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Chapter 8 Microbial Genetics © 2013 Pearson Education, Inc. Lectures prepared by Christine L. Case

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Page 1: Microbial Genetics - Duncan Rossduncanross.net/MIC2010/_ch_08_lecture_presentation.pdf · DNA polymerase. DNA polymerase digests RNA primer DNA . polymerase . DNA polymerase Okazaki

Copyright © 2013 Pearson Education, Inc. Lectures prepared by Christine L. Case

Chapter 8

Microbial Genetics

© 2013 Pearson Education, Inc. Lectures prepared by Christine L. Case

Page 2: Microbial Genetics - Duncan Rossduncanross.net/MIC2010/_ch_08_lecture_presentation.pdf · DNA polymerase. DNA polymerase digests RNA primer DNA . polymerase . DNA polymerase Okazaki

© 2013 Pearson Education, Inc.

Insert Fig CO 8

Page 3: Microbial Genetics - Duncan Rossduncanross.net/MIC2010/_ch_08_lecture_presentation.pdf · DNA polymerase. DNA polymerase digests RNA primer DNA . polymerase . DNA polymerase Okazaki

© 2013 Pearson Education, Inc.

Structure and Function of Genetic Material

8-1 Define genetics, genome, chromosome, gene, genetic code, genotype, phenotype, and genomics.

8-2 Describe how DNA serves as genetic information. 8-3 Describe the process of DNA replication. 8-4 Describe protein synthesis, including

transcription, RNA processing, and translation. 8-5 Compare protein synthesis in prokaryotes and

eukaryotes.

Learning Objectives

Page 4: Microbial Genetics - Duncan Rossduncanross.net/MIC2010/_ch_08_lecture_presentation.pdf · DNA polymerase. DNA polymerase digests RNA primer DNA . polymerase . DNA polymerase Okazaki

© 2013 Pearson Education, Inc.

Terminology

Genetics: the study of what genes are, how they carry information, how information is expressed, and how genes are replicated

Gene: a segment of DNA that encodes a functional product, usually a protein

Chromosome: structure containing DNA that physically carries hereditary information; the chromosomes contain the genes

Genome: all the genetic information in a cell

Page 5: Microbial Genetics - Duncan Rossduncanross.net/MIC2010/_ch_08_lecture_presentation.pdf · DNA polymerase. DNA polymerase digests RNA primer DNA . polymerase . DNA polymerase Okazaki

© 2013 Pearson Education, Inc.

Terminology

Genomics: the molecular study of genomes Genotype: the genes of an organism Phenotype: expression of the genes

Page 6: Microbial Genetics - Duncan Rossduncanross.net/MIC2010/_ch_08_lecture_presentation.pdf · DNA polymerase. DNA polymerase digests RNA primer DNA . polymerase . DNA polymerase Okazaki

© 2013 Pearson Education, Inc.

Insert table from Clinical Focus, p. 220, bottom If possible on this slide, include a title: Determine Relatedness

Determine Relatedness

Page 7: Microbial Genetics - Duncan Rossduncanross.net/MIC2010/_ch_08_lecture_presentation.pdf · DNA polymerase. DNA polymerase digests RNA primer DNA . polymerase . DNA polymerase Okazaki

© 2013 Pearson Education, Inc.

Determine Relatedness

Strain % Similar to Uganda

Kenya 71%

U.S. 51%

Which strain is more closely related to the Uganda strain?

Page 8: Microbial Genetics - Duncan Rossduncanross.net/MIC2010/_ch_08_lecture_presentation.pdf · DNA polymerase. DNA polymerase digests RNA primer DNA . polymerase . DNA polymerase Okazaki

© 2013 Pearson Education, Inc.

Insert Fig 8.1a

Figure 8.1a A prokaryotic chromosome.

Chromosome

Disrupted E. coli cell

Page 9: Microbial Genetics - Duncan Rossduncanross.net/MIC2010/_ch_08_lecture_presentation.pdf · DNA polymerase. DNA polymerase digests RNA primer DNA . polymerase . DNA polymerase Okazaki

© 2013 Pearson Education, Inc.

Insert Fig 8.1b

Amino acid metabolism DNA replication and repair Lipid metabolism

Carbohydrate metabolism Membrane synthesis

KEY

Figure 8.1b A prokaryotic chromosome.

Genetic map of the chromosome of E. coli

Page 10: Microbial Genetics - Duncan Rossduncanross.net/MIC2010/_ch_08_lecture_presentation.pdf · DNA polymerase. DNA polymerase digests RNA primer DNA . polymerase . DNA polymerase Okazaki

© 2013 Pearson Education, Inc.

Insert Fig 8.2

Parent cell

DNA

expression

Genetic information is used within a cell to produce the proteins needed for the cell to function.

Transcription

Translation

recombination

Genetic information can be transferred between cells of the same generation.

New combinations of genes

Cell metabolizes and grows Recombinant cell Daughter cells

replication

Genetic information can be transferred between generations of cells.

Figure 8.2 The Flow of Genetic Information.

