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Genetics of Viruses and Bacteria






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Genetics of Viruses and Bacteria. 0.5 m. Figure 18.1. Microbial Model Systems. Viruses called bacteriophages can infect and set in motion a genetic takeover of bacteria, such as Escherichia coli
Genetics of Viruses and Bacteria

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Slide 1

Genetics of Virusesand Bacteria

Slide 2

0.5 m

Figure 18.1

Microbial Model Systems

  • Viruses called bacteriophages can infect and set in motion a genetic takeover of bacteria, such as Escherichia coli

  • E. coli and phage model systems frequently use by researchers in studies that reveal broad biological principles

  • Viruses and bacteria have unique genetic mechanisms

Slide 3

Virus

Bacterium

Animalcell

0.25 m

Animal cell nucleus

Characteristics of Viruses

  • Recall that bacteria are prokaryotes with cells much smaller and more simply organized than those of eukaryotes

  • Viruses are smaller and simpler still

    • Smallest viruses are only 20 nm in diameter

    • The virus particle, or virion, is just nucleic acid enclosed by a protein coat

Slide 4

Characteristics of Viruses

  • A virus has a genome but can reproduce only within a host cell

  • Scientists detected viruses indirectly long before they could see them

  • The story of how viruses were discovered begins in the late 1800s

    • Tobacco mosaic disease stunts growth of tobacco plants and gives their leaves a mosaic coloration

    • In the late 1800s, researchers hypothesized that a particle smaller than bacteria caused the disease

    • In 1935, Wendell Stanley confirmed this hypothesis by crystallizing the infectious particle, now known as tobacco mosaic virus (TMV)

Slide 5

Characteristics of Viruses

  • Viruses are very small infectious particles consisting of

    • Nucleic acid - genome

    • Protein coat which encloses the genome

    • And in some cases, a membranous envelope

  • Viral genomes may consist of

    • Double- or single-stranded DNA

    • Double- or single-stranded RNA

Slide 6

Capsomereof capsid

RNA

DNA

Capsomere

Glycoprotein

70–90 nm (diameter)

18  250 mm

20 nm

50 nm

(b) Adenoviruses

(a) Tobacco mosaic virus

Capsids

  • A capsid is the protein shell that encloses the viral genome, it can have various structures

  • May be rod-shaped, polyhedral or complex

  • Composed of capsomeres – protein subunits; from one or a few types of proteins

  • Spikes or glycoproteins like the herpes shown

Slide 7

Membranous Envelope

  • Some viruses have envelopes which are membranous coverings derived from the membrane of the host cell

  • Maybe a single layer or double layer envelope

  • Bilipid bilayer with glycoproteins spikes protruding from the outer layer

Slide 8

Membranousenvelope

Capsid

RNA

Glycoprotein

80–200 nm (diameter)

50 nm

(c) Influenza viruses

Membranous Envelope

  • Many animal viruses have a membranous envelope

  • The membrane cloaks the viral capsid, helps viruses infect their host

  • Derived from host cell membrane which is usually virus-modified

  • Viral glycoproteins on the envelope bind to specific receptor molecules on the surface of a host cell

Slide 9

Head

DNA

Tail sheath

Tail fiber

80  225 nm

50 nm

(d) Bacteriophage T4

Bacteriophages

  • Also called phages (T2, T4, T6) have the most complex capsids found among viruses

  • Icosohedral head encloses the genetic material; the protein tailpiece w/tail fibers attaches the phage to its bacterial host and injects its DNA into the bacterium

Slide 10

Viral Reproductive Cycles

  • Although a virus has a genome it can only reproduce within a host cell

  • Viruses are obligate intracellular parasites

  • Each virus has a host range - a limited number of host cells that it can infect

  • Recognize host cells by a complementary fit between external viral proteins and specific cell surface receptor sites

  • Viruses use enzymes, ribosomes, and small molecules of host cells to synthesize progeny viruses

Slide 11

VIRUS

DNA

Entry into cell and

uncoating of DNA

Capsid

Transcription

Replication

HOST CELL

Viral DNA

mRNA

Viral DNA

Capsid proteins

Self-assembly of new virus particles and their exit from cell

Viral Reproduction

Slide 12

Reproductive Cycles of Phages

  • Phages are the best understood of all viruses

  • They through two alternative reproductive mechanisms: the lytic cycle and the lysogenic cycle

    • Lytic cycle - culminates in the death of the host

    • Lysogenic cycle - replicates the phage genome without destroying the host

Slide 13

1

Attachment. The T4 phage usesits tail fibers to bind to specificreceptor sites on the outer surface of an E. coli cell.

