Topic 11: Mechanisms of Gene Variation

• Bacteria has an “immune system”
  • Phenotype changes - making capsules
• Exogenous DNA fragments
  • ex: antibiotics resistance
  • chemical resistance, temperature resistance, resistance to dry condition, pH resistance
• Mutations
  • stable, hertiable changes in sequence of bases in DNA
  • in prokaryotes usually produce phenotypic changes
  • ex: pathogenic vs. non-pathogenic, smooth vs. rough
  • can occur spontaneously or be induced by chemical mutagens or radiation
• How genetic variation occurs
  • although the living cell is the best example of organization and efficiency, metabolic errors do occur = mutations
  • spontaneous mutation rate = around 1 in 10^8
  • mutation per million replicated genes
  • mutagens increase mutation rate 10 - 1000x
    • radiation on plants
    • mutagens: an agent, such as radiation or a chemical substance, which causes genetic mutation
  • ex: what if one survive in a rough environment and doubling time is 30 minutes, in 12 hours, there is 32 million cells
  • ex: mature culture = 10^9 cells / ml (US population x 4)
  • 10 cells = one gene mutation / ml
  • 40,000 mutatns in the mature culture
  • all 40,000 are good? or bad? or no effect? → don’t know
  • may be neutral (silent), beneficial, or harmful
• point mutations: base pair subsitution (silent, missense, nonsene)
  • sickle-cell anemia
• frameshift mutations: insertion or deletion of one or more nucelotide pairs
• chemical mutagens: nuceloside analogs have altered base-pairing properties; they can be …
  • randomly incorporated into growing cells (cancer drugs)
  • HIV: only used by viral enzymes (e.g. AZT - azidothymidine antiretroviral - prevent DNA replication
  • nuceloside analog: nucleic acid analog and sugar
  • nucelotide analog: nucleic acid analog, sugar, and 1-3 phosphate group
  • frameshift mutagens such as intercalating (distortion) agents (e.g. fungal aflatoxin, ethidium bromide, flame retardant)
  • fire fighters have 3 x higher level in blood - kidney and other cancers
  • teflon - we all have in our blood, cause kidney/testicular cancer, ulcerative colitis, thyroid disease, high cholesterol, pregnancy-induced hypertension (only 6 according to class action lawsuit investigation)
  • potent carinogens: distortion due to intercalating agent will lead to one or more base-pairs inserted or deleted during replication
• Spontaneous Mutations
  • arise without exposure to external agents
  • may result from errors in DNA replication
  • due to base tautomerization resulting in transition and transversion mutations
  • due to insertion or deletion of nucleotides
  • may also results from the action of mobile genetic elements such as transposons (jumping genes)
• Tautomerization Mutations
  • Tautomerization: organic compounds interconverts
  • ex: single bond to double bond, normally A=T, G (triple bond) C
  • Transitions: A & G and C &T (normal pairing)
  • Transversions: A & C and G & T (not normal pairing)
• Induced Mutations
  • caused by agents that directly damage NA
  • Base analogs
  • structurally similar to normal bases
  • mistakes occur when they are incorporated into growing polynucleotide chain
  • ex: 5-bromouracil
  • DNA modifying agents
  • alter a base causing it to mispair
  • ex: methyl nitrosoguanidine
    • changes are “subtle” so mismatch repair will miss it; use in cancer treatment
  • Intercalating agents
  • distort DNA to induce single nucelotide pair insertions and deletions
• Ultraviolet (UV) damage of DNA - UV Radiation
  • results in formation of thymine dimers
  • the resulting DNA can no longer serve as a template
  • (wear sunscreen!)