Page 11: Microbial Genetics - Duncan Rossduncanross.net/MIC2010/_ch_08_lecture_presentation.pdf · DNA polymerase. DNA polymerase digests RNA primer DNA . polymerase . DNA polymerase Okazaki

© 2013 Pearson Education, Inc.

DNA

Polymer of nucleotides: adenine, thymine, cytosine, and guanine

Double helix associated with proteins “Backbone” is deoxyribose-phosphate Strands are held together by hydrogen bonds

between AT and CG Strands are antiparallel

Page 12: Microbial Genetics - Duncan Rossduncanross.net/MIC2010/_ch_08_lecture_presentation.pdf · DNA polymerase. DNA polymerase digests RNA primer DNA . polymerase . DNA polymerase Okazaki

© 2013 Pearson Education, Inc.

Insert Fig 8.3b

The two strands of DNA are antiparallel. The sugar-phosphate backbone of one strand is upside down relative to the backbone of the other strand. Turn the book upside down to demonstrate this.

3′ end

5′ end

5′ end

3′ end

Figure 8.3b DNA replication.

Page 13: Microbial Genetics - Duncan Rossduncanross.net/MIC2010/_ch_08_lecture_presentation.pdf · DNA polymerase. DNA polymerase digests RNA primer DNA . polymerase . DNA polymerase Okazaki

© 2013 Pearson Education, Inc.

Insert Fig 8.3a

1

The double helix of the parental DNA separates as weak hydrogen bonds between the nucleotides on opposite strands break in response to the action of replication enzymes.

1

2

Hydrogen bonds form between new complementary nucleotides and each strand of the parental template to form new base pairs.

The replication fork

Daughter strand

Enzymes catalyze the formation of sugar-phosphate bonds between sequential nucleotides on each resulting daughter strand.

3

2

3

2

1

Replication fork

5′ end Parental strand

3′ end Parental strand Daughter

strand forming

Parental strand Parental

strand

5′ end 3′ end

Figure 8.3a DNA replication.

Page 14: Microbial Genetics - Duncan Rossduncanross.net/MIC2010/_ch_08_lecture_presentation.pdf · DNA polymerase. DNA polymerase digests RNA primer DNA . polymerase . DNA polymerase Okazaki

© 2013 Pearson Education, Inc.

Insert Fig 8.4

1

2

New Strand

Template Strand

Sugar

Phosphate

When a nucleoside triphosphate bonds to the sugar, it loses two phosphates.

Hydrolysis of the phosphate bonds provides the energy for the reaction.

Page 15: Microbial Genetics - Duncan Rossduncanross.net/MIC2010/_ch_08_lecture_presentation.pdf · DNA polymerase. DNA polymerase digests RNA primer DNA . polymerase . DNA polymerase Okazaki

© 2013 Pearson Education, Inc.

DNA Synthesis

DNA is copied by DNA polymerase In the 5' → 3' direction Initiated by an RNA primer Leading strand is synthesized continuously Lagging strand is synthesized discontinuously Okazaki fragments RNA primers are removed and Okazaki fragments joined

by a DNA polymerase and DNA ligase

Page 16: Microbial Genetics - Duncan Rossduncanross.net/MIC2010/_ch_08_lecture_presentation.pdf · DNA polymerase. DNA polymerase digests RNA primer DNA . polymerase . DNA polymerase Okazaki

© 2013 Pearson Education, Inc.

Insert Table 8.1

Table 8.1 Important Enzymes in DNA Replication, Expression, and Repair

Page 17: Microbial Genetics - Duncan Rossduncanross.net/MIC2010/_ch_08_lecture_presentation.pdf · DNA polymerase. DNA polymerase digests RNA primer DNA . polymerase . DNA polymerase Okazaki

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Insert Fig 8.5

1 Enzymes unwind the parental double helix.

Proteins stabilize the unwound parental DNA.

DNA polymerase

The leading strand is synthesized continuously by DNA polymerase.

3'

Replication fork RNA primer

The lagging strand is synthesized discontinuously. Primase, an RNA polymerase, synthesizes a short RNA primer, which is then extended by DNA polymerase.

DNA polymerase digests RNA primer and replaces it with DNA.

DNA polymerase

DNA polymerase

Okazaki fragment

DNA ligase joins the discontinuous fragments of the lagging strand.

DNA ligase

Primase

REPLICATION

3'

5'

5'

Figure 8.5 A summary of events at the DNA replication fork.

Parental strand

3'

5'

Page 18: Microbial Genetics - Duncan Rossduncanross.net/MIC2010/_ch_08_lecture_presentation.pdf · DNA polymerase. DNA polymerase digests RNA primer DNA . polymerase . DNA polymerase Okazaki

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1 An E. coli chromosome in the process of replicating

Replication forks

REPLICATION

Origin of replication

Parental strand Daughter strand

Replication fork Replication fork

Termination of replication

Bidirectional replication of a circular bacterial DNA molecule

Figure 8.6 Replication of bacterial DNA.