2

Entry of phage DNA and degradation of host DNA.The sheath of the tail contracts,injecting the phage DNA intothe cell and leaving an emptycapsid outside. The cell’sDNA is hydrolyzed.

5

Release. The phage directs productionof an enzyme that damages the bacterialcell wall, allowing fluid to enter. The cellswells and finally bursts, releasing 100 to 200 phage particles.

Phage assembly

3

Synthesis of viral genomes and proteins. The phage DNAdirects production of phageproteins and copies of the phagegenome by host enzymes, usingcomponents within the cell.

4

Assembly. Three separate sets of proteinsself-assemble to form phage heads, tails,and tail fibers. The phage genome ispackaged inside the capsid as the head forms.

Head

Tail fibers

Tails

The Lytic Cycle

  • A phage reproductive cycle that culminates in the death of the host cell

  • Produces new phages and digests the host’s cell wall, releasing the progeny viruses

  • A phage that reproduces only by the lytic cycle is called a virulent phage

  • Bacteria have defenses against phages, including restriction enzymes that recognize and cut up certain phage DNA

Slide 14

Phage

DNA

The phage attaches to a

host cell and injects its DNA.

Many cell divisions produce a large population of bacteria infected with the prophage.

Phage DNA

circularizes

Phage

Occasionally, a prophage exits the bacterial chromosome,

initiating a lytic cycle.

Bacterial

chromosome

Lysogenic cycle

Lytic cycle

Certain factors

determine whether

The bacterium reproduces

normally, copying the prophage

and transmitting it to daughter cells.

The cell lyses, releasing phages.

Prophage

Lytic cycle

is induced

Lysogenic cycle

is entered

or

New phage DNA and proteins are synthesized and assembled into phages.

Phage DNA integrates into the bacterial chromosome,becoming a prophage.

The Lysogenic Cycle

  • The lysogenic cycle replicates the phage genome without destroying the host

  • The viral DNA molecule is incorporated by genetic recombination into the host cell’s chromosome

  • This integrated viral DNA is known as a prophage

  • Every time the host divides, it copies the phage DNA and passes it to the daughter cells

  • Phages that use both the lytic and lysogenic cycles are called temperate phages

Slide 15

Viral Classification

  • The nature of the genome is the basis for the common classification of animal viruses

Slide 16

3 patterns of viral replication

  • DNA  DNA: If viral DNA is double-stranded, DNA replication resembles that of cellular DNA, and the virus uses DNA polymerase produced by the host.

  • RNA  RNA: Since host cells lack the enzyme to copy RNA, most RNA viruses contain a gene that codes for RNA replicase, an enzyme that uses viral RNA as a template to produce complementary RNA.

  • RNA  DNA  RNA: Some RNA viruses encode reverse transcriptase, an enzyme that transcribes DNA from a RNA template.

Slide 17

Glycoprotein

Viral envelope

Capsid

RNA(two identicalstrands)

Reversetranscriptase

RNA As Genetic Material - Retroviruses / Proviruses

  • The broadest variety of RNA genomes is found among the viruses that infect animals

  • Retroviruses, such as HIV, use the enzyme reverse transcriptase to copy their RNA genome into DNA

  • The viral DNA that is integrated into the host genome is called a provirus

  • Unlike a prophage, a provirus remains a permanent resident of the host cell

Slide 18

Glycoproteins on the viral envelope bind to specific receptor molecules(not shown) on the host cell, promoting viral entry into the cell.

Capsid

RNA

1

2

Envelope (with

glycoproteins)

Capsid and viral genome

enter cell

3

HOST CELL

The viral genome (red)

functions as a template forsynthesis of complementary

RNA strands (pink) by a viral

enzyme.

Viral genome (RNA)

Template

5

Complementary RNA

strands also function as mRNA,

which is translated into both

capsid proteins (in the cytosol)and glycoproteins for the viral

envelope (in the ER).

mRNA

New copies of viral

genome RNA are made

using complementary RNA

strands as templates.

4

Capsid

proteins

ER

Copy of

genome (RNA)

Glyco-

proteins

6

Vesicles transport

envelope glycoproteins to

the plasma membrane.

7

New virus

8

A capsid assembles

around each viral

genome molecule.