• Radiation as a Mutagen
  • Ionizing radiation (x-rays and y-rays) - lead to deletion mutations (ds breaks)
  • UV rays (UV B) lead to thymine dimers (intrastrand bonding)
  • Photolyases: light repair enzymes (use energy from visible light to fix UV light damage)
  • Nucelotide excision repair for repair of all mutations
• Repair
  • Photolyases separate thymine dimers
  • Nucleotide excision repair
• Other Types of Mutations
  • Subsitution: during DNA synthesis, an incorrect nucelotide on the new strand pairs with a nucelotie on the template
  • Deletion: during DNA synthesis, a nucelotide is omitted in the formation of the new strand
  • Insertion: during DNA synthesis, a nucelotide is inserted at a point in the formation of the new strand where there was no opposite nucelotide in the template
  • substitution is called a point mutation
  • insertion and deletion are called frameshift mutations
• Point mutations
  • silent mutation: change nuceloside sequence of codon but not the encoded amino acid
  • missense mutation: a single base substitution that changes codon for one amino acid into codon for another amino acid
  • nonsense mutation: converts a sense codon to a stop codon
• Other Types of Mutations
  • conditional mutations: expressed only under certain environmental conditions
  • auxotrophic mutant: unable to make an essential macromolecule such as amino acid or nucleotide
• Detection and Isolation of Mutants
  • mutations are generally rare
  • one per 10^7 to 10^11 cells
  • finding mutants requires sensitive detection methods and/or methods to increase frequency of mutations
• Mutant Detection
  • observation of changes in phenotype
  • replica plating technique
  • used to detect autxotrophic mutants
  • stamp master plate with velvet surface and stamp on replica plates (one regular and one without certain nutrients)
• Mutagen Identification: Ames Test
  • Histidine auxotroph vs. prototroph
  • screen for frameshift or point mutation
  • combine animal liver cell extracts with salmonella auxotroph
  • exposure mixture to test substance
  • examine for signs of mutation in salmonells, ie. look for cells (colonies) that have reverted from his- to his+
  • ames reverse gene mutation test
• DNA repair mechanism
• DNA Repair
  • proofreading
  • correction of errors in base pairing made during replication
  • errors corrected by DNA polymerase
  • other DNA repair mechanisms also exist
• Excision Repair
  • corrects damage that causes distorations in double helix
  • two types of repair systems are known
  • nucleotide excision repair NER
  • base excision repair BER
    • both remove the damaged portion of the DNA strand and use the intact complementary strand as a template to synethesize new DNA
• Thymine Dimers - NER pathway
  • two sub pathway
  • GG-NER: global genomic NER
  • TC-NER: transcription coupled NER
  • recognize/repair damage differently
  • ex: xeroderma pigmentosum (XP) - individuals who can’t be in direct sun
• BER pathway
  • Apyrimidinic (TC) or Apurinic (AG)
• DNA mismatch repair (MMR) pathway
  • DNA polymerase fill in the correct bases using non-damaged strand, DNA ligase connect the strands
  • e.g. mismatch repair system in E. coli
  • mismathc correction enzyme scans newly synthesized DNA for mismatched pairs
  • mismatched pairs removed and replaced by DNA polymerase and DNA ligase
• Creating Genetic Variability
  • mutations are subject to selective pressure
  • each mutant form that survives becomes an allele, an alternate for of a same gene
  • recombination is the process in which one or more nucleic acids are rearranged or combined to produce a new nucleotide sequence
• Recombinant DNA
  • A DNA molecule that is created when differnet DNA molecules are broken and separate pieces joined together to form a new DNA molecule
  • This new recombinant DNA (rDNA) molecule possesses a different genetic message from the orginial molecules
• Genetic Recombination
  • exchange of genes between two DNA molecules
  • crossing over occurs when two chromosomes break and rejoin
  • vertical gene transfer: occurs during reproduction between generations of cells
  • ex: human
  • horizontal gene transfer: the transfer of genes between cells of the same generation; leads to genetic recombination
  • ex: bacteria
  • three mechanisms of horizontal gene transfer: transformation, conjugation, transduction
• Bacterial Transformation
  • uptake of naked DNA by a competent cell followed by incorporation of the DNA into the recipient cell’s genome
  • competent cell: high density and/or limited nutrition state - end of long phase/stationary phase
• Bacterial Transformation
  • “naked” DNA transfer
  • recipient cells have to be “competent”
  • occurs naturally amoung very few genera (G+ and G-)
  • simple laboratory treatment will make E.coli competent → workhouse for genetic engineering
  • Griffith’s historical experiement in 1928 (discussed before)
• Recombination in Eukaryotes