Page 19: Microbial Genetics - Duncan Rossduncanross.net/MIC2010/_ch_08_lecture_presentation.pdf · DNA polymerase. DNA polymerase digests RNA primer DNA . polymerase . DNA polymerase Okazaki

© 2013 Pearson Education, Inc.

ANIMATION DNA Replication: Replication Proteins

ANIMATION DNA Replication: Overview

ANIMATION DNA Replication: Forming the Replication Fork

Replication of Bacterial DNA

Page 20: Microbial Genetics - Duncan Rossduncanross.net/MIC2010/_ch_08_lecture_presentation.pdf · DNA polymerase. DNA polymerase digests RNA primer DNA . polymerase . DNA polymerase Okazaki

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Check Your Understanding Give a clinical application of genomics. 8-1 Why is the base pairing in DNA important? 8-2 Describe DNA replication, including the functions of

DNA gyrase, DNA ligase, and DNA polymerase. 8-3

Page 21: Microbial Genetics - Duncan Rossduncanross.net/MIC2010/_ch_08_lecture_presentation.pdf · DNA polymerase. DNA polymerase digests RNA primer DNA . polymerase . DNA polymerase Okazaki

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Transcription

DNA is transcribed to make RNA (mRNA, tRNA, and rRNA)

Transcription begins when RNA polymerase binds to the promoter sequence

Transcription proceeds in the 5' → 3' direction Transcription stops when it reaches the

terminator sequence

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Insert Fig 8.7

1

TRANSCRIPTION

DNA

mRNA

Protein

RNA polymerase

DNA

RNA polymerase bound to DNA

1 RNA polymerase binds to the promoter, and DNA unwinds at the beginning of a gene.

RNA is synthesized by complementary base pairing of free nucleotides with the nucleotide bases on the template strand of DNA.

2

3 The site of synthesis moves along DNA; DNA that has been transcribed rewinds.

4

5

Transcription reaches the terminator.

RNA and RNA polymerase are released, and the DNA helix re-forms.

Promoter (gene begins)

RNA polymerase Template strand of DNA

RNA RNA nucleotides

RNA synthesis

Terminator (gene ends)

Figure 8.7 The process of transcription.

Page 23: Microbial Genetics - Duncan Rossduncanross.net/MIC2010/_ch_08_lecture_presentation.pdf · DNA polymerase. DNA polymerase digests RNA primer DNA . polymerase . DNA polymerase Okazaki

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ANIMATION Transcription: Overview

ANIMATION Transcription: The Process

The Process of Transcription

Page 24: Microbial Genetics - Duncan Rossduncanross.net/MIC2010/_ch_08_lecture_presentation.pdf · DNA polymerase. DNA polymerase digests RNA primer DNA . polymerase . DNA polymerase Okazaki

© 2013 Pearson Education, Inc.

Insert Fig 8.11

1

RNA polymerase

DNA

Met

Met

Met

Met

Met

DNA

Exon Intron Exon Intron Exon

1 In the nucleus, a gene composed of exons and introns is transcribed to RNA by RNA polymerase.

RNA transcript

mRNA

2 Processing involves snRNPs in the nucleus to remove the intron-derived RNA and splice together the exon- derived RNA into mRNA.

Nucleus

Cytoplasm

3 After further modification, the mature mRNA travels to the cytoplasm, where it directs protein synthesis.

Figure 8.11 RNA processing in eukaryotic cells.

Page 25: Microbial Genetics - Duncan Rossduncanross.net/MIC2010/_ch_08_lecture_presentation.pdf · DNA polymerase. DNA polymerase digests RNA primer DNA . polymerase . DNA polymerase Okazaki

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Translation

mRNA is translated in codons (three nucleotides) Translation of mRNA begins at the start codon:

AUG Translation ends at nonsense codons: UAA, UAG,

UGA

Page 26: Microbial Genetics - Duncan Rossduncanross.net/MIC2010/_ch_08_lecture_presentation.pdf · DNA polymerase. DNA polymerase digests RNA primer DNA . polymerase . DNA polymerase Okazaki

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Insert Fig 8.2

Parent cell

DNA

expression

Genetic information is used within a cell to produce the proteins needed for the cell to function.

Transcription

Translation

recombination

Genetic information can be transferred between cells of the same generation.

New combinations of genes

Cell metabolizes and grows Recombinant cell Daughter cells

replication

Genetic information can be transferred between generations of cells.

Figure 8.2 The Flow of Genetic Information.

Page 27: Microbial Genetics - Duncan Rossduncanross.net/MIC2010/_ch_08_lecture_presentation.pdf · DNA polymerase. DNA polymerase digests RNA primer DNA . polymerase . DNA polymerase Okazaki

© 2013 Pearson Education, Inc.

The Genetic Code

64 sense codons on mRNA encode the 20 amino acids

The genetic code is degenerate tRNA carries the complementary anticodon

Page 28: Microbial Genetics - Duncan Rossduncanross.net/MIC2010/_ch_08_lecture_presentation.pdf · DNA polymerase. DNA polymerase digests RNA primer DNA . polymerase . DNA polymerase Okazaki

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Insert Fig 8.8

Figure 8.8 The genetic code.

