Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings DNA technology has many useful applications –The Human Genome Project –The production

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings

• DNA technology has many useful applications– The Human Genome Project

– The production of vaccines, cancer drugs, and pesticides

– Engineered bacteria that can clean up toxic wastes


MICROBIAL GENETICS

Copyright © 2009 Pearson Education, Inc.


• Viruses provided some of the earliest evidence that genes are made of DNA

• Molecular biology studies how DNA serves as the molecular basis of heredity


• The Hershey-Chase experiment showed that certain viruses reprogram host cells to produce more viruses by injecting their DNA

THE STRUCTURE OF THE GENETIC MATERIAL

10.1 Experiments showed that DNA is the genetic material

Head

Tail

Tailfiber

DNA

Figure 10.1A


• Phage reproductive cycle

Figure 10.1C

Phage attaches to bacterial cell.

Phage injects DNA.

Phage DNA directs host cell to make more phage DNA and protein parts. New phages assemble.

Cell lyses and releases new phages.


• The Hershey-Chase Experiment

Figure 10.1B

Mix radioactivelylabeled phages with bacteria. The phages infect the bacterial cells.

Phage

Bacterium

Radioactiveprotein

DNA

Emptyprotein shell

1 2 Agitate in a blender to separate phages outside the bacteria from the cells and their contents.

3 Centrifuge the mixture so bacteria form a pellet at the bottom of the test tube.

4 Measure the radioactivity in the pellet and liquid.

Batch 1Radioactiveprotein

Batch 2RadioactiveDNA

RadioactiveDNA

PhageDNA

Centrifuge

Pellet

Radioactivityin liquid

Radioactivityin pelletPellet

Centrifuge


• Many viruses have RNA, rather than DNA, as their genetic material

– Example: flu viruses

10.18 Connection: Many viruses cause disease in animals

Figure 10.18A

Membranousenvelope

RNA

Proteincoat

Glycoprotein spike


• Most plant viruses have RNA

– Example: tobacco mosaic disease

10.19 Connection: Plant viruses are serious agricultural pests

Figure 10.19

Protein RNA


• The deadly Ebola virus causes hemorrhagic fever

– Each virus is an enveloped thread of protein-coated RNA

• Hantavirus is another enveloped RNA virus

10.20 Connection: Emerging viruses threaten human health

Figure 10.20A, B


VIRUSES: GENES IN PACKAGES

10.17 Viral DNA may become part of the host chromosome

Phage

New phage DNA andproteins are synthesized

Phage DNA inserts into the bacterialchromosome by recombination

Attachesto cell

Phage DNA

Bacterialchromosome

Phage injects DNA

Occasionally a prophagemay leave the bacterialchromosome

Many celldivisions

Lysogenic bacteriumreproduces normally,replicating the prophageat each cell division

Prophage

Phage DNAcircularizes

LYSOGENIC CYCLE

Cell lyses,releasing phages

Phagesassemble

LYTIC CYCLE

OR


• HIV is a retrovirus

10.21 The AIDS virus makes DNA on an RNA template

Figure 10.21A

EnvelopeGlycoprotein

Proteincoat

RNA(two identicalstrands)

Reversetranscriptase


• Inside a cell, HIV uses its RNA as a template for making DNA to insert into the host chromosome

Figure 10.21B

Viral RNA

1

2 3

5

4

6

DNAstrand

Double-strandedDNA

ViralRNAandproteins

CYTOPLASM

NUCLEUS

ChromosomalDNA

ProvirusDNA

RNA


• Virus studies help establish molecular genetics

• Molecular genetics helps us understand viruses

– such as HIV, seen here attacking a white blood cell

10.22 Virus research and molecular genetics are intertwined

Figure 10.22


10.19 EVOLUTION CONNECTION: Emerging viruses threaten human health

– Examples of emerging viruses

– HIV

– Ebola virus

– West Nile virus

– RNA coronavirus causing severe acute respiratory syndrome (SARS)

– Avian flu virus



• Research on E. coli revealed that these bacteria have a sexual mechanism that can bring about the combining of genes from two different cells

• This discovery led to the development of recombinant DNA technology

– a set of techniques for combining genes from different sources

From E.Coli to a Map of Our Genes


10.22 Bacteria can transfer DNA in three ways

– Transformation is the uptake of DNA from the surrounding environment

– Transduction is gene transfer through bacteriophages

– Conjugation is the transfer of DNA from a donor to a recipient bacterial cell through a cytoplasmic bridge

