutants the Environment affects Phenotype. Conditional mutations are those that express their associated phenotype only under some conditions (restrictive conditions) and not others (permissive conditions). Conditional lethal mutations are common. Temperature-sensitive conditional mutations are invaluable in genetic research.

NNN AUG UUU AGC UUU AGC UUU AGC NNN Met Phe Ser Phe Ser Phe Ser Frameshift mutations A single base-pair deletion or insertion results in a change in the reading frame NNN AUG UUU AGC UUU AGC UUU AGC NNN 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

NNNAUGUUUAGCUUUAGCUUUAGCNNN …MetPheSerPheSerPheSer… Frameshift mutations A single base-pair deletion or insertion results in a change in the reading frame NNNAUGUUUAGCUUUAGCUUUAGCNNN …MetPheSerPheSerPheSer… …CysLeuAlaLeuAlaLeu… …ValStpLeuStpLeuStp… 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

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

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

Missense mutations UUU UUU UGC UUU UUU WT Phe Phe Cys Phe Phe 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

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

AUG UUU AGC UUU AGC UUU AGC WT Met Phe Ser Phe Ser Phe Ser 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 Mutations that occur in introns are also silent Mutations that occur in non-genic regions are often silent

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

UUU UUU UGC UUU UUU Phe Phe Cys Phe Phe UUU UUU UGA UUU UUU 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

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

Nonsense suppressor mutations! 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. 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 ? AUG Trp AUG Trp AUC Trp ---UAC---UAG---UAA --UAC---UAG---UAA Normal tRNA Mutant tRNA

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 UAA-----3’ AUC Trp AAA Met Ala Phe --- Phe Phe Trp Phe Phe | A mutant protein that is larger than normal will be synthesized!!

Nonsense and Nonsense suppressor --- UUU UUU CAG UUU UUU ----- --- Phe Phe Gln Phe Phe --- Nonsense mutation --- UUU UUU UAG UUU UUU ----- --- Phe Phe STOP AUC Trp ---UAG--- 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. 5’--- UUU UUU UAG UUU UUU -----3’ AUC Trp AAA Met Ala Phe Phe Phe Trp Phe Phe

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