Page 29: Microbial Genetics - Duncan Rossduncanross.net/MIC2010/_ch_08_lecture_presentation.pdf · DNA polymerase. DNA polymerase digests RNA primer DNA . polymerase . DNA polymerase Okazaki

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Insert Fig 8.10

1

RNA polymerase

DNA

Met

Met

Met Met

Met

Met

Met Met

TRANSLATION

DNA

mRNA

Protein

RNA Ribosome Peptide

Direction of transcription RNA polymerase

DNA

5′

Peptide

Polyribosome

Ribosome

Direction of translation mRNA

Figure 8.10 Simultaneous transcription and translation in bacteria.

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Insert Fig 8.9 (1) and (2)

1

RNA polymerase

DNA

TRANSLATION

DNA

mRNA

Protein

Ribosomal subunit tRNA

Anticodon

Ribosomal subunit

Met

1 Components needed to begin translation come together.

2 On the assembled ribosome, a tRNA carrying the first amino acid is paired with the start codon on the mRNA. The place where this first tRNA sits is called the P site. A tRNA carrying the second amino acid approaches.

Met Leu

Ribosome

P Site

Start codon

Second codon mRNA mRNA

Figure 8.9.1-2 The process of translation.

Page 31: Microbial Genetics - Duncan Rossduncanross.net/MIC2010/_ch_08_lecture_presentation.pdf · DNA polymerase. DNA polymerase digests RNA primer DNA . polymerase . DNA polymerase Okazaki

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Insert Fig 8.9 (3) and (4)

1

DNA

Met

Met Met

Met

Peptide bond forms

Leu Leu Gly

Phe

mRNA

A site E site

mRNA Ribosome moves along mRNA

3 4 The ribosome moves along the mRNA until the second tRNA is in the P site. The next codon to be translated is brought into the A site. The first tRNA now occupies the E site.

The second codon of the mRNA pairs with a tRNA carrying the second amino acid at the A site. The first amino acid joins to the second by a peptide bond. This attaches the polypeptide to the tRNA in the P site.

Figure 8.9.3-4 The process of translation.

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Insert Fig 8.9 (5) and (6)

tRNA released Met Met

Leu

Leu Growing polypeptide chain

Gly Phe

Gly

Phe Met

mRNA

mRNA

5 The second amino acid joins to the third by another peptide bond, and the first tRNA is released from the E site.

6 The ribosome continues to move along the mRNA, and new amino acids are added to the polypeptide.

Figure 8.9.5-6 The process of translation.

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Insert Fig 8.9 (7) and (8)

Met Met

Met Met Polypeptide released

Arg

Gly

Phe

Gly

mRNA

Stop codon

7 When the ribosome reaches a stop codon, the polypeptide is released.

New protein

mRNA

8 Finally, the last tRNA is released, and the ribosome comes apart. The released polypeptide forms a new protein.

Gly

Phe

Arg

Figure 8.9.7-8 The process of translation.

Page 34: Microbial Genetics - Duncan Rossduncanross.net/MIC2010/_ch_08_lecture_presentation.pdf · DNA polymerase. DNA polymerase digests RNA primer DNA . polymerase . DNA polymerase Okazaki

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ANIMATION Translation: Overview

ANIMATION Translation: The Genetic Code

ANIMATION Translation: The Process

The Genetic Code

Page 35: Microbial Genetics - Duncan Rossduncanross.net/MIC2010/_ch_08_lecture_presentation.pdf · DNA polymerase. DNA polymerase digests RNA primer DNA . polymerase . DNA polymerase Okazaki

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Check Your Understanding What is the role of the promoter, terminator, and

mRNA in transcription? 8-4 How does mRNA production in eukaryotes differ

from the process in prokaryotes? 8-5

Page 36: Microbial Genetics - Duncan Rossduncanross.net/MIC2010/_ch_08_lecture_presentation.pdf · DNA polymerase. DNA polymerase digests RNA primer DNA . polymerase . DNA polymerase Okazaki

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The Regulation of Gene Expression

8-6 Define operon. 8-7 Explain pre-transcriptional regulation of gene

expression in bacteria. 8-8 Explain post-transcriptional regulation of gene

expression.

Learning Objectives

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Regulation

Constitutive genes are expressed at a fixed rate Other genes are expressed only as needed Inducible genes Repressible genes Catabolite repression

Page 38: Microbial Genetics - Duncan Rossduncanross.net/MIC2010/_ch_08_lecture_presentation.pdf · DNA polymerase. DNA polymerase digests RNA primer DNA . polymerase . DNA polymerase Okazaki

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ANIMATION Operons: Overview

Operon

Page 39: Microbial Genetics - Duncan Rossduncanross.net/MIC2010/_ch_08_lecture_presentation.pdf · DNA polymerase. DNA polymerase digests RNA primer DNA . polymerase . DNA polymerase Okazaki

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Structure of the operon. The operon consists of the promoter (P) and operator (O) sites and structural genes that code for the protein. The operon is regulated by the product of the regulatory gene (I ).

