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)
- 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