The Reproductive Cycle Of An Enveloped RNA Virus

  • The host’s RNA polymerase transcribes the proviral DNA into RNA molecules

  • The RNA molecules function both as mRNA for synthesis of viral proteins and as genomes for new virus particles released from the cell

Slide 19

1

Reverse transcriptase

catalyzes the synthesis of a

DNA strand complementary

to the viral RNA.

2

The virus fuses with the

cell’s plasma membrane.

The capsid proteins are

removed, releasing the viral proteins and RNA.

Membrane of white blood cell

HIV

Reverse transcriptase

catalyzes the synthesis ofa second DNA strand

complementary to the first.

3

Reverse transcriptase

HOST CELL

Viral RNA

4

The double-stranded DNA is incorporated

as a provirus into the cell’s DNA.

RNA-DNAhybrid

0.25 µm

HIV entering a cell

DNA

ChromosomalDNA

NUCLEUS

Provirus

5

Proviral genes are transcribed into RNA molecules, which serve as genomes for the next viral generation and as mRNAs for translation into viral proteins.

RNA genomefor the nextviral generation

mRNA

6

The viral proteins include capsid proteins and reverse transcriptase (made in the cytosol) and envelope glycoproteins (made in the ER).

7

Capsids are

assembled around

viral genomes and

reverse transcriptase

molecules.

Vesicles transport the

glycoproteins from the ER to

the cell’s plasma membrane.

8

9

New viruses bud

off from the host cell.

New HIV leaving a cell

The Reproductive Cycle Of HIV, A Retrovirus

Slide 20

(b) The SARS-causing agent is a coronavirus like this one (colorized TEM), so named for the “corona” of glycoprotein spikes protruding from the envelope.

(a) Young ballet students in Hong Kong wear face masks to protect themselves from the virus causing SARS.

Viral Diseases in Animals

  • Viruses, viroids, and prions are formidable pathogens in animals and plants

  • Viruses may damage or kill cells by causing the release of hydrolytic enzymes from lysosomes

  • Some viruses cause infected cells to produce toxins that lead to disease symptoms

  • Emerging viruses are those that appear suddenly or suddenly come to the attention of medical scientists

  • Outbreaks of “new” viral diseases in humans are usually caused by existing viruses that expand their host territory

  • Severe acute respiratory syndrome (SARS) recently appeared in China

Slide 21

Bacterial Genetics

  • Rapid reproduction, mutation, and genetic recombination contribute to the genetic diversity of bacteria

  • Bacteria allow researchers to investigate molecular genetics in the simplest true organisms

  • The bacterial chromosome is usually a circular DNA molecule with few associated proteins

  • In addition to the chromosome, many bacteria have plasmids, smaller circular DNA molecules that can replicate independently of the bacterial chromosome

Slide 22

Mixture

Mutantstrainarg+trp–

Mutantstrainarg–trp+

RESULTS

EXPERIMENT

No colonies(control)

No colonies(control)

Researchers had two mutant strains, one that could make arginine but not tryptophan (arg+ trp–) and one that could make tryptophan but not arginine (arg trp+). Each mutant strain and a mixture of both strains were grown in a liquid medium containing all the required amino acids. Samples from each liquid culture were spread on plates containing a solution of glucose and inorganic salts (minimal medium), solidified with agar.

CONCLUSION

Mixture

Only the samples from the mixed culture, contained cells that gave rise to colonies on minimal medium, which lacks amino acids.

Mutantstrainarg+trp–

Mutantstrainargtrp+

Coloniesgrew

Because only cells that can make both arginine and tryptophan (arg+ trp+ cells) can grow into colonies on minimal medium, the lack of colonies on the two control plates showed that no further mutations had occurred restoring this ability to cells of the mutant strains. Thus, each cell from the mixture that formed a colony on the minimal medium must have acquired one or more genes from a cell of the other strain by genetic recombination.