DNA

Control region Structural genes

Operon

Regulatory gene Promoter Operator

Figure 8.12.1 An inducible operon.

1

I P O Z Y A

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RNA polymerase

Repressor active, operon off. The repressor protein binds with the operator, preventing transcription from the operon.

Transcription

Repressor mRNA

Translation

Active repressor protein

Figure 8.12.2 An inducible operon.

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Transcription

Translation

Operon mRNA

Transacetylase Permease

β-Galactosidase

Inactive repressor protein

Allolactose (inducer)

Repressor inactive, operon on. When the inducer allolactose binds to the repressor protein, the inactivated repressor can no longer block transcription. The structural genes are transcribed, ultimately resulting in the production of the enzymes needed for lactose catabolism.

Figure 8.12.3 An inducible operon.

Page 42: Microbial Genetics - Duncan Rossduncanross.net/MIC2010/_ch_08_lecture_presentation.pdf · DNA polymerase. DNA polymerase digests RNA primer DNA . polymerase . DNA polymerase Okazaki

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Structure of the operon. The operon consists of the promoter (P) and operator (O) sites and structural genes that code for the protein. The operon is regulated by the product of the regulatory gene (I ).

Control region Structural genes

Operon

DNA

Regulatory gene

Promoter Operator

Figure 8.13.1 A repressible operon.

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Repressor inactive, operon on. The repressor is inactive, and transcription and translation proceed, leading to the synthesis of tryptophan.

Transcription

Translation

Inactive repressor protein

Polypeptides comprising the enzymes for tryptophan synthesis

Repressor mRNA Operon

mRNA

RNA polymerase Figure 8.13.2 A repressible operon.

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Repressor active, operon off. When the corepressor tryptophan binds to the repressor protein, the activated repressor binds with the operator, preventing transcription from the operon.

Tryptophan (corepressor)

Active repressor protein

Figure 8.13.3 A repressible operon.

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ANIMATION Operons: Induction

ANIMATION Operons: Repression

Repression

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Glucose

Lactose

Bacteria growing on glucose as the sole carbon source grow faster than on lactose.

Log 1

0 of n

umbe

r of c

ells

Time

Bacteria growing in a medium containing glucose and lactose first consume the glucose and then, after a short lag time, the lactose. During the lag time, intracellular cAMP increases, the lac operon is transcribed, more lactose is transported into the cell, and β-galactosidase is synthesized to break down lactose.

Log 1

0 of n

umbe

r of c

ells

Glucose used

Lag time

Lactose used

Time

All glucose consumed

Figure 8.14 The growth rate of E. coli on glucose and lactose.

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DNA

Promoter

lacI lacZ

Operator RNA polymerase can bind and transcribe

cAMP

Active CAP

Inactive CAP

Inactive lac repressor

Lactose present, glucose scarce (cAMP level high). If glucose is scarce, the high level of cAMP activates CAP, and the lac operon produces large amounts of mRNA for lactose digestion.

DNA

Promoter lacI lacZ

CAP-binding site Operator RNA polymerase can't bind

Inactive CAP

Inactive lac repressor

Lactose present, glucose present (cAMP level low). When glucose is present, cAMP is scarce, and CAP is unable to stimulate transcription.

Figure 8.15 Positive regulation of the lac operon.

CAP-binding site

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Epigenetic Control

Methylating nucleotides Methylated (off) genes are passed to offspring cells Not permanent Biofilm behavior

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Applications of Microbiology 3.1 Paenibacillus.

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Applications of Microbiology 3.3 A fruiting body of a myxobacterium.

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Transcription of miRNA occurs.

miRNA binds to target mRNA that has at least six complementary bases.

mRNA is degraded.

DNA

miRNA

mRNA

Figure 8.16 MicroRNAs control a wide range of activities in cells.

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Check Your Understanding Use the following metabolic pathway to answer the

questions that follow it. 8-6

Substrate A Intermediate B End-product C

a. If enzyme a is inducible and is not being synthesized at present, a (1) __________ protein must be bound tightly to the (2) __________ site. When the inducer is present, it will bind to the (3) __________ so that (4) __________ can occur.

b. If enzyme a is repressible, end-product C, called a (1) __________, causes the (2) __________ to bind to the (3) __________. What causes derepression?

enzyme a enzyme b

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Check Your Understanding What is the role of cAMP in catabolite repression?

8-7 How does miRNA stop protein synthesis? 8-8

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Mutation: Change in the Genetic Material

8-9 Classify mutations by type. 8-10 Describe two ways mutations can be repaired. 8-11 Describe the effect of mutagens on the mutation

rate. 8-12 Outline the methods of direct and indirect

selection of mutants. 8-13 Identify the purpose of and outline the procedure

for the Ames test.

