Bacterial Genetics

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created 4 years ago by Michele_Schweers_Simms
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1

Haploid

cell has one copy of every chromosome

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Diploid

cell has two copies of every chromosome

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mutation

change in the DNA sequence; random/chance event; no control over it happening

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Auxotroph

mutant that is no longer able to synthesize a nutrient for itself; must acquire it from the environment

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wild type

organism that does not have a mutation

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genotype

actual pieces of DNA; the genes; inherited

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phenotype

expression of a gene; expression of the gene to make protein

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Three ways bacteria adapt to their environment

Mutation; Gene Transfer, Regulating Gene Expression

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Mutation

gives different bacteria different properties; some may be better able to survive in certain environments than others

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Gene Transfer

bacter can transfer copies of their DNA to other bacteria to share

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Regulating Gene Expression

turning genes on and off as necessary with repressors/activator

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Types of Mutation

Base Substitution, Insertion, Deletion, Transposable Element

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Base substitution

one, more than one, incorrect base is added during DNA synthesis

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Types of base subsitutions

Point mutation, Missense mutation, Nonsense mutation, Knockout mutation

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Point mutation

if only one base is incorrect; protein is made, does not function as well as wild type

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Missense mutation

a different amino acid is inserted in the protein; protein is made, does not function as well as wild type

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Nonsense mutation

a stop codon is inserted in the middle of the sequence; doesn't make and entire protein, cannot function

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Knockout mutation

inactivates the entire gene; no transcription or translation; no protein is made at all

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Deletion

one, or more than one base is removed; can cause frameshift mutation

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Frameshift mutation

causes completely different amino acids to be inserted into the protein; may or may not be functional; makes a different protein

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Insertion

one or more bases are added; also can cause a frameshift mutation

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Transposable Element

Transposon/Jumping Gene; large segment of DNA that removes itself from its original position and inserts itself somewhere else in the chromosome; causes a large insertion mutation and a functional protein is NOT made

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Why do scientists use bacteria to study mutation?

Haploid; easy to grow; cheap to purchase and maintain; grow in large numbers in a short time; no ethical issues with mutating bacteria

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mutagens

agents of mutation; things that can cause mutations to occur; chemicals, transposons and radiation used in the lab to stimulate mutation

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Chemical mutagents

Nitrous acid, alkylating agents, base analogs, intercalating agents

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Nitrous acid

causes Cytosine to be converted to Uracil; DNA not supposed to have uracil

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alkylating agents

add short chains of carbon to bases which changes how they hydrogen bond

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Base analogs

chemicals with structures similar to nitrogenous bases; look like A,T,C,G; drug AZT works this way to make DNA of HIV non-functional

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Intercalating agents

insert themselves at the replication fork during DNA synthesis and cause insertion mutations; extra C, G, etc that are not supposed to be there

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Transposons as a mutagen

scientists can add transposons to bacteria on purpose to cause mutations

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Radiation mutagens

UV light and Xrays

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UV light

causes thymine dimer formation

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Thymine dimers

covalent bond between two adjacent thymines causes DNA to bend and polymerase cannot read; no DNA replication or transcription

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Xrays

causes single and double stranded breaks in the DNA

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Proofreading

DNA polymerase can correct its own mistakes; but if it misses mistake, cannot go backwards to fix it

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Endonuclease

mismatch repair; enzyme that goes behind DNA Polymerase to check its work; if it finds mismatch it will cut out the wrong base and calls another DNA polymerase in to add correct base

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3 ways of thymine dimer repair

Light repair, Dark repair and SOS repair

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Light repair

photoreactivation of UV light activates enzyme that can break the covalent bond between adjacent thymines

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Dark repair

excision repair; does not require use of light; different enzyme cuts out the section of DNA that contains the thymine dimer and calls in DNA polymerase to add new bases; 2 new thymines attached

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SOS repair

only used by bacteria that have suffered heavy UV light damage and are in danger of cell death; set of 20 genes activated with new DNA polymerase. New DNA polymerase can read past thymine dimer BUT cannot proofread so increase chances of mutation can occur

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Direct selection

use a media that only the mutated bacteria will grow on; only works if media exists and is known; ex: penicillin enrichment

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Indirect selection

used for identifying auxotrophic bacteria

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Replica plating

Uses two plates; one with NA and one with NA + nutrient to carbon copy with sterile velvet to determine which bacteria are auxotrophic because they do not appear on NA plate, only on NA + nutrient plate. Wild type will appear on both plates

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What are the 3 principles of the Ames test?

Reversion to wild type can be measured, mutagens will increase the rate of reversion, carcinogens are mutagens

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What are the mechanisms of Gene Transfer?

DNA mediated transformation, Transduction, Conjugation

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What do all three mechanisms of Gene Transfer have in common?

They only transfer a small piece of the chromosome (not the entire chromosome) and they all use homologous recombination

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Vertical gene transfer

from parent bacteria to offspring bacteria through binary fission

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Lateral (horizontal) gene transfer

from one adult bacteria to another adult bacteria

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Homologous recombination

when a single stranded piece of DNA from the donor bacteria replaces a similar but not identical genes in the recipient cell

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DNA Mediated Transformation

Donor DNA is naked; free floating in the environment; recipient cell must be competent; recipient cell takes in the naked DNA and is incorporated by homologous recombination

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Competent

not all bacteria are; not for their entire life cycle; only able to take in naked DNA at specific points in their lifecycle

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Forced competency

Electroporation and calcium chloride

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Electroporation

mild electric shock causes the cell to become leaky; can now take in naked DNA

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Calcium chloride

weak solutions of CaCl2 also causes the cell to become leaky and take in naked DNA

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Transduction

donor DNA is brought into the recipient cell by a virus

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Bacteriophage

virus that only infects bacteria; infects the donor cell and assembles some bacterial DNA that is included in new virus; new virus infects recipient cell and brings donor bacterial DNA that is incorporated by homologous recombination

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Conjugation

involves plasmids and requires two bacteria to physically touch

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Plasmid

small extrachromosomal piece of DNA that does not carry any genes necessary for life; only has genes that may be helpful

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Narrow host range plasmid

can only be transferred between specific types of bacteria

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Wide host range plasmid

can be transferred between many types of bacteria

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R plasmids

resistance plasmids; have genes for antibiotic resistance

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F plasmid

has the instructions for making the sex pilus

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Hfr cells

High Frequency of Transfer; occurs with transfer of bacterial chromosome through sex pilus; chromosome is incorporated into recipient cell by homologous recombination; F plasmid not transmitted in its entirety. Recipient cell remains F-

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natural selection

an organism's need to adapt in order to survive and multiply

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prototroph

does not require growth factors; wild type

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spontaneous mutations

genetic changes that result from normal cell processes

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silent mutation

although a base substitution has occurred, the resulting codon still codes for the same amino acid

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Replicon

origin of replication; plasmids and chromosomes