Mutation and Genetic Recombination

  • Since bacteria can reproduce rapidly new mutations can quickly increase a population’s genetic diversity

  • Further genetic diversity can arise by recombination of the DNA from two different bacterial cells

Slide 23

Mechanisms of Gene Transfer and Genetic Recombination in Bacteria

  • Three processes bring bacterial DNA from different individuals together

    • Transformation - Is the alteration of a bacterial cell’s genotype and phenotype by the uptake of naked, foreign DNA from the surrounding environment

    • Transduction - Phages carry bacterial genes from one host cell to another

    • Conjugation - Is the direct transfer of genetic material between bacterial cells that are temporarily joined

Slide 24

Mixture of heat-killed

S cells and living R cells

Heat-killed

S cells (control)

Living R cells

(control)

Living S cells

(control)

RESULTS

Mouse dies

Mouse healthy

Mouse healthy

Mouse dies

Living S cells

are found in

blood sample

Transformation

  • Transformation is the alteration of a bacterial cell’s genotype and phenotype by the uptake of naked, foreign DNA from the surrounding environment

  • For example, harmless Streptococcus pneumoniae bacteria can be transformed to pneumonia-causing cells

Slide 25

A+

A+

B+

B+

2

Transduction

Phage DNA

1

Phage infects bacterial cell that has alleles A+ and B+

Host DNA (brown) is fragmented, and phage DNA and proteins are made. This is the donor cell.

Donorcell

A bacterial DNA fragment (in this case a fragment withthe A+ allele) may be packaged in a phage capsid.

3

A+

Crossingover

Phage with the A+ allele from the donor cell infects

a recipient A–B– cell, and crossing over (recombination)

between donor DNA (brown) and recipient DNA

(green) occurs at two places (dotted lines).

4

A+

A–

B–

Recipientcell

The genotype of the resulting recombinant cell (A+B–) differs from the genotypes of both the donor (A+B+) and the recipient (A–B–).

5

A+

B–

Recombinant cell

Slide 26

Conjugation and Plasmids

  • Conjugation is the direct transfer of genetic material between bacterial cells that are temporarily joined

  • The transfer is one-way: One cell (“male”) donates DNA, and its “mate” (“female”) receives the genes

  • “Maleness,” the ability to form a sex pilus and donate DNA, results from an F (for fertility) factor as part of the chromosome or as a plasmid

  • Plasmids, including the F plasmid, are small, circular, self-replicating DNA molecules

Slide 27

A single strand of the F plasmid breaks at a specific point (tip of blue arrowhead) and begins tomove into the recipient cell. As transfer continues, the donor plasmid rotates(red arrow).

DNA replication occurs inboth donor and recipientcells, using the single parental strands of the F plasmid as templates to synthesize complementary strands.

The plasmid in the recipient cell circularizes. Transfer and replication result in a compete F plasmid in each cell. Thus, both cells are now F+.

A cell carrying an F plasmid(an F+ cell) can form amating bridge with an F– celland transfer its F plasmid.

4

2

3

1

F plasmid

Bacterial chromosome

F+ cell

F+ cell

Mating

bridge

F– cell

F+ cell

Bacterial

chromosome

Conjunction and transfer of an F plasmid from and F+ donor to an F– recipient

The F Plasmid and Conjugation

  • Cells containing the F plasmid, designated F+ cells, function as DNA donors during conjugation

  • F+ cells transfer DNA to an F recipient cell

  • Chromosomal genes can be transferred during conjugation when the donor cell’s F factor is integrated into the chromosome

Slide 28

Hfr cell

F+ cell

F factor

2

1

The circular F plasmid in an F+ cellcan be integrated into the circularchromosome by a single crossoverevent (dotted line).

The resulting cell is called an Hfr cell

(for High frequency of recombination).

B+

D+

C+

C+

Hfr cell

A+

D+

A+

B+

D+

A+

C+

B+

A+

B+

C–

C–

F– cell

C–

A+

B–

D–

A+

B–

D–

D+

B–

D–

C+

B+

A–

A–

A–

C–

The location and orientation of the F factor in the donor chromosome determine the sequence of gene transfer during conjugation. In this example, the transfer sequence for four genes is A-B-C-D.

The mating bridgeusually breaks well before the entire chromosome andthe rest of the F factor are transferred.

A single strand of the F factorbreaks and begins to move through the bridge. DNA replication occurs in both donor and recipient cells, resulting in double-stranded DNA

B+

D–

Since an Hfr cell has all the F-factor genes, it can form a mating bridge with an F– cell and transfer DNA.

B–

3

4

6

5

A–

A+

Temporary

partial

diploid

Recombinant F–

bacterium

C–

C–

B+

B–

D–

D–

B+

B–

A–

A–

A+

A+

Two crossovers can result in the exchange of similar (homologous) genes between the transferred chromosome fragment (brown) and the recipient cell’s chromosome (green).