  • during meiosis, crossing over between homologous chromosomes creates new combinations of alleles
  • followed by chromosomal segregation into gametes and zygote formation
  • transfer of genes from parents to progeny is called vertical gene transfer
• Horizontal (lateral) gene transfer (HGT) in prokaryotes
  • occurs in the three mechanisms evolved by bacteria to create recombinants
  • site specific recombination
  • transposition
  • homologous recombination
• HGT history
  • First described in 1951, virulent vs non-virulent strain Corynebacterium diphtheriae via viral gene transfer
  • antibiotic resistance inter-bacterial gene transfer described in 1959
  • mid 1980’s, HGT prediciton was made - suggesting biological significance/evolutionary history of Earth
  • 2007 HGT - played a major role in bacterial evolution, the role remains unclear in multicellular eukaryotes
• HGT in bacteria
  • common, genes can be transferred to the same or even distant and different species (inter-, intra-)
  • spread/rise of antibiotic resistance; MRSA, and others = primary reason for bacterial antibiotic resistance
  • spread of virulence factor - exotoxin adaptation in E. coli from Shigella via transduction (virus)
  • synthetic man-made chemicals (pesticides) degradation via adaptation - bacteria symbioetic relation to insects = live longer
• HGT
  • Bacteria to some fungi, and yeast
  • Aphid contains genes from fungi
  • Bacteria to insects
  • Bacteriophage-mediated = prokaryotes to eukaryotes
  • plants are capable of receiving genetic info via viruses
  • approx. 100/20,000 human genes - results of HGT according to one study - highly refuted
• HGT terminology
  • transfer of genes from donor to recipient
  • exogenote: DNA that is transferred to recipient
  • endogenote: genome of recipient
  • merozygote: recipent cell that is temporarily diploid as results of transfer process
• Site-specific recombination
  • important in insertion of viral genome into host chromosomes
  • there is only a small region of homology between inserted genetic material and host chromosome
• Transposable Elements
  • segments of DNA that move about the genome in a process called transposition
  • can be integrated into different sites in the chromosome
  • are someties called “jumping genes”
  • the simplest transposable elements are insertion sequences
  • transposable elements which contain genes other than those used for transposition are called composite transposons
• Types of Transposition
  • Class I (retrotransposons): first they are transcribed fro DNA to RNA, and the RNA produced is then reversed transcribed to DNA. this copied DNA is then inserted at a new position into the genome
  • Class II (DNA transposons): the cute and paste transposition mechanism of class II does not involve an RNA intermediate. a staggered cute is made at the site of transposon; the segment is removed and placed at another site with the same staggered cut. a specific restriction enzyme is used
• Bacterial Plasmids - replicative transposition
  • small, automously replicating DNA molecules that can exist independently or, as episomes, integrate reversibly into the host chromosome
  • number varies: 1-30
  • conjugative plasmids such as the F (fertility) plasmid can transfer copies of themselves to other bacteria during conjugation
• Bacterial conjugation
  • the transfer of genes between bacteria that depends on
  • direct cell to cell contact mediated by the F pilus (sex pilus) or mating bridge
  • pilin (fibrous bacterial protein) make up F pilus
• F factor
  • F=fertility, name came first before fully understood
  • episomes = integrated into chromosome
  • only 1 copy or non / bacteria; F = +/-
  • Fertility factor allows genes to be transferred
• HFr conjugation
  • HFr cell = F plasmid integrated (F+ cell)
  • donor HFr cell has F factor integrated into its chromosome
  • donor genes are transferred to recipient cell (F- cell)
  • a complete copy of the F factor is usually not transferred
  • HFr=high frequency recombinant
• Transduction
  • the transfer of bacterial genes by viruses
  • viruses (bacteriophages) can carry out the lytic cycle in which the host cell is destroyed, or the viral DNA can integrate into the host genome, becoming a latent pro-phage (lysogenic cycle)
  • temperate phage = lytic/lysogenic cycle
• Generalized Transduction
  • any part of bacterial genome can be transferred
  • occurs during lytic cycle
  • during viral assembly, fragements of host DNA mistakenly packaged into phage head
  • generalized transfucing particle
• Specialized Transduction
  • carried out only by temperate phages that have established lysogeny
  • only specific portion of becterial genome is transferred
  • occurs when prophage is incorrectly excised
• Genetic Recombination in Bacteriophages
  • recombination frequency determined when cells infected simultaneously with two different viruses

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