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Lecture 3

BIOC15Fall2013 Lecture 3 and Lecture 4 Notes 2.13.29 PM.docx

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Biological Sciences
Karen Williams

BIOC15Fall2013 Lecture 3 and Lecture 4 Notes Lecture 3: Mutants Mutations classified by their effect on DNA o Wild type allele is the most common variants o Forward mutation  WT allele is changed to a different allele o Reverse mutation or reversion  novel mutatnt allele reverts back to WT o Substitution occurs when a base at a certain position in one strand of the DNA molecule is replaced by one of the other 3 bases o Substitutions can be transitions (purine is replaced by purine – A, G, or pyrimidine replaced pyrimidine – T, C) or transversions (pyrimidine changed to purine and vice versa) o Transition mutation example: A-T to G-C o Transversion mutation example: C-G to G-C o o Deletion  one or more nucleotide pairs is lost form DNA molecule o Insertion  block of one or more nucleotide pairs is inserted into DNA o Inversion  180 degree rotations of segment of DNA molecule o Reciprocal transolocations  parts of 2 non homologous chromosomes change places o Mutations happen very infrequently How do mutations affect polypeptide structure and function? o Base pair substitution mutation defined by their action on the DNA  transition mutation or transversion mutation o Mutations defined by their action on the protein product o Missense mutation  changes from one amino acid to another o o Nonsense mutation  change in codon does not code for an amino acid, so incomplete polypeptide is formed due to premature termination of translation o Neutral mutation (Silent mutation) change from one amino acid to another with similar chemical properties (example from Lysine to Arginine – both basic amino acids) o Amino acids with non polar side groups: o o amino acids with uncharged R groups: 1 o amino acids with charged R groups: o o Frameshift mutation  addition or deletion of one or a few base pairs leads to a change in reading frame so all the amino acids afterwards are most likely wrong o Most mutations outside the coding sequence such as in promoters and transcription termination signals, affect the amount but not the nature of the protein product o Rare exceptions include mutations that lead to incorrectly spliced mRNAs, or the convert to a stop codon into a codon for an amino acid Alkaptonuria o Mutations in homogenistic oxidase gene (HGO) result in a deficiency of the enzyme HA oxidase and the accumulation of homogenistic acid o Homogenisitc acid builds up and thus turns urine black o Phenyalanine hydroxylase (PAH) o most PAH alleles inactivate the protein and of the two classes, pp produces no active protein o However PP and Pp are indistinguishable because a single dose of P_ is sufficient to produce WT phenotype o The PAH gene is said to be haplosufficient (half is enough) o The reason the defective PAH allele is recessive is that one P is sufficient to produce the WT phenotype Reversion o One effect of a point mutation on the phenotype may be to restore the mutant gene to normal function by reverse mutation o Mutation occurs in the gene that causes the mutation to go back to being normal (mutant gene  normal gene) o Reversion may also occur by a point mutation at another site or in another gene, that restores WT function by suppressing the mutant phenotype (occurs in the suppressor gene) 2 o o mutations = DNA damage – DNA repair o most times the mutation is repaired The Ames test identifies potential carcinogens o test for mutagenicity o assay uses his mutants in S.typhimurium o uses rat liver enzymes to simulate the action of mammalian metabolism  can convert a harmless compound into a mutagen o detects mutations that cause his to his reversion o if the left plate has more bacteria than the right plate, then the compound is considered to be mutagenic o o there can be nutritional mutants , example Neurospora crassa o conditional mutants, example yeast and bacteria o resistance mutants, example antibiotic resistance in bacteria Lethal gene mutations o some mutations we are not able to find because it kills the organism which has the mutation 3 o Human example  Tay-Sachs: single base pair substitution in HEXA gene change the amino acid of an enzyme essential for the removal of gangliosides o Mouse example  mouse coat colour mutation yellow is homozygous lethal allele of Agouti (A) o Yellow x yellow  2/3 yellow, 1/3 WT progeny o What happens? o A A x A A  A A , 2A A, AA o Since this is a homozygous lethal allele, the A A individual dies, leaving only 3 possible progenies left  out of which the heterozygous ones will be yellow, and the homozygous dominant ones will be white (WT) ClB chromosome o 1928 Muller  devised method of searching for any lethal mutation on the X chromosome (chromosome I in Drosophila) o he constructed a chromosome called ClB which carries an inversion cross-over supressor (C), a lethal (l) and a dominant mutation Bar-eye(B) marker o o F1 females have irradiated paternal X chromosome (Red) and a Bar-marked balancer maternal X chromosome (wavy blue line) o These 2 chromosome cannot recombine because the balancer chromosome has multiple inversions o Single F1 females, are then individually mated with WT males o If the paternal X chromosome in any one F1 female has an Xray induced recessive lethal mutation she can produce only Bar-eyed sons (left) o If the X chrosmosome has no such mutation, this F1 female produces both Bar eyed and non Bar eyes sonsa (right) Mutation hunt or screen o Choice of mutagen: o Fruit flies  EMS  and alkylating agent that produces small point mutations  X-rays, gamma rays, ionizing radiation that produces large scale changes