The piece of DNA ending up outside thebacterial chromosome will eventually be degraded by the cell’s enzymes. The recipient cell now contains a new combination of genes but no F factor; it is a recombinant F– cell.

7

8

The F Plasmid and Conjugation

  • A cell with a built-in F factor is called an Hfr cell

  • The F factor of an Hfr cell brings some chromosomal DNA along when transferred to an F– cell

  • Thr transfer of part of the bacterial chromosome from an Hfr donor to an F– recipient results in recombination

Slide 29

R plasmids and Antibiotic Resistance

  • R plasmids confer resistance to various antibiotics

  • When a bacterial population is exposed to an antibiotic, individuals with the R plasmid will survive and increase in the overall population

Slide 30

Transposition of Genetic Elements

  • The DNA of a cell can also undergo recombination due to movement of transposable elements within the cell’s genome

  • Transposable elements:

    • Can move around within a cell’s genome

    • Are often called “jumping genes”

    • Contribute to genetic shuffling in bacteria

Slide 31

Insertion sequence

3

5

3

5

A T C C G G T…

A C C G G A T…

T A G G C C A …

T G G C C T A …

Transposase gene

Inverted

repeat

Inverted

repeat

(a) Insertion sequences, the simplest transposable elements in bacteria, contain a single gene that encodes transposase, which catalyzes movement within the genome. The inverted repeats are backward, upside-down versions of each other; only a portion is shown. The inverted repeat sequence varies from one type of insertion sequence to another.

Figure 18.19a

Insertion Sequences

  • The simplest transposable elements, called insertion sequences, exist only in bacteria

  • An insertion sequence contains a single gene for transposase, an enzyme that catalyzes movement of the insertion sequence from one site to another within the genome

Slide 32

Transposon

Antibioticresistance gene

Insertion sequence

Insertion sequence

5

3

5

3

Inverted repeats

Transposase gene

(b) Transposons contain one or more genes in addition to the transposase gene. In the transposon shown here, a gene for resistance to an antibiotic is located between twin insertion sequences. The gene for antibiotic resistance is carried along as part of the transposon when the transposon is inserted at a new site in the genome.

Figure 18.19b

Transposons

  • Transposable elements called transposons are longer and more complex than insertion sequences

  • In addition to DNA required for transposition, transposons have extra genes that “go along for the ride,” such as genes for antibiotic resistance

Slide 33

Control of Gene Expression

  • Every cell contains thousands of genes which code for proteins.

  • However, every gene is not actively producing proteins at all times.

  • To be expressed, a gene must be transcribed into m-RNA, the m-RNA must be translated into a protein, and the protein must become active.

  • Gene regulation can theoretically occur at any step in this process

Slide 34

Control of Gene Expression

  • Two categories of gene regulation:

    • Transcriptional controls - factors that regulate transcription

    • Post-transcriptional controls – factors that regulate any step in gene expression after transcription is complete

  • It is most efficient to regulate genes during transcription.

  • Both prokaryotes and eukaryotes rely primarily on transcriptional controls.

Slide 35

(a) Regulation of enzyme activity

(b) Regulation of enzyme production

Precursor

Feedback

inhibition

Enzyme 1

Gene 1

Regulation

of gene

expression

Enzyme 2

Gene 2

Gene 3

Enzyme 3

Gene 4

Enzyme 4

Gene 5

Enzyme 5

Tryptophan

Regulating Prokaryotic Gene Expression

  • Prokaryotes can quickly turn genes on and off in response to environmental conditions.

  • This metabolic control occurs on two levels

    • Adjusting the activity of metabolic enzymes already present

    • Regulating the genes encoding the metabolic enzymes

Slide 36

Prokaryotic Gene Regulation

  • Response is facilitated by:

    • Simultaneous transcription and translation

    • Short-lived m-RNAs

    • Operons

  • Functionally related genes are often located next to each other and are transcribed as a unit.

  • For example E. coli,

    • 5 different enzymes are needed to synthesize the amino acid tryptophan

    • The genes that code for these enzymes are located together

Slide 37

Operons: The Basic Concept

  • In bacteria, genes are often clustered into operons, composed of

    • An operator, an “on-off” switch

    • A promoter

    • Genes for metabolic enzymes

  • An operon

    • Is usually turned “on”

    • Can be switched off by a protein called a repressor

Slide 38

trp operon

Promoter

Promoter

Genes of operon

RNA polymerase

Start codon Stop codon

trpR

trpD

trpC

trpB

trpE

trpA

DNA

Operator

Regulatory

gene

3

mRNA 5

mRNA

5

Polypeptides that make up

enzymes for tryptophan synthesis

C

E

D

B

A

Inactiverepressor

Protein

(a) Tryptophan absent, repressor inactive, operon on. RNA polymerase attaches to the DNA at the promoter and transcribes the operon’s genes.