Learning Objectives

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Mutation

A change in the genetic material Mutations may be neutral, beneficial, or harmful Mutagen: agent that causes mutations Spontaneous mutations: occur in the absence of

a mutagen

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Mutation

Base substitution (point mutation) Change in one base

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Insert Fig 8.17

During DNA replication, a thymine is incorporated opposite guanine by mistake.

Parental DNA

Replication

Daughter DNA

Daughter DNA In the next round of replication, adenine pairs with the new thymine, yielding an AT pair in place of the original GC pair.

Daughter DNA

Replication

Granddaughter DNA

When mRNA is transcribed from the DNA containing this substitution, a codon is produced that, during translation, encodes a different amino acid: tyrosine instead of cysteine.

Transcription

mRNA

Amino acids Cysteine Tyrosine

Translation

Cysteine Cysteine

Figure 8.17 Base substitutions.

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Mutation

Missense mutation Base substitution results in change in amino acid

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Translation

DNA (template strand)

Transcription

mRNA

Amino acid sequence Met Lys Phe Gly Stop

Normal DNA molecule

Figure 8.18a-b Types of mutations and their effects on the amino acid sequences of proteins.

DNA (template strand)

Amino acid sequence Lys Phe Ser

mRNA

Met Stop

Missense mutation

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Mutation

Nonsense mutation Base substitution results in a nonsense codon

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Translation

DNA (template strand)

Transcription

mRNA

Amino acid sequence Met Lys Phe Gly Stop

Normal DNA molecule

Figure 8.18ac Types of mutations and their effects on the amino acid sequences of proteins.

Nonsense mutation

Met Stop

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Mutation

Frameshift mutation Insertion or deletion of one or more nucleotide pairs

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Translation

DNA (template strand)

Transcription

mRNA

Amino acid sequence Met Lys Phe Gly Stop

Normal DNA molecule

Figure 8.18ad Types of mutations and their effects on the amino acid sequences of proteins.

Frameshift mutation

Met Lys Leu Ala

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Spontaneous mutation rate = 1 in 109 replicated base pairs or 1 in 106 replicated genes

Mutagens increase to 10–5 or 10–3 per replicated gene

ANIMATION Mutations: Types

The Frequency of Mutation

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Insert Fig 8.19a

Met Stop

Met Stop

Stop Stop

Met Stop Adenosine nucleoside normally base-pairs by hydrogen bonds with an oxygen and a hydrogen of a thymine or uracil nucleotide.

Altered adenine will hydrogen bond with a hydrogen and a nitrogen of a cytosine nucleotide.

Figure 8.19a Oxidation of nucleotides makes a mutagen.

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Insert Fig 8.19b

Met Stop

Met Stop

Stop Stop

Stop

The altered adenine pairs with cytosine instead of thymine.

Normal parent DNA Altered parent DNA

Replication

Normal daughter DNA

Altered daughter DNA

Replication

Mutated granddaughter DNA

Altered granddaughter DNA

Figure 8.19b Oxidation of nucleotides makes a mutagen.

HNO2

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ANIMATION Mutagens

Chemical Mutagens

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Insert Fig 8.20

Met Stop

Met Stop

Stop Stop

Stop

The 2-aminopurine is incorporated into DNA in place of adenine but can pair with cytosine, so an AT pair becomes a CG pair.

Normal nitrogenous base Analog

Thymine nucleoside 5-Bromouracil nucleoside

Adenine nucleoside 2-Aminopurine nucleoside

The 5-bromouracil is used as an anticancer drug because it is mistaken for thymine by cellular enzymes but pairs with cytosine. In the next DNA replication, an AT pair becomes a GC pair.

Figure 8.20 Nucleoside analogs and the nitrogenous bases they replace.

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Radiation

Ionizing radiation (X rays and gamma rays) causes the formation of ions that can react with nucleotides and the deoxyribose-phosphate backbone

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Radiation

UV radiation causes thymine dimers

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Ultraviolet light

Thymine dimer

New DNA

Exposure to ultraviolet light causes adjacent thymines to become cross-linked, forming a thymine dimer and disrupting their normal base pairing.

An endonuclease cuts the DNA, and an exonuclease removes the damaged DNA.

DNA polymerase fills the gap by synthesizing new DNA, using the intact strand as a template.

DNA ligase seals the remaining gap by joining the old and new DNA.

Figure 8.21 The creation and repair of a thymine dimer caused by ultraviolet light.

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Photolyases separate thymine dimers Nucleotide excision repair

Repair

ANIMATION Mutations: Repair

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Selection

Positive (direct) selection detects mutant cells because they grow or appear different

Negative (indirect) selection detects mutant cells because they do not grow Replica plating

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Master plate with medium containing histidine

Velvet surface (sterilized)

Handle

Sterile velvet is pressed on the grown colonies on the master plate.

Cells from each colony are transferred from the velvet to new plates.

Petri plate with medium lacking histidine

Colony missing Auxotrophic mutant

Petri plate with medium containing histidine

Plates are incubated.

Growth on plates is compared. A colony that grows on the medium with histidine but could not grow on the medium without histidine is auxotrophic (histidine-requiring mutant).