such as intragenic deletions  Mobilization of transposable elements o Mouse or zebra fish  ENU Rates of Spontaneous mutation 6 o Rates of recessive forward mutations at five coat colour genes in mice  11 mutations per gene, in every 10 gametes o Mutation rates in other organisms  2-12 mutations per gene every 10 gametes o Spontaneous mutations are a rare event o Different genes have different mutation rates  depends on size and susceptibility to mechanisms that can cause mutations o The rate of forward mutation (a disrutption in gene function) is alsmost always higher than the rate of reversion o Fluctuation test  observation of substantial fluctuation in the number of resistant colonies in different pteri plates, scientists concluded that bacterial resistance arises from mutations that exist before exposure to the bacteriophage Lecture 4: Genomes Alkaptonuria – Insertion/Frameshift mutation in HGO o L119insG  Leucine at sequence position 119 has a G neucleotide inserted in the DNA sequence 4 o So the amino acid is Leucine instead of Cysteine o Framshift mutation  So all amino acids afterwords are changed o Transposase Gene o transposase gene allows for mobility of insertion elements o Unequal crossing over can occur between homologous chromosomes o pairing between homologs during meiosis can be out of register during meiosis and produce gametes with either a deletion or a reciprocal duplication o unequal crossing over results in a deletion on one homolog and a duplication in another homolog o example: colourblindness  unequal crossing over between the nearby and highly similar genes for red and green photoreceptors o o we can use this to create site specific deletions Transposable elements (TEs) move around the genome and alter DNA o these elements occur naturally o definition  all DNA segments that move about in the genome, no matter of the mechanism o TEs can jump into a gene and disrupt its function o Two mechanisms of TE movement (transposition) o o TEs that move by the second mechanism are exception  forward rate of muatation is not higher than that of reversion because excision from the gene where the TE inserted, reverts the gene to its normal state Insertional mutagenesis o mutations may be described by how they affect the proteins o Loss of function mutation (null) Mutation inside or outside a coding region that reduces or abolishes protein activity o Amorphic mutations  loss of function alleles that completely block the function of a protein o Hypomorphic mutation (leaky)  loss of function mutation that produces either much less of a protein or a protein with very weak but detectable function o Incomplete dominance is caused by hypomorphic mutations  phenotypes vary with the amount of functional protein that is present  having one dominant allele is not enough for full function of the necessary enzyme to have the same phenotype as homozygous dominant o This is called haploinsufficiency  one WT allele does not provide enough of a gene product 5 o Antimorphic or dominant negative alleles  some allales of genes encode subunits of multimers that block the activity of the WT subunits, therefore gaining dominance over the WT allele o Hypermorphic mutation is a gain of function mutation that generates withr more protein thatn WT or the same amount of a more efficient protein o Neomorphic mutation  generate novel phenotype o Extopic expression  production of protein outside of its normal place or time o Nonsense suppressor tRNAs  mutant tRNAs can insert an amino acid in the polypeptide even after a stop codon, therefore suppressing the effects of nonsense mutations o Null  the proteins they encode completely lack function o Leaky  the proteins they encode have some residual function o Insertions sequence (IS) elements that are inserted into a gene may disrupt the function o Transposons in serted into the promoter region of a gene may activate gene expression Tyrosinase-negative Oculocutaneous Albinism (Tyr) o Oculocutaneous Albinism: complete lack of pigment in eyes and skin o In patient S.S the singe insertion in exon 2 caused a disruption of the tyrosinase gene and the production of tyrosinase o o you can see at the bottom what amino acid the sequence encodes o you can see the difference between normal and insertion (amino acids were changed) so that the gene is completely changed, and there is no longer production of Tyrosinase McClintock: Transposable elements in maize o corn is often purple or yellow o purple (C) kernels  the pigment is produced o but not pigment is produced in colourless (c) kernels o AND there also is mottled kernels  purple and white kernels o You can cross a yellow x purple kernel o C_ x cc  Cc and _c but we don’t know if heterozygous is yellow or purple o If heterozygous = purple then the offspring would be either yellow or purple  so how do we get mottled kernels??? o GENES MOVE! o o o Ac activates the transposable elemenet Ds o Ds inserted into C  disrupts production of colour pigment so you get the colourless yellow kernel Excision of inserted element – Reversion o One effect of a point mutation on the phenotype may be to restore the mutatnt gene to normal function by reverse mutation  same gene changed at the same time 6 o o so all the cells where the insertion hopped out are purple and all the cells where the transposable element did not hop out are yellow o this is called reversion from colourless to colour in certain spots Rice(Oryza sativa) o genome was sequenced in 2005 o 15000 of 56000 genes are from transposable elements How natural processes can change the information stored in DNA o X-rays break the sugar –phosphate backbone of DNA  causes deletions o UV light causes adjacent
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