Transcript Chapter 12
Chapter 12 Opening photo. Peacock [© Photos.com] © 2006 Jones and Bartlett Publishers Mutations: altered information heritable change in the DNA (?) Classification spontaneous vs. induced random unpredictable rates radiation chemical mutagen Ames test Classification somatic vs. germ line body cells “mosaics” most cancers gametes passed on Classification conditional on off vs. unconditional permissive conditions restrictive conditions expressed all the time e.g., temperature sensitive mutants Cats: enzyme for melanin deposition is temperature sensitive Off at normal body temperature On at cooler temperatures (face, paws, tail) Fig. 12.1. A Siamese cat showing the characteristic pattern of pigment deposition [Courtesy of Jen Vertullo] © 2006 Jones and Bartlett Publishers Classification based on their affect on gene recessive dominant loss of function(knockout) hypomorhphic (leaky) below above hypermorphic gain of function (also ectopic exp.) Classification based on molecular changes base substitution 5’-GAG-3’ 5’-GAG-3’ 3’-CTC-5’ 3’-CAC-5’ 5’-GAG-3’ unmutated 3’-CTC-5’ 5’-GTG-3’ mutant 3’-CAC-5’ Classification based on molecular changes base substitution transition: pyrimidine pyrimidine T 4 C, C 4 T: transversion: How many? purine purine A 4 G, G 4 A pyrimidine to purine purine to pyrimidine Classification based on molecular changes base insertions or deletions (wait three slides) (position) base substitutions in coding region: missense change of amino acid silent doesn’t change amino acid nonsense make a new stop codon frameshift small insertion of deletion shifts reading frame Chapter 8 Gene Expression missense ? Table 1.1 silent ? nonsense codons are linear and non-overlapping Fig. 8.23. Reading bases in an RNA molecule © 2006 Jones and Bartlett Publishers insertion reading frame frameshift mutation Fig. 8.24. Change in an amino acid sequence of a protein caused by the addition of an extra base © 2006 Jones and Bartlett Publishers Fig. 8.25. Interpretation of the rll frameshift mutations © 2006 Jones and Bartlett Publishers Table 12.1. Major types of mutations and their distinguishing features © 2006 Jones and Bartlett Publishers One of the “classic” mutations is sickle cell anemia single base substitution (missense) amino acid #6 from glutamic acid to valine Fig. 12.3. Base substitution mutation in sickle-cell anemia © 2006 Jones and Bartlett Publishers © 2006 Jones and Bartlett Publishers -hemoglobinA normal protein folding normal RBC’s -hemoglobinS forms long needle like crystals RBC’s become sickle shaped © 2006 Jones and Bartlett Publishers HbA HbA “normal” HbA HbS sickle cell trait some symptoms HbS HbS sickle cell disease often die young dynamic mutations X chromosome instability in region of CGG repeat replication slippage Fig. 12.4. Pedigree showing transmission of the fragile-X syndrome. [After C. D. Laird. 1987. Genetics 117: 587] © 2006 Jones and Bartlett Publishers 12.4 mutations are random, but… …they also happen at characteristic rates (they do not arise in response to conditions) agar plates with antibiotic plate antibiotic-sensitive bacteria some colonies grow (have resistance) induced? or random? replica plating (Lederberg’s) mutants grow in the same place, therefore the mutations occurred before they were plated Fig. 12.13. Replica plating © 2006 Jones and Bartlett Publishers Selective techniques merely select mutants that pre-exist in a population development of resistance to: antibiotics (pesticides, etc.,) DDT resistant insects MRSA TB in Russian prisons Mutations random can’t predict where they happen consistent they happen at a measurable frequency variable rate varies from gene to gene and organism to organism Mutational hotspots places where mutations are more likely to occur trinucleotide repeats methylated cytosine Fig. 12.15. Loss of the amino group in 5-methylcytosine and from normal cytosine © 2006 Jones and Bartlett Publishers What are the physical causes of mutations? Table 12.3. Major agents of mutation and their mechanism of action © 2006 Jones and Bartlett Publishers Water depurination removal of base from purine nucleotide Fig. 12.16. Depurination © 2006 Jones and Bartlett Publishers Water depurination removal of base from purine nucleotide can be repaired mutation rate (in air?): 3 depurinations 109 purines per minute nitrous acid can deaminate A C H U G thymine cytosine Fig. 12.17. Deamination of adenine results in hypoxanthine © 2006 Jones and Bartlett Publishers nitrous acid can deaminate A C G H U ? C base analogs can substitute for normal base more likely to mis-pair than normal bases 5-bromouracil (Bu) similar to thymidine ketoenol- Fig. 12.18. Mispairing mutagenesis by 5-bromouracil © 2006 Jones and Bartlett Publishers Fig. 12.18. Mispairing mutagenesis by 5-bromouracil © 2006 Jones and Bartlett Publishers Fig. 12.19. Two pathways for mutagenesis by 5-bromouracil © 2006 Jones and Bartlett Publishers alkylating agents EMS ethyl methanesulfonate nitrogen mustard Fig. 12.20. Chemical structures of two highly mutagenic alkylating agents mispairing Fig. 12.21. Mutagenesis of guanine by ethyl methanesulfonate (EMS) © 2006 Jones and Bartlett Publishers Intercalating agents interfere with topoisomerase II (gyrase) leaves nicks in DNA leads to deletions or insertions (EtBr) http://en.wikipedia.org/wiki/Ethidium_bromide UV light causes formation of T-T dimers distorts double helix interferes with transcription translation Fig. 12.22. (A) Formation of a thymine dimer (B) distortion of the DNA helix caused by two thymines © 2006 Jones and Bartlett Publishers Ionizing radiation X-rays particles/radiation from radioactive decay Over a wide range of X-ray doses: frequency of mutation proportional to radiation dose Fig. 12.23. Relationship between the percentage of x-linked recessive lethals and x-ray dose in D. melanogaster © 2006 Jones and Bartlett Publishers Three types of damage from ionizing radiation single-strand breakage usually repaired double-strand breakage nucleotide alteration chromosome breaks radiation therapy Fig. 12.24. Annual exposure of human beings in the United States to various forms of ionizing radiation. [Source: National Research Council] © 2006 Jones and Bartlett Publishers Chernobyl 1986 10X radiation of bombing of Hiroshima but little damage was expected 1996 mutations rates were 2X (for some loci) Fig. 12.25. Mutation rates of five tandem repeats among people of Belarus who were exposed to radiation from Chernobyl and among unexposed British people. [Data from Y. E. Dubrova, et. al. 1996. Nature 380: 183.] © 2006 Jones and Bartlett Publishers 12.5 Fixing DNA damage 1 mutation billion bp per minute every cell would have damage at 10,000 sites every 24 hours Exam 3 Next Friday 4/4 Mendelian genetics (2) Modifications to Mendel Sex determination and sex-linkage (3) Linkage and crossing over including two and three point test crosses (4) and including lab material Quantitative genetics (15) Mutation and DNA repair (12) Problem set #1 Problem set #2 No class (tentatively) 4/18 DNA ligase * uracil glycosylase* mismatch repair AP endonuclease enzymatic reversal excision repair postreplication repair Table 12.6 Types of DNA damage and mechanisms of repair © 2006 Jones and Bartlett Publishers mismatch repair “last-chance” error correction for mistakes mismatch repair detect mismatch cut one strand on either side of mistake Which strand? strand that is least methylated mismatch repair detect mismatch cut one strand on either side of mistake remove that strand in area of mismatch fill in missing strand mismatch repair mutS protein from mutL protein from protein from mutH exonuclease DNA Pol DNA Ligase in prokaryotes recognize and bind to mismatches makes a nick in bad strand removes strand past mistake fills in new compliment seals the nick Fig. 12.27. Mismatch repair © 2006 Jones and Bartlett Publishers mismatch repair mutS protein from mutL protein from protein from mutH exonuclease DNA Pol DNA Ligase in prokaryotes recognize and bind to mismatches makes a nick in bad strand removes strand past mistake fills in new compliment seals the nick mismatch repair mutS mutL in prokaryotes bacteria with defects in either of these have high rates of spontaneous mutations four homologous genes in humans mutations in any may lead to HNPCC (human nonpolyposis colorectal cancer) AP endonuclease filling in gaps deamination C to U hydrolysis A,T to -OH (apyrimdinic site) (apurinic site) Fig. 12.28. Action of AP endonuclease © 2006 Jones and Bartlett Publishers UV damage (cross-links) enzymes that reverse linkage excision repair endonuclease cut on either side of damage DNA pol. displaces damaged segment fills in new compliment DNA ligase joins ends together endonuclease Fig. 12.29. Mechanism of excision repair of damage to DNA © 2006 Jones and Bartlett Publishers postreplication repair (PRR) Fig. 12.30. Postreplication repair © 2006 Jones and Bartlett Publishers 12.7 Testing for mutagens The Ames test histidine requiring (His-) mutants of Salmonella test for reversion (to His+) sensitive, quantitative Fig. 12.31. Linear dose– response relationships obtained with various chemical mutagens in the Ames test.. [Data from B. N. Ames. 1974. Science 204: 587–593.] © 2006 Jones and Bartlett Publishers 4.6 Recombination chiasmata (chapter 3) physical manifestation of crossing-over 4.6 Recombination chiasmata (chapter 3) physical manifestation of crossing-over help homologous chromosomes align at equator 4.6 Recombination chiasmata (chapter 3) are preceeded by DSB’s (double-strand breaks) occur at “hot spots” certain positions where breaks are more like to occur repair ? Fig. 4.30. Molecular mechanism of recombination. [After D. K. Bishop and D. Zickler. 2004. Cell 117: 9.] © 2006 Jones and Bartlett Publishers 4.6 Recombination non crossing-over 3’ broken end invades intact chromosome base-pairs with complimentary strand other strand forms “D-loop” 3’ end is elongated eventually ejected from template (?) base pair with other end of break fill in missing nucleotides 4.6 Recombination crossing-over 3’ broken end invades intact chromosome base-pairs with complimentary strand other strand forms “D-loop” D-loop expands acts as template for other broken strand base pair with other end of break fill in missing nucleotides Holliday junction-resolving enzyme = Fig. 4.31. Two Holliday junctions in a pair of DNA molecules undergoing recombination [EM, © 1997 from Essential Cell Biology, 1st Edition by Dr. Bruce Alberts. Reproduced by permission of Garland Science/Taylor & Francis Books, Inc.] © 2006 Jones and Bartlett Publishers 12.3 Transposable elements Discovered in corn by Babara McClintock (noble prize, 1983) Pieces of DNA that could move around Many encode their own transposase 12.3 Transposable elements Different classes of transposons DNA transposons LTR retrotransposons non LTR retrotransposons LINE long interspersed elements SINE short 12.3 Transposable elements DNA transposons (cut and paste transposition) have terminal inverted repeats binding sites for transposase Fig. 12.8. Sequence arrangement of a cut-and-paste transposable element and the changes that take place when it inserts into the © 2006 Jones and Bartlett Publishers genome LTR retrotransposons long terminal repeats direct repeats same orientation Fig. 12.9. Sequence in direct and inverted repeats © 2006 Jones and Bartlett Publishers LTR retrotransposons long terminal repeats direct repeats same orientation inverted repeats inverse orientation Fig. 12.9. Sequence in direct and inverted repeats © 2006 Jones and Bartlett Publishers LTR retrotransposons long terminal repeats direct repeats inverted repeats both use RNA intermediate (transcription, then RT) Fig. 12.10. Sequence organization of a copia retrotransposable element of Drosophila melanogaster © 2006 Jones and Bartlett Publishers non-LTR retrotransposons no long terminal repeats long interspersed elements short interspersed elements Alu I family 300 bp 106 copies 11% Human DNA Transposable elements can cause mutations insertion into a gene loss of function (knockout) recombination between trans. elements deletions, inversions or duplications Fig. 12.11. Recombination between transposable elements in the same chromosome © 2006 Jones and Bartlett Publishers Fig. 12.12. Unequal crossing-over between homologous transposable elements present in the same orientation in different chromatids © 2006 Jones and Bartlett Publishers Transposable elements make up much of the human genome (45%) Table 12.2. Transposable elements in the human genome © 2006 Jones and Bartlett Publishers Transposable elements make up much of the human genome (45%) function ? SINEs mariner transcribed under stress horizontal transmission (species to species)