Figure 8.22 Replica plating.

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Each sample is poured onto a plate of medium lacking histidine. The plates are then incubated at 37°C for two days. Only bacteria whose histidine-dependent phenotype has mutated back (reverted) to histidine synthesizing will grow into colonies.

The numbers of colonies on the experimental and control plates are compared. The control plate may show a few spontaneous histidine-synthesizing revertants. The test plates will show an increase in the number of histidine synthesizing revertants if the test chemical is indeed a mutagen and potential carcinogen. The higher the concentration of mutagen used, the more revertant colonies will result.

The suspected mutagen is added to the experimental sample only; rat liver extract (an activator) is added to both samples.

Two cultures are prepared of Salmonella bacteria that have lost the ability to synthesize histidine (histidinedependent).

Control plate

Incubation

Rat liver extract

Experimental plate

Incubation

Rat liver extract

Suspected mutagen

Control (no suspected mutagen added)

Experimental sample

Colonies of revertant bacteria

Media lacking histidine

Cultures of histidine-dependent Salmonella

Figure 8.23 The Ames reverse gene mutation test.

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Check Your Understanding How can a mutation be beneficial? 8-9 How can mutations be repaired? 8-10 How do mutagens affect the mutation rate? 8-11 How would you isolate an antibiotic-resistant

bacterium? An antibiotic-sensitive bacterium? 8-12 What is the principle behind the Ames test? 8-13

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Genetic Transfer and Recombination

8-14 Differentiate horizontal and vertical gene transfer.

8-15 Compare the mechanisms of genetic recombination in bacteria.

8-16 Describe the functions of plasmids and transposons.

Learning Objectives

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Genetic Recombination

Vertical gene transfer: occurs during reproduction between generations of cells

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Insert Fig 8.2

Parent cell

DNA

expression

Genetic information is used within a cell to produce the proteins needed for the cell to function.

Transcription

Translation

recombination

Genetic information can be transferred between cells of the same generation.

New combinations of genes

Cell metabolizes and grows Recombinant cell Daughter cells

replication

Genetic information can be transferred between generations of cells.

Figure 8.2 The Flow of Genetic Information.

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Genetic Recombination

Horizontal gene transfer: the transfer of genes between cells of the same generation

ANIMATION Horizontal Gene Transfer: Overview

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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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Insert Fig 8.24

The result is that the recipient’s chromosome contains new DNA. Complementary base pairs between the two strands will be resolved by DNA polymerase and ligase. The donor DNA will be destroyed. The recipient may now have one or more new genes.

RecA protein catalyzes the joining of the two strands.

DNA from the donor aligns with complementary base pairs in the recipient’s chromosome. This can involve thousands of base pairs.

DNA from one cell aligns with DNA in the recipient cell. Notice that there is a nick in the donor DNA.

RecA protein

Recipient chromosome

Donor DNA

Figure 8.24 Genetic recombination by crossing over.

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ANIMATION Transformation

Genetic Transformation

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RECOMBINATION

Mouse died. Mouse died. Mouse remained healthy. Mouse remained healthy.

Colonies of encapsulated bacteria were isolated from dead mouse.

Colonies of encapsulated bacteria were isolated from dead mouse.

No colonies were isolated from mouse.

A few colonies of non-encapsulated bacteria were isolated from mouse; phagocytes destroyed nonencapsulated bacteria.

Living encapsulated bacteria injected into mouse.

Living nonencapsulated bacteria injected into mouse.

Heat-killed encapsulated bacteria injected into mouse.

Living nonencapsulated and heat-killed encapsulated bacteria injected into mouse.

Figure 8.25 Griffith’s experiment demonstrating genetic transformation.

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Degraded unrecombined DNA

Genetically transformed cell

Recombination occurs between donor DNA and recipient DNA.

Donor DNA aligns with complementary bases.

Recipient cell takes up donor DNA.

Chromosomal DNA

Recipient cell

DNA fragments from donor cells

Figure 8.26 The mechanism of genetic transformation in bacteria.

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Insert Fig 8.27

Sex pilus Mating bridge

Sex pilus

F+ cell

F– cell

Mating bridge

Figure 8.27 Bacterial conjugation.

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Insert Fig 8.28a

When an F factor (a plasmid) is transferred from a donor (F+) to a recipient (F–), the F– cell is converted to an F+ cell.

F+ cell F+ cell F+ cell F– cell

Replication and transfer of F factor

RECOMBINATION

Mating bridge Bacterial chromosome

F factor

Figure 8.28a Conjugation in E. coli.

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Insert Fig 8.28b

When an F factor becomes integrated into the chromosome of an F+ cell, it makes the cell a high frequency of recombination (Hfr) cell.

F+ cell Hfr cell

Insertion of F factor into chromosome

Integrated F factor

Recombination between F factor and chromosome, occurring at a specific site on each

Figure 8.28b Conjugation in E. coli.

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Insert Fig 8.28c

When an Hfr donor passes a portion of its chromosome into an F– recipient, a recombinant F– cell results.