– Recombination of the transferred DNA with the host bacterial chromosome leads to new combinations of genes



DNA enterscell

Bacterial chromosome(DNA)

Fragment ofDNA fromanotherbacterial cell


Phage

Fragment ofDNA fromanotherbacterial cell(former phagehost)


Mating bridge

Sex pili

Donor cell(“male”)

Recipient cell(“female”)


Donated DNA

Recipient cell’schromosome

Crossovers

Recombinantchromosome

Degraded DNA


10.23 Bacterial plasmids can serve as carriers for gene transfer

Plasmids are small circular pieces of DNA separate from the main DNA of the bacteria and key tools for DNA technology

– R plasmids transfer genes for antibiotic resistance by conjugation

– Researchers use plasmids to insert genes into bacteria



Figure 12.3

Plasmidisolated

1Bacterium

Bacterialchromosome

Plasmid

2DNAisolated

Cell containing geneof interest

DNAGene ofinterest

3 Gene inserted into plasmid

Recombinant DNA(plasmid)

4 Plasmid put intobacterial cell

Recombinantbacterium

5

Copies of gene Copies of protein

Clones of cellGene for pestresistanceinserted intoplants

Gene used to alter bacteriafor cleaning up toxic waste

Protein used to dissolve bloodclots in heart attack therapy

Protein used to make snow format highertemperature

Cell multiplies withgene of interest


• Restriction enzymes cut DNA at specific points

• DNA ligase “pastes” the DNA fragments together

• The result is recombinant DNA

12.4 Enzymes are used to “cut and paste” DNA

Figure 12.4

DNA1

Restriction enzymerecognition sequence

Restriction enzymecuts the DNA intofragments

Sticky end

2

3

4

5

Restriction enzymecuts the DNA intofragments

Addition of a DNAfragment fromanother source

Two (or more)fragments sticktogether bybase-pairing

DNA ligasepastes the strand

Recombinant DNA molecule


• Bacteria take the recombinant plasmids and reproduce

• This clones the plasmids and the genes they carry

– Products of the gene can then be harvested

12.5 Genes can be cloned in recombinant plasmids: A closer look


Figure 12.5

Isolate DNAfrom two sources

1E. coli

Cut both DNAs with the same

restrictionenzyme

2

Plasmid

Human cell

DNA

Gene VSticky ends

Mix the DNAs; they joinby base-pairing

3

Add DNA ligaseto bond the DNA covalently

4

Recombinant DNAplasmid Gene V

Put plasmid into bacteriumby transformation

5

Clone the bacterium6

Bacterial clone carrying manycopies of the human gene


• Recombinant DNA technology allows the construction of genomic libraries

– Genomic libraries are sets of DNA fragments containing all of an organism’s genes

• Copies of DNA fragments can be stored in a cloned bacterial plasmid or phage

12.6 Cloned genes can be stored in genomic libraries

Figure 12.6

Genome cut up with restriction enzyme

Recombinantplasmid

OR

Recombinantphage DNA

Bacterialclone

Phageclone

Phagelibrary

Plasmidlibrary


• Reverse transcriptase can be used to make smaller cDNA libraries

– These contain only the genes that are transcribed by a particular type of cell

12.7 Reverse transcriptase helps make genes for cloning

OTHER TOOLS OF DNA TECHNOLOGY


Figure 12.7

Transcription1

CELL NUCLEUS

DNA ofeukaryoticgene

RNAtranscript

mRNA

Exon Intron Exon Intron Exon

TEST TUBE

Reverse transcriptase

cDNA strand

cDNA of gene(no introns)

RNA splicing(removes introns)

2

Isolation of mRNAfrom cell and additionof reverse transcriptase;synthesis of DNA strand

3

Breakdown of RNA4

Synthesis of secondDNA strand

5


• A nucleic acid probe can tag a desired gene in a library

12.8 Nucleic acid probes identify clones carrying specific genes

Figure 12.8A

Radioactiveprobe (DNA)

Mix with single-stranded DNA fromvarious bacterial(or phage) clones

Single-strandedDNA

Base pairingindicates thegene of interest


• DNA probes can identify a bacterial clone carrying a specific gene

Figure 12.8B

Bacterial colonies containingcloned segments of foreign DNA

Radioactive DNA

Transfercells tofilