4 I. Mutation 10.1 Mutations and Mutants 10.2 Molecular Basis of Mutation 10.3 Mutation Rates 10.4 Mutagenesis 10.5 Mutagenesis and Carcinogenesis: The Ames Test
5 10.1 Mutations and Mutants Mutation: A heritable change in the genome. Mutant: a strain carrying mutation. Genotype: the sequence of the genome. Phenotype: the observable properties of a strain, relative to the parental strain. Wild-type: the natural isolate. hisc HisC hisc1, hisc2 gene protein mutations Biosynthesis of the amino acid histine Phenotype: His + His -
6 10.1 Mutations and Mutants Selectable: advantageous under certain conditions; Non-selectable mutation: maybe apparent in phenotypes Mutant Selectable mutation Non-selectable mutation Wild-type
7 10.1 Mutations and Mutants All colonies grow Mutants do not grow All colonies grow Mutants do not grow Incubate Master plate; growth on complete medium Velveteen; sterilized Press plate onto velveteen Complete medium Transfer imprint of colonies to fresh media Minimal medium Plastic hoop Wooden block Velveteen with imprint of all colonies Screening for auxotrophs (a mutant with a nutritional requirement for growth; vs. prototroph) : The replica plating method (outdated). Now use toothpicks. Penicillin-selection: kill actively growing cells
8 10.1 Mutations and Mutants Spontaneous: mutations occur naturally. Cellular microorganisms: 10-6 to 10-7 per kb per round of replication; Higher organisms: 10 times lower; DNA viruses: 100 to 1000 times higher; RNA viruses: 10 5 times higher (why?). Induced: mutations made deliberately.
9 10.2 Molecular Basis of Mutation DNA M U T A T I O N Normal DNA replication Base-pair substitution: Missense; Nonsense; Silent. DNA Transition: purine Transcription purine or pyrimidine Asparagine codon Stop codon Tyrosine codon Tyrosine codon mrna Translation pyrimidine; Transversion: purine pyrimidine or Faulty protein Incomplete protein Normal protein Normal protein Protein pyrimidine purine Missense mutation Nonsense mutation Silent mutation Wild type Point mutations: one base pair change mutations
10 10.2 Molecular Basis of Mutation Frameshift: mutations different reading frame. DNA Transcription of light green strand mrna Reading frame +1 Insertion Codons 0 Deletion 1 Insertion: base pair(s) added into DNA sequence. Deletion: base pair(s) removed from DNA sequence. Possible consequences: In-frame or frameshift mutations
11 10.2 Molecular Basis of Mutation Site-directed mutagenesis: mutation at specific sites. Reversions (back mutations): point and insertion are reversible. Deletion (>3 bp) is generally regarded to be irreversible. True: the same site revertant, restores the original sequence. Suppressor: a second mutation compensates for the original mutation, restores the original phenotype.
12 10.4 Mutagenesis Induced mutations: Mutations occur at a greatly increased rate in the presence of mutagens (chemical, physical, and biological agents)
13 10.4 Mutagenesis Analog 5-Bromouracil 2-Aminopurine Substitutes for Thymine Adenine Nucleotide base analogs: a. 5-Bromouracil basepair with G: AT GC; b. 2-Aminopurine basepair with C: AT GC. Chemical modifications: Alkylating agents, Nitrosoguanidine, extremely efficient, modifies DNA. Intercalating agents: Acridine, Ethidium bromide (EB) Animation: Mutagens
14 10.4 Mutagenesis Analog Substitutes for 5-Bromouracil Thymine Nitrosoguanidine, acts by adding alkyl groups to the O 6 of guanine and O 4 of thymine, which can lead to transition mutations between GC and AT. 2-Aminopurine Introduce changes in nonreplicating DNA. Adenine
15 10.4 Mutagenesis Intercalating agents: Acridine, planar molecules able to insert DNA frameshift. Ethidium bromide (EB)
16 10.4 Mutagenesis Wavelength (nm) Electromagnetic spectrum Ionizing Cosmic Gamma 200 X-rays Microwave Radar Television Radio Ultraviolet 400 Visible Infrared Radiation: 1) Nonionizing, UV, 260nM (maximum absorption) pyrimidine dimers. A dose of UV (50-90% death rate) for mutagenesis. 2) Ionizing radiation (X-rays, g) OH on macromolecules.