Figure 18.21a

Prokaryotic Gene Regulation

  • A single promoter serves all 5 genes. (region where RNA polymerase binds to DNA and begins transcription)

  • The genes are transcribed as a unit, - one long mRNA molecule which contains the code to make all 5 enzymes

Slide 39

trp operon

Promoter

Promoter

Genes of operon

Start codon Stop codon

RNA polymerase

trpR

trpD

trpC

trpB

trpE

trpA

DNA

Operator

Regulatory

gene

3

mRNA 5

mRNA

5

Polypeptides that make up

enzymes for tryptophan synthesis

C

E

D

B

A

Inactiverepressor

Protein

(a) Tryptophan absent, repressor inactive, operon on. RNA polymerase attaches to the DNA at the promoter and transcribes the operon’s genes.

Prokaryotic Gene Regulation

  • There is also a single regulatory switch, called the operator.

  • The operator is positioned within the promoter, or between the promoter and the protein coding genes. It controls access of RNA polymerase to the genes.

Slide 40

DNA

No RNA made

mRNA

Active

repressor

Protein

Tryptophan

(corepressor)

Tryptophan present, repressor active, operon off. As tryptophan

accumulates, it inhibits its own production by activating the repressor protein.

(b)

Prokaryotic Gene Regulation

  • Transcription of the 5 coding genes in the tryptophan operon is blocked when a transcriptional repressorbinds to the operator.

  • The repressor binds to the operator only when there is a high level of tryptophan present:

Slide 41

Two Types of Negative Gene Regulation

  • In a repressible operon, binding of a specific repressor protein to the operator shuts off transcription

  • Repressible enzymes usually function in anabolic pathways

  • In an inducible operon, binding of an inducer to a repressor inactivates the repressor and turns on transcription

  • Inducible enzymes usually function in catabolic pathways

Slide 42

Promoter

Regulatorygene

Operator

DNA

lacl

lacZ

NoRNAmade

RNApolymerase

3

mRNA

5

Activerepressor

Protein

(a)

Prokaryotic Gene Regulation: Inducible Operon

  • The lactose operon in E. coli is an inducible operon

  • It controls the production of 3 enzymes needed to digest lactose (catabolism of a disaccharide made of glucose and galactose)

  • When lactose is absent, the repressor is active and the operon is off.

  • The lac repressor is innately active, and in the absence of lactose it switches off the operon by binding to the operator.

Slide 43

DNA

lacl

lacz

lacY

lacA

lac operon

RNApolymerase

3

mRNA

5

mRNA 5

mRNA 5'

-Galactosidase

Permease

Transacetylase

Protein

Inactiverepressor

Allolactose(inducer)

lac Operon

  • If lactose is present, the repressor is inactivated and the operon is on

  • Allolactose, an isomer of lactose, turns on the operon by inactivating the repressor. In this way, the enzymes for lactose utilization are induced.

Slide 44

Positive Gene Regulation

  • Regulation of both the trp and lac operons involves the negative control of genes, because the operons are switched off by the active form of the repressor protein

  • Some operons are also subject to positive control via a stimulatory activator protein, such as catabolite activator protein (CAP)

  • CAP (catabolite activator protein) stimulates transcription of genes that allow E. coli to use other food sources when glucose is not present such as lactose

Slide 45

Operator

RNA

polymerase

can bindand transcribe

Promoter

DNA

lacl

lacZ

CAP-binding site

ActiveCAP

cAMP

Inactive lac

repressor

InactiveCAP

(a)

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

Positive Transcriptional Control

  • In E. coli, when glucose is scarce, the lac operon is activated by the binding of a regulatory protein, catabolite activator protein (CAP)

  • Low levels of glucose lead to high levels of cAMP

  • cAMP binds to CAP, CAP binds to CAP binding site, and transcription of lac mRNA is stimulated for catabolism of lactose

Slide 46

Promoter

Operator

DNA

lacl

lacZ

CAP-binding site

RNA

polymerase

can’t bind

InactiveCAP

Inactive lac

repressor

Lactose present, glucose present (cAMP level low): little lac mRNA synthesized.When glucose is present, cAMP is scarce, and CAP is unable to stimulate transcription.

