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IN THE NAME OF GOD Islamic Azad University Falavarjan Branch Falavarjan Branch School of Biological Sciences Department of Microbiology
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Microbial Genetics By: Keivan Beheshti Maal
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Genetic Mutations
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Mutations: Changes in DNA Why are mutations in DNA important to humans? 2 types of mutations: Spontaneous Mutations: –occur in the natural environment without the addition of mutagens (agents that cause mutations) –Occur randomly and spontaneously Induced Mutations: –Mutations that are created by the addition of mutagens
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Spontaneous Mutations Two types: 1. Base substitutions 2. Frameshift mutations
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Spontaneous Mutations: Base substitutions Most common type of substitution Mistake during DNA replication, incorrect base incorporated into DNA Three types: 1. Silent mutation: no effect on protein (remember- several codons code for the same amino acid) 2. Missense mutation: codon has changed and different amino acid is incorporated 3. Nonsense mutation: codon has changed to a stop codon
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Figure 8.15 - Overview
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Spontaneous Mutation: Base-pair deletion or insertion Insert or delete a nucleotide- very disastrous Shifts codons of DNA when transcribed into RNA (also called frameshift mutation) All nucleotides downstream of mutation will be grouped into improper codons, and wrong amino acids will be added Protein will be non-functional
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Mutation Mutation –Change in the base sequence of DNA may cause change in the product coded by the gene –Beneficial –Lethal –Neutral Occur commonly Degeneracy
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Mutations Types of mutations –Base substitution (point mutation) AT substituted for CG mRNA carries incorrect base Translation –Insertion of incorrect amino acid into protein –Missense mutation, nonsense mutation, frame shift mutation, and spontaneous mutations
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Base substitution
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Mutations Normal –No mutations –DNA strand properly transcribed by mRNA –Correct sequence of amino acids for protein
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Mutations Mis sense mutation –Base substitution results in an amino acid substitution in protein –Sickle cell anemia A to T Glutamic acid to valine Hb shape changed during low oxygen
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Mutations Non sense mutation –Base substitution creates a nonsense or stop codon –Protein is not produced –Only a fragment of protein is produced
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Mutations Frame shift mutation –One or a few nucleotide pairs are deleted or inserted in the DNA –Shifts the translation reading frame –Almost always result in a long stretch of altered amino acids –Inactive protein
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Mutations Insertion of extra bases into a gene –Huntington's disease Spontaneous mutations –Occur occasionally in DNA replication Mutagens –Chemically of physically alters DNA and effects a change is called a mutagen Radiation, ultraviolet light
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Mutagens Chemical Mutagens –Nitrous acid Converts adenine (A) to a form that doesn’t bind with thymine (T), but instead binds with cytosine (C) Alters base pair on DNA, works on random locations
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Mutagens Chemical mutagens (cont) –Nucleoside analogs Structurally similar to normal nitrogenous bases 2 - aminopurine –Adenine 5 – bromouracil –Thymine analog –Will bind with guanine
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Mutagens Chemical mutagens (cont) –During replication analogs cause base pairing mistakes –Antiviral and antitumor drugs AZT (azidothymi dine)
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Mutagens Chemical mutagens (cont) –Other chemicals cause deletions, frameshifts, or insertions Benzyprene – present in smoke and soot –Frameshift Aflatoxin – Aspergillus flavus –Frameshift
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Mutagens Radiation mutagens –X – rays –Gamma rays –Ultraviolet Forms covalent bond between certain bases Thymine dimers –Death of damage to cell Light repair enzymes –Photolyases Use visible light energy to separate dimer
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Mutagens Ultraviolet damage –Nucleotide excision repair Enzymes cut out distorted thymines Creates gap Gap is filled with newly synthesized DNA DNA ligase joins strand to surrounding backbone
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Mutation frequency Mutation rate –Probability that a gene will mutate when a cell divides –Expressed in power of 10 10 -4 mutation rate (1 in 10,000 chance of mutation) 10 -6 ( 1 in 1,000,000) –Mutagens Increase spontaneous mutation by 10 – 10,000 times 10 -6 becomes 10 -3 to 10 -5
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Identifying Mutants Positive (direct) selection –Detection of mutant cells by rejection of unmutated parent cells Penicillin in agar Unmutated parental cell will not grow Only mutated cells grow
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Identifying Mutants Negative (indirect) selection –Replica plating technique
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Replica Plating
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Replica plating Auxotroph –A mutant microorganism having a nutritional requirement that is absent in the parent.
