Bacterial Genetics

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Mutation

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A heritable change in DNA sequence.

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Genetic Recombination

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Process by which DNA from 2 genomes are brought together into a single genome.

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Genotype

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Genetic identity of an organism. Genotypes are not observable traits but can be monitored at the DNA level.

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Nomenclature for Genes?

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Genes are written in 3 lowercase symbols followed by a capital letter. Example- hisC, a gene involved in histidine biosynthesis.

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Gene products

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are written in non-italicized script with the first and fourth letter capitalized. Example- HisC, the protein product encoded by the gene hisC.

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Phenotype

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the observable traits possessed by an organism resulting from its genotype. Example His+ refers to an organism capable of synthesizing its own histidine. His- phenotype refers to an organism which must be grown on media containing histidine.

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Auxotroph

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mutation resulting in the requirement of a growth factor.

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Prototroph

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the parent (non-mutant) of an auxotroph.

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Wild Type

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Non mutated genome

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Mutations

Causes of mutations?

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1. Spontaneous, 10(6)-10(10) cellular division

2. Environment

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Mutation

Base Pair Substitution (Point Mutation)

Missense Mutation

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1. Single codon is altered so that one amino acid in a protein is replaced with a different amino acid

Resuls in the formation of a faulty protein. Includes Temperature Sensitive Mutants

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Mutation

Base Pair Substitution (Point Mutation)

Nonsense Mutation

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1. A mutation in which the codon for an amino acid is changed to a stop codon

2. Results in an incomplete protein.

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Mutation

Base Pair Substitution (Point Mutation)

Silent Mutation

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1. Change in the DNA sequence that has no effect on the phenotype

2. Results in a normal functioning protein.

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Mutations

Frameshift Mutation

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1. results in the shifting of the reading frame of the genetic code.

2. Occur through either the insertion or deletion of a base pair.

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Mutations

Frameshift Mutation

Microdeletions or Microinsertions

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When a small number of nucleotides are deleted or inserted such changes

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Reversions

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An alteration in DNA that reverses the effects of a prior mutation

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Reversions

Same-Site Revertant

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contains a second mutation at the same site where the original mutation occurred.

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Reversions

Same-Site Revertant

True Revertant

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same-site revertant that now possesses a wild type genotype this is referred to as a

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Reversions

Second-Site Revertant

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involves a second mutation at a site other than that of the original mutation

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Reversions

Second-Site Revertant

Supressor Mutation

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second-site revertant which restores wild type phenotype

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Reversions

Second-Site Revertant

Types of Changes in Organisms caused by Second-Site

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1. If the second mutation is in the same gene then possibly restore enzyme activity. (Compensatory Mutants)
2. The second mutation may occur in a separate gene which restores function of original mutated gene. (Compensatory Mutants)
3. Mutation in a second gene results creation of a new metabolic pathway which overrides the effect of the original mutation. In such situations the activity of the original mutant enzyme is not restored.

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Deletion Mutations

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involves the removal of a large stretch of nucleotides, possibly even multiple genes

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Insertion Mutations?

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involves the addition of a large stretch of nucleotides.

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Insertion Mutations?

How do they occur?

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1. may involve noncoding sequences of DNA (insertion sequences) OR

2. actual movement of host DNA or foreign gene containing DNA called Transposons

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Insertion Mutations?

What can the result be?

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Insertion mutations may involve Translocations, Inversions and Duplications of genes.

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Chemical and Physical Mutagens

Base Analogs? What can be the result?

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1. compounds which act like DNA nucleotides and are mistakenly incorporated into a growing DNA strand during replication.

2. form incorrect base pairing resulting in a base pair substitution after a second round of DNA replication.

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Chemical and Physical Mutagens

Base Analogs

5 Bromouracil

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incorporated like Thymine. Occasionally forms faulty base pairs with Guanosine. AT->GC. Occasionally GC-> AT.

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Chemical and Physical Mutagens

Base Analogs

2 aminopurine

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incorporated like adenosine, forms faulty pairing with Cytosine. TA->CG

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Energy as a Mutagen

Result if come in contact with Ultraviolet Light

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purine and pyrimidine bases absorb UV light strongly. This energy has multiple effects on DNA but one well studied result is the formation of pyrimidine dimers. (Thymine/Cytosine). Dimerization of bases results in improper transcription. In addition repair may lead to errors or deletion in the DNA.