(b)

Positive Transcriptional Control

  • When the glucose level is high, cAMP is low. CAP is not activated and transcription is not stimulated:

  • When glucose levels in an E. coli cell increase, CAP detaches from the lac operon, turning it off

Slide 47

Lab 9A/B - How Are Plasmids Used In Recombinant DNA Technology

Slide 48

Recombinant DNA

  • Formed by joining DNA from 2 different individuals into a single molecule.

  • Various natural mechanisms can combine DNA from 2 individuals of the same species

  • Scientists have also developed techniques to combine DNA from any 2 individuals.

Slide 49

Recombinant DNA

  • Two key enzymes are used to make artificially recombined DNA.

    • Restriction enzymes (also called restriction endonucleases) cut DNA into fragments – so called “molecular scissors”

      • Each one recognizes and cuts DNA only where a specific sequence of base pairs occurs

      • A restriction enzyme will usually make many cuts in a DNA molecule yielding a set of restriction fragments

      • The most useful restriction enzymes cut DNA in a staggered way leaving unpaired bases at both ends.

      • These fragments are called “sticky ends” and can bond with complementary “sticky ends” of other fragments

    • DNA ligase is used to join DNA fragments together. This is the “molecular glue”

Slide 50

1

3

2

Restriction site

5

3

DNA

G A A T T C

3

5

C T T A A G

Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow

A A T T C

G

C T T A A

G

Sticky end

A A T T C

G

G

DNA fragment from another source is added. Base pairing of sticky ends produces various combinations.

C T T A A

Fragment from differentDNA molecule cut by thesame restriction enzyme

G

A A T T

C

A A T T C

G

C T T A A

G

G

T T A A

C

One possible combination

DNA ligaseseals the strands.

Recombinant DNA molecule

Procedure for Recombining DNA

  • Isolate DNA from 2 different sources

  • Cut the DNA from both sources into fragments using the same restriction enzyme.

  • Mix the DNA fragments together. Since they were cut with the same restriction enzyme, fragments from different sources will have the same “sticky ends” and can pair up.

  • Use the enzyme DNA ligase to join the paired fragments together

Slide 51

Recombinant Plasmids

  • Recombinant DNA technology can be used to create recombinant plasmids (or other agents such as viruses) used to insert foreign genes into recipient cells.

  • Plasmids (or other recombinant agents) used to insert foreign DNA into recipient cells are called vectors

  • Recombinant plasmids can then be used to produce multiple copies of the DNA fragment

Slide 52

Lab 9-A

  • Transformation – bacteria absorb fragments of DNA from surrounding media

  • Transform E. Coli with 3 unknown media samples

    • One solution contains no DNA at all

    • One solutuion contains normal pUC18 plasmid

      • Gene for ampicillin resistance

      • Lac Z gene which codes for -galactosidase, lactose digestion enzyme

    • One solution contains recombinant pUC18

      • Contains a fragment of foreign DNA from  phage

      • Inserted in to middle of Lac Z gene, inactivating it

Slide 53

Transformation Procedure

  • Add E. Coli to all three unknown solutions

  • Chill then “heat shock” samples to facilitate uptake of plasmid

  • Incubate then inoculate agar plates

  • Agar plates contain nutrients, ampicillin, Xgal (analog of lactose that release blue color when digested)

  • Results?

Slide 54

Using Restriction Enzyme EcoRI

  • Procedure will cut the plasmids in the three unknown samples with the restriction enzyme EcoRI

  • Add EcoRI to the three unknown plasmid stock solutions and incubate

  • Separate the DNA fragments using gel electrophresis

    • Small fragments move faster farther

    • Similar to proteins except instead of MW we use base pairs (bp) to reference size

  • Results?

Slide 55

Plasmid pUC18

  • 2686 base pairs in size

bp 1

Laz Z gene: bp 236 - 469

EcoRI site: bp 396

Ampicillin resistance gene: bp 1626 - 2486

bp 2014

bp 671

bp 1343

Slide 56

Plasmid pUC18

  • 2686 base pairs in size

Laz Z gene: bp 236 - 469

bp 1

Phage DNA inserted

(Not to scale!)

Ampicillin resistance gene: bp 1626 - 2486

bp 2014

bp 671

bp 1343


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