microbial genetics s

© 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 the functions of plasmids and transposons.

Objectives

Chapter 8 Microbial Genetics

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TerminologyC

om

ple

men

tary

bu

t an

tipara

llel

DNA

Genetics

Genome

Gene

Chromosome

Base pairs

Genetic code

Genomics

Genotype

Phenotype

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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)

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E. coli

Figure 8.1a

Fig 8.1

Figure 8.1b

Chromosome Map of Chromosome Map of E. coliE. coli

Chromosome length: 1mmCell length ?

Flow of Genetic Information

Fig 8.2 – Foundation Figure

DNA Replication DNA polymerase

initiated by RNA primer

bidirectional

origin of replication

leading strand: continuous DNA synthesis

lagging strand: discontinuous DNA synthesis Okazaki fragments

semiconservative

2

Replication forkReplication in 5' Replication in 5' 3' direction 3' direction

Fig 5.8

Fig 8.6

Replication 1; 2; 3 of circular bacterial

Chromosome

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Protein SynthesisGenetic code: universal and

degenerate (or redundant)

Transcription produces 3 types of RNA (?) Enzyme necessary ? Promoters and terminators

Translation produces the protein Sense codons vs. nonsense codons anticodons

Fig 8.9

Fig 8.7

Fig 8.8

Compare to Fig 8.8

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Transcription RNA polymerase binds to promotor sequence proceeds

in 5' 3' direction

stops whenit reaches terminator sequence

Fig 8.7

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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

Compare to Fig 8.9

Continuous Transcription and TranslationSimultaneous Transcription and Translation in Prokaryotes

Compare to Fig 8.10

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MutationsChange in genetic material.1. Point mutations = base pair substitution (silent,

missense, nonsense)2. Frameshift mutations = Insertion or deletion of one

or more nucleotide pairs

Review Fig 8.17

Various Point Mutations

Silent

Missense

Nonsense

Fig 8.17

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Mutations cont.

May be neutral (silent), beneficial, or harmful.

Spontaneous mutation rate 10-6 1 mutation per million replicated genes

Mutagens increase mutation rate 10 – 1000x

Chemical mutagens 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)

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

Fig 8.19a

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

Potent carcinogens!

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Radiation as a Mutagen

1. Ionizing radiation (x-rays and -rays) lead 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

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Fig 8.20

Repair

ANIMATION Mutations: 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. 8.22

Ames Reverse Gene Mutation TestAmes Reverse Gene Mutation Test

Positive or negative Ames test?

Explain what happened

Professor Richard A. Muller of UC Berkeley on the Ames Test and Natural Foods

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Genetic Transfer and Recombination

Vertical gene transfer: Occurs during reproduction between generations of cells.

Horizontal (lateral) gene transfer: Transfer of genes between cells of the same generation. Leads to genetic recombination

Three mechanisms of horizontal gene transfer: Transformation Conjugation Transduction

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Vertical gene transfer: Occurs during reproduction between generations of cells.

Horizontal gene transfer: The transfer of genes between cells of the same generation. Leads to genetic recombination.

Three mechanisms of horizontal gene transfer:

1. Transformation2. Conjugation3. Transduction

ANIMATION Horizontal Gene Transfer: Overview

Genetic Recombination

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Genetic Recombination

Exchange of genes between two DNA molecules

Crossing over occurs when two chromosomes break and rejoin

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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

ANIMATION Transformation

Griffith’s Experiment to Demonstrate Genetic Transformation

Fig 8.24

Fig 8.25

Transformation and Recombination

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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 8.26

Fig 8.27A

NIM

AT

ION

s

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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

Fig 8.27

ANIMATION Generalized Transduction

ANIMATION Specialized Transduction

Transduction by a Bacteriophage

Fig 8.28