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Energy as a Mutagen

Result if come in contact with Short waves of energy

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may cause water to ionize, forming a hydroxyl radical ( •OH ). Free radicals are very unstable and will react with other molecules damaging them.

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Ames Test

Process

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1. HIS- Salmonella strain in presence of chemical to be tested

2. Media lacks histidine

3. Look for reversion mutants (HIS+)

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Ames Test

Advantages and Disadvantages

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Advantages-
1. quick
2. screen large numbers of chemicals

Disadvantages-
1. false negatives
2. correlation between prokaryotic and eukaryotic systems

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DNA Repair Mechanisms

Mismatch Repair

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1. Def - Single base error during replication

2. 3’->5’ exonuclease activity of DNA POL III removes wrong nucleotide

3. 5’->3’ polymerase activity of DNA POL III adds proper nucleotide using opposing strand as template

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DNA Repair Mechanisms

Excision Repair

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1. Nucleases identify damaged region

2. Create nicks, generate SS DNA

3. SS DNA (containing damaged region) removed by a DNA Nuclease

4. DNA Polymerase I generates new DNA 5’->3’

5. DNA Ligase seals nicks

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DNA Repair Mechanisms

SOS Regulatory System (Errors During DNA Repair)

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1. Induced when there is severe damage to the DNA

2. Procedure
a. SOS system is normally kept off by the repressor protein LexA.

b. During times of cellular distress expression of RecA will result in cleavage of LexA allowing for expression of the SOS system.

c. Deactivation of LexA allows for error-prone repair by DNA Poly IV and V. There is no template for them to make repairs, but better to be error than blank.

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DNA Recombination

Def?

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Genetic exchange between two genomes.

1. Recombination occurs in a donor to recipient manner.

2. Recombination involving a non-replicating DNA fragment must result in the integration of that DNA fragment into the host genome.

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Recombination

Process

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1. Nuclease- creates a single stranded DNA fragment (Invading Strand)

2. Single Strand Binding Protein

3. Rec A- facilitates strand displacement by binding to Invading Strand

4. Strand displacement with homologous stretch of recipient DNA

5. Integration of donor strand into recipient genome

6. Creation of compliment to invading strand to recreate double helix

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Steps of Transformation

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1. A DS DNA binds to the host cell by the DNA-binding proteins.
2. Nuclease degrades one strand while the other strand is taken up by the host
3. Competent specific proteins attach themselves to the single strand
4. RecA proteins then assist in integrating into the genome of the host cell, by recombenation

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Transformation

Factors Affecting Transformation

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1, Membrane Permeability

2. Surface rectors

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Steps of Conjugation

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1. F+ organisms attach to F- recipients with a pilus
2. The pilus is retracted, pulling the organisms together
3. Once together their membrans infuse creating an opening that allows of transfer of genetic matierial
4. The transfer occurs in a rolling circle replication, allowing the host to receive a SS copy of DNA that will be duplicated, leaving the donor with a copy to duplicate as well.
5. Successful conjugation results in transformation F-F+

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Hfr Strains

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1. F Plasmid integrated into a host chromosome.

2. Can transfer both the F plasmid and chromosome of host because of the F Plasmid.

3. Does not make F+, but transfer usually stay seperate.

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Transduction

Generalized

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1. accidental incorporation of host DNA into viral capsid in place of virus genome

2. single event incapable of independent replication

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Transduction

Specialized

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1. Improper excision of viral DNA

2. Template contains virus plus host DNA

3. Error can be transmitted into offspring

4. Rare event

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Transposable Genetic Elements

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Genetic sequences which have the ability to “move around” a DNA molecule

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Transposable Genetic Elements

Insertion Sequences

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1. 1000 nucleotides

2. Sequence of DNA flanked by Long Terminal Repeat and Inverted Repeat

3. No genetic information other than the ability to integrate and excise from chromosome (Transposase Gene)

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Transposable Genetic Elements

Transposons (Jumping Genes)

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1.sequence of DNA (gene) flanked by LTR, IR

2. sequences excise and later reintegrate