17 10.4 Mutagenesis Repair systems 1) Error-free a) Direct reversal, photolyase, alkylated bases; b) ssdna damage, cut the damaged and synthesize new; 2) Error-prone, dsdna recombination mechanism; -- SOS system
18 10.4 Mutagenesis The SOS System (error-prone): Large-scale damage. A regulon by LexA and RecA Repression of the regulon by LexA is relieved by self-cleavage. Error-prone DNA polymerase (UmuCD).
19 10.4 Mutagenesis The SOS System (errorprone): Large-scale damage. Error-prone DNA polymerase (UmuCD).
20 Changes in Mutation Rate Genetic stability Metabolic cost Benefit Evolutionary improvement dnaq, subunit of DNA polymerase III, proofreading Mutator strains Increased mutation rate Increased evolutionary fitness
21 10.5 Mutagenesis and Carcinogenesis: The Ames Test forward mutation Wild type his + Auxotroph his - Back mutation Spontaneous revertants (negative control) The chemical increased the mutation rate
22 Nitrosamines ( 亚硝胺类 ) mutation detection by Ames assay
23 II. Gene Transfer 10.6 Genetic Recombination 10.7 Transformation 10.8 Transduction 10.9 Conjugation: Essential Features The Formation of Hfr Strains and Chromosome Mobilization Complementation Gene Transfer in Archaea Mobile DNA: Transposable Elements
24 II. Gene Transfer Transformation by free DNA (fragment or plasmid); Transduction, mediated by a virus; Conjugation, DNA exchange via cell-cell contact.
25 The fate of the transferred DNA 1) Degraded by restriction enzymes; 2) Replicate by itself (but only if it possesses its own origin of replication such as a plasmid or phage genome); 3) Combine with the host chromosome.
26 10.6 Genetic Recombination Homologous Recombination: a process that results in genetic exchange between homologous DNA sequences from two different sources.
27 10.6 Genetic Recombination Homologous Recombination: 1) Single Crossover, whole plasmid (circular) integration to the chromosome; 2) Double Crossover, the fragment of linear DNA between two crossover sites exchanged.
28 10.6 Genetic Recombination Detection of recombination Agar lacking tryptophan DNA from Trp + cells Agar lacking tryptophan Trp cells No growth Trp cells Recombinants form colonies Using a selective medium to detect rare genetic recombinants Phenotypically different from both parents. Selectable markers: drug resistance, nutritional requirements, etc.
29 10.7 Transformation Heat-killed S cells Live S cells Live R cells Live R cells + heat-killed S cells Griffith in 20s and Avery in 40s Animation: Transformation
30 10.7 Transformation Transformation: Free DNA (self-replicable or Not; Transfection if DNA from a virus); Host cells must be competent (natural or induced); Methods: chemical induced, electroporation.
31 10.8 Transduction Transduction: DNA transfer mediated by a virus. 1) Generalized transduction, any portion of the host genome; 2) Specialized transduction, DNA from a specific region of the host genome.
32 10.8 Transduction Specialized: phage DNA integrates into the host chromosome at specific sites, facilitating the nearby genes transfer. Animation: Specialized Transduction
33 10.9 Conjugation IS3 Tn1000 tra region 99.2kbp/0 kb IS3 IS2 Pilus with attached phage virions F plasmid 75 kbp 25 kbp orit 50 kbp Replication and segregation Conjugation: Genetic transfer via direct cell-to-cell contact mediated by pili (F pili).
