Ch 8

Microbial Genetics

© 2004 by Jones and Bartlett Publishers

Define genetics, genome, chromosome, gene, genetic code, genotype, phenotype, and genomics.

Describe the process of DNA replication.Describe protein synthesis, including transcription, RNA

processing, and translation.Classify mutations by type, and describe how mutations are

prevented and repaired.Define mutagen.Describe two ways mutations can be repaired.Outline methods of direct and indirect selection of mutants.Identify the purpose and outline the procedure for the Ames

test.Compare the mechanisms of genetic recombination in

bacteria.Differentiate between horizontal and vertical gene transfer.Describe plasmids and their functions.

Student Learning Outcomes

Terminology•Genetics

•Genome

•Gene

•Chromosome

•Base pairs

•Genetic code

•Genomics

•Genotype

•Phenotype

Fig 8.3b

DNA•Polymer of nucleotides: ___________________

•Double helix associated with proteins

•"Backbone" composed of ___________________

•Strands are held together by H bonds between ____ and ____

•Strands are antiparallel

The Bacterial DNA

•Mostly single circular chromosome

•Attached to plasma membrane

•DNA is supercoiled

•Number of genes in E. coli

•Extra-chromosomal bacterial DNA: _________(1-5% of chromosome size)

Flow of Genetic Information

Fig 8.2 – Foundation Figure

DNA Replication•DNA polymerase

initiated by RNA primer

•bidirectional

•leading strand: continuous DNA synthesis

•lagging strand: discontinuous DNA synthesis Okazaki fragments

•semiconservative

2

Fig 8.3a

Replication forkReplication in 5' 3' direction

Fig 8.5

Fig 8.6

Replication of circular bacterial Chromosome - Three YouTube

animations 1; 2; 3

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Protein SynthesisTranscription ▫produces 3 types of RNA (?)▫Enzyme necessary ?▫Promoters and terminators

Translation ▫produces the protein▫Sense codons vs. nonsense codons▫anticodons

Genetic code: universal and degenerate (or redundant)

Fig 8.8: Review but do not memorize

More Details on Transcription•RNA polymerase binds to promotor sequence

•proceeds in 5' 3' direction

•stops whenit reaches terminator sequence

Fig 8.7Fig 8.7

More Details on Translation

•Nucleotide sequence of mRNA is translated into amino acid sequence of protein using “three letter words” = codons

•Translation of mRNA begins at the start codon: AUG

•Translation ends at a stop codon: UAA, UAG, UGA

•Requires various accessory molecules and 3 major components: ?

•In Prokaryotes: Simultaneous transcription and translation Polyribosomes

The Translation Process in Protein Synthesis

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Simultaneous Transcription and Translation in Prokaryotes

Compare to Fig 8.10

Mutations•Changes in the genetic material

•May be neutral, beneficial, or harmful

•Mutagen: Agent that causes mutations

•Spontaneous mutations: Occur in the absence of a mutagen

Types of Mutations:1. Point mutation = base substitution (silent,

missense, nonsense, readthrough)

2. Frameshift mutation = Insertion or deletion of one or more nucleotide pairs

Various Point Mutations

Silent

Missense

Nonsense

TAA

What type of mutation?

Review Fig 8.17

1. Nonsense mutation2. Missense mutation3. Silent mutation4. Point mutation5. Frameshift mutation

Mutation Rate and Mutagens•Spontaneous mutation rate = 1 in 109 replicated

base pairs or 1 in 106 replicated genes

•Mutagens increase mutation rate 10 – 1000x

Chemical mutagens, examples:

1. Nucleoside (base) analogs have altered base-pairing properties. They can be

randomly incorporated into growing cells (cancer drugs) only used by viral enzymes (e.g. AZT)

2. Frameshift mutagens such as intercalating agents (e.g.:, aflatoxin, ethidium bromide)

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Fig 8.20 a

Nucleoside Analogs

Distortion due to intercalating agent will lead to one or more base-pairs inserted or deleted during replication.

Potent carcinogens!

Intercalation

Radiation as a Mutagen

1. Ionizing radiation (x-rays and -rays) causes formation of ions that can react with nucleotides and backbone leads to deletion mutations (ds breaks)

2. UV rays lead to thymine dimers (intrastrand bonding)

Photolyases = light repair enzymes (use energy from visible light to fix UV light damage)

Nucleotide excision repair for repair of all mutations

Fig 8.21

Repair•Photolyases separate thymine dimers•Nucleotide excision repair

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Mutagen Identification: Ames Test

•Wild type vs. mutant•Auxotroph vs. prototroph

•Many mutagens are carcinogens

Combine animal liver cell extracts with Salmonella auxotroph

Expose mixture to test substance

Examine for signs of mutation in Salmonella, i.e. Look for cells (colonies) that have reverted from his– to his+

Fig. 8.23

Ames Reverse Gene Mutation Test

Positive or negative Ames test?

Explain what happened

Fig 8.24

Genetic Recombination

•Exchange of genes between two DNA molecules

•Crossing over occurs when two chromosomes break and rejoin

Vertical gene transfer: During reproduction between cell generations.

Horizontal gene transfer: Gene transfer between cells of same generation. Leads to genetic recombination.

Three mechanisms of horizontal gene transfer:

1. Transformation2. Conjugation3. Transduction

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1) Transformation

“Naked” DNA transfer

Recipient cells have to be “competent”

Occurs naturally among very few genera (G+ and G–)

Simple laboratory treatment will make E. coli competent workhorse for genetic engineering

Griffith’s historical experiment in 1928

Griffith’s Experiment to Demonstrate Genetic Transformation

Fig 8.25

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Fig 8.26

Transformation and Recombination

2) ConjugationPlasmid and chromosomal DNA transfer via direct cell to cell contact

High efficiency

F+ = donor cell. Contains F plasmid (factor) and produces conjugation (F) pilus (aka “sex pilus”)

Recipient cell (F– ) becomes F+

In some cells F factor integrates into chromosome Hfr cell R plasmids (R factors) are also transferred via conjugation

Fig 8.27

Review Fig 8.28

3) TransductionDNA Transfer from donor to recipient cell with help of bacteriophage (= transducing phage)

2 types of phage-bacteria interaction:

1. Generalized transduction happens via lytic cycle caused by virulent phages

2. Specialized transduction will be covered in Ch 13

Generalized Transduction

Fig 8.29

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