F– cell Hfr cell Hfr cell Recombinant F– cell

Replication and transfer of part of the chromosome

In the recipient, recombination between the Hfr chromosome fragment and the F– chromosome

Figure 8.28c Conjugation in E. coli.

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ANIMATION Conjugation: Hfr Conjugation

ANIMATION Conjugation: F Factor

ANIMATION Conjugation: Overview

Conjugation in E. coli

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Genetic Map of the Chromosome of E. coli

ANIMATION Conjugation: Chromosome Mapping

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Insert Fig 8.1b

Amino acid metabolism DNA replication and repair Lipid metabolism

Carbohydrate metabolism Membrane synthesis

KEY

Figure 8.1b A prokaryotic chromosome.

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ANIMATION Transduction: Generalized Transduction

Transduction by a Bacteriophage

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Insert Fig 8.29

RECOMBINATION

Many cell divisions

Recombinant cell reproduces normally

Donor bacterial DNA

Recipient bacterial DNA

Bacterial DNA

Phage DNA

Donor cell

Phage protein coat

Phage DNA Bacterial chromosome

Recipient cell

A phage infects the donor bacterial cell.

Phage DNA and proteins are made, and the bacterial chromosome is broken into pieces.

Occasionally during phage assembly, pieces of bacterial DNA are packaged in a phage capsid. Then the donor cell lyses and releases phage particles containing bacterial DNA.

A phage carrying bacterial DNA infects a new host cell, the recipient cell.

Recombination can occur, producing a recombinant cell with a genotype different from both the donor and recipient cells.

5

Figure 8.29 Transduction by a bacteriophage.

© 2013 Pearson Education, Inc.

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ANIMATION Transduction: Specialized Transduction

Transduction by a Bacteriophage

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Insert Fig 13.13

1

6

5

3

4

2

Prophage exists in galactose-using host (containing the gal gene).

Phage genome excises, carrying with it the adjacent gal gene from the host.

Phage matures and cell lyses, releasing phage carrying gal gene.

Phage infects a cell that cannot utilize galactose (lacking gal gene).

Along with the prophage, the bacterial gal gene becomes integrated into the new host's DNA.

Lysogenic cell can now metabolize galactose.

Prophage gal gene

gal gene

Galactose-negative recipient cell

Galactose-positive donor cell

Bacterial DNA

Galactose-positive recombinant cell

Figure 13.13 Specialized transduction.

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Plasmids

Conjugative plasmid: carries genes for sex pili and transfer of the plasmid

Dissimilation plasmids: encode enzymes for catabolism of unusual compounds

R factors: encode antibiotic resistance

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Insert Fig 8.30

Pilus and conjugation proteins

Origin of transfer tet

cml

str sul

mer Origin of replication

Figure 8.30 R factor, a type of plasmid.

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Segments of DNA that can move from one region of DNA to another

Contain insertion sequences for cutting and resealing DNA (transposase)

Complex transposons carry other genes

Transposons

ANIMATION Transposons: Overview

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(a) An insertion sequence (IS), the simplest transposon, contains a gene for transposase, the enzyme that catalyzes transposition. The tranposase gene is bounded at each end by inverted repeat sequences that function as recognition sites for the transposon. IS1 is one example of an insertion sequence, shown here with simplified IR sequences.

IS1

Inverted repeat

Transposase gene

Inverted repeat

Figure 8.31a-b Transposons and insertion.

(b) Complex transposons carry other genetic material in addition to transposase genes. The example shown here, Tn5, carries the gene for kanamycin resistance and has complete copies of the insertion sequence IS1 at each end.

IS1 IS1

Tn5

Kanamycin resistance

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Transposons

ANIMATION Transposons: Complex Transposons

ANIMATION Transposons: Insertion Sequences

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Transposase cuts DNA, leaving sticky ends.

Sticky ends of transposon and target DNA anneal.

Transposase gene

IS1

IS1

(c) Insertion of the transposon Tn5 into R100 plasmid

Figure 8.31c Transposons and insertion.

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Check Your Understanding Differentiate horizontal and vertical gene transfer.

8-14 Compare conjugation between the following pairs:

F+ × F–, Hfr × F–. 8-15 What types of genes do plasmids carry? 8-16

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Genes and Evolution

8-17 Discuss how genetic mutation and recombination provide material for natural selection to act upon.

Learning Objective

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Genes and Evolution

Mutations and recombination provide diversity Fittest organisms for an environment are selected by

natural selection

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Insert West Nile Virus figure from Clinical Focus, p. 220

Clinical Focus, unnumbered figure, page 220.

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West Nile virus

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Insert Table from Clinical Focus, p. 220, bottom

Clinical Focus, unnumbered table, page 220

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Evolution

Strain % Similar to Uganda

Kenya 71%

U.S. 51%

Which strain is more closely related to the Uganda strain?

How did the virus change?

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Check Your Understanding Natural selection means that the environment favors

survival of some genotypes. From where does diversity in genotypes come? 8-17