34 10.9 Conjugation Bacterial chromosome F plasmid Pilus DNA is synthesized by rolling circle replication. F cell (donor) Pilus retracts F cell (recipient) Cell pairs stabilized. F plasmid nicked in one strand Very fast, 100 kbp can be done in 5 min. Transfer of one strand from F cell to F cell. F plasmid simultaneously replicated in F cell Synthesis of complementary strand begins in recipient cell Completion of DNA transfer and synthesis. Cells separate F cell F cell
35 10.9 Conjugation TraI, encoded by the tra operon of the F plasmid, has helicase activity; DNA polymerase; Membrane proteins. Plasmid loss may happen spontaneously if no selection pressure.
36 10.10 Chromosome transfer Transfer to F recipient orit rep rep orit tra F plasmid IS3 pro tra IS3 leu Recombination thr Inserted F plasmid IS3 lac pro lac Chromosome Chromosome IS3 gal pro IS3 rep orit tra IS3 lac arg his trp Hfr (High frequency of recombination) strain. F + strain (F plasmid) Hfr when F integrated into chromosome (??? Crossover) Transferable chromosome.
37 10.10 Chromosome transfer Hfr + F - Hfr X + F + or two Hfr strains
38 10.10 Chromosome transfer Hfr 1 Hfr 3 Hfr 4 Hfr 2 Hfr 1 Hfr 2 Hfr 3 Hfr 4 Gene C donated first; clockwise order Gene L donated first; counterclockwise order Gene X donated first; clockwise order Gene G donated first; counterclockwise order Chromosome Mapping: organization of a chromosome
39 Number of recombinants per 100 Hfr bacteria Chromosome transfer 0 8 min Thr + Leu + Gal + Trp Thr + Leu + Gal + Trp Time, after mixing parental cultures (min) Time of gene entry in a mating culture (interrupted mating) Chromosome Mapping: organization of a chromosome
40 Hfr donor: F recipient: Thr + Leu + Lac + Str s X Thr Leu Lac Str r Mating, to allow conjugation, followed by plating onto agar media Thr + Leu + Str r recombinants Lac + Str r recombinants Selection for recombinants Agar minimal medium with streptomycin and glucose; selects for markers Thr + Leu + ; does not select for Lac Agar minimal medium with streptomycin, lactose, threonine, leucine; selects for marker Lac + ; does not select for Thr or Leu
41 10.11 Complementation Wild-type cell; both genes A and B are functional and cell is Trp + Mutant strain 3 cell contains mutation 3 and is Trp Mutant strain 1 cell contains mutation 1 and is Trp (requires tryptophan for growth) Trans test of mutations 1 and 2; complementation occurs (cell is Trp + ), therefore mutations are in different genes Mutant strain 2 cell contains mutation 2 and is also Trp Trans test of mutations 2 and 3; no complementation occurs (cell is Trp ), therefore mutations are in the same genes Complementation: rescue defective phenotype caused by mutation. in trans: in the different molecules; or in cis: in the same molecules.
42 10.12 Gene transfer in Archaea The unusual growth conditions needed by many Archaea make genetic experimentation difficult. Many antibiotics are ineffective against Archaea, making it difficult to select recombinants effectively. Penicillins? Why? The genetic transfer systems (transformation, transduction, and conjugation) are all know in Archaea.
43 IS2 Tn5 tnp IS50L kan Mobile DNA str bleo Target DNA sequence Insertion Duplicated target sequence IS50R tnp Transposable element Mobile DNA: discrete segments of DNA that moves as units from one location to another within other DNA molecules. This process called transposition. Transposable elements: two essential components: short inverted terminal repeats and transposase. 1) IS (insertion sequences), 2) transposons, contain other genes.
44 10.13 Mobile DNA Conservative transposition Replicative transposition Target sequence Mechanisms: Conservative and replicative. Site-specific Donor DNA with break Transposon in new location Donor DNA undamaged recombination
45 10.13 Mobile DNA Mutagenesis with transposons: 1) gene interrupted by the insertion; 2) gene untranscribe by the insertion Gene A Gene 1 Gene 2 Gene 3 Transposon Transposition Disrupted gene 2 Chromosome Gene 1 Gene A Gene 3 Chromosome