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Identifying Chemical Carcinogens Carcinogen –A substance found to cause cancer in animals –Often mutagens are carcinogens as well –Previously used animal testing Time consuming Expensive
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Ames test Ames test utilizes bacteria to act as carcinogen indicator Based on observation that exposure to mutant bacteria to mutagenic substance may reverse effect of the original mutation
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Ames test These are called reversions –Back mutations Measures the reversion of Salmonella –Auxotrophs Have lost there ability to synthesize histidine (his - ) (his + ) bacteria have ability to synthesize histidine 90% of substances that cause reversion have been shown to be carcinogens
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Ames Test
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Induced Mutation Mutations are induced by either certain chemical mutagens or physical mutagens Sometimes scientists intentionally mutate DNA to study it
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Physical Mutagens: Radiation
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Repair of thymine dimers
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Effects of Mutation
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Gene Transfer
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Three methods of horizontal gene transfer: 1. Transformation 2. Transduction 3. Conjugation
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Gene Transfer Vertical Gene Transfer= When genes are passed from an organism to it’s offspring Horizontal Gene Transfer= Occurs between bacteria
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Horizontal Gene Transfer Two types of cells: 1. Donor: transfers DNA to recipient 2. Recipient: receives the DNA
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Genetic transfer and recombination Eukaryotes –Meiosis Prophase I Prokaryotes –Numerous different ways
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Genetic Transfer and Recombination Vertical gene transfer –Genetic information passed from an organism to its offspring Plants and animals Horizontal gene transfer –Bacteria transfer genetic information form one organism to another in the same generation –Genetic information passed laterally
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Horizontal Gene Transfer Horizontal gene transfer –Donor cell Organism gives up its entire DNA Part goes to recipient cell Part is degraded by cellular enzymes –Recipient cell Receives portion of donor cells DNA Incorporates donor DNA into its own DNA –Recombinant DNA –Less than 1 % of population
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Transformation Genes transferred from one bacterium to another in solution –Naked DNA –Discovered by Griffith –Used Streptococcus pneumoniae Two strains –Virulent (pathologic) strain Had a polysaccharide capsule resists phagocytosis –Avirulent (non- pathogenic) strain Lacked a capsule
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Griffith’s Experiment
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Transformation Bacteria after cell death and lysis could release DNA into environment Recipient cell can take up DNA fragments and incorporate into their own DNA –Resulting in a hybrid (recombinant cell) –Recombinant cell must be competent Able to alter cell wall to allow DNA (large molecule) to enter Bacillus, Haemophilus, Neisseria, Acinetobacter, and some Staph and Strep
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Genetic Transformation
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Conjugation Conjugation –Involves plasmid Circular piece of DNA Replicates independent of chromosome Non essential for growth genes –Requires cell to cell contact –Opposite mating type Donor cell carries plasmid Recipient cell lacks plasmid
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Conjugation Gram positive –Sticky surfaces cause bacteria to come in contact with one another Gram negative –Utilize sex pili
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Conjugation E coli model –F factor plasmid Fertility factor Donors (F + ) Recipients (F - ) –Converted to (F + ) –F + factor integrated into chromosome Becomes Hfr (high frequency of recombination) cell
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Conjugation Hfr conjugates with F - cell Chromosomal strand replicates and transferred to recipient Incomplete transfer of donor DNA Recipient integrates new DNA –Acquires new versions of chromosome –Remains F - cell
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Conjugation Minutes and conjugation –Identify locations of various genes –Hfr His, pro, thr, leu, and F (+) –F( - ) His, pro, thr, leu, and F( - )
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Conjugation In some cells carrying F factors, the F factor integrates into the host chromosome Now called Hfr cell Conjugation between Hfr and F - –Chromosome replicates –Transferred to F - cell –Usually chromosome breaks off before completely transferred –Generally remains F - because does not receive F factor
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Transduction in Bacteria Transfer of bacterial DNA transferred via bacteriophage Bacteriophage –Virus that infects bacteria
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Transduction Steps of transduction –1- bacteriophage infects donor bacterial cell –2- Phage DNA and proteins, and bacterial chromosome is broken into pieces
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Transduction Steps of transduction –3- during phage reassembly, bacterial DNA incorporated in capsid of bacteriophage –4 – donor cell lysis releasing new bacteriophage particles
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Transduction Steps in transduction –5- phage carrying donor DNA infects new recipient cell –6- recombination can occur Producing bacteria with genotype different than donor and recipient
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Transduction Generalized transduction –Previously explained Specialized transduction –Only certain genes are transferred –i.e. phage codes for toxins to be produced Cornybacterium diphtheriae – diphtheria toxin Streptococcus pyogenes – erythrogenic toxin Escherichia coli – Shiga toxin (hemorrhagic diarrhea)
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Plasmids Plasmids –Self replicating rings of DNA –1-5% size of chromosomal DNA –Non – essential genes –Conjugative plasmid F factor –Dissimilation plasmids Code for enzymes to breakdown unusual sugars and hydrocarbons Help in survival of unusual environments
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Plasmids Other plasmids –Toxins (Anthrax, tetanus, Staph) –Bacterial attachment –Bacteriocins Toxic proteins that kill other bacteria –Resistance factors (R factors) Resistance to antibiotics, heavy metals, cellular toxins
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Plasmids Resistance factors –Two groups RTF – resistance transfer factor –Includes genes for plasmid replication and conjugation r-determinant –Resistance genes –Codes for production of enzymes that inactivate drugs or toxic substances Bacteria can conjugate and transfer plasmids between species –Neisseria Penicillinase resists penicillin
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R factor Plasmids
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R plasmid Resistance plasmid- confer antibiotic resistance Two parts: 1. Resistance genes (R genes) 2. Resistance transfer factor (RTF)
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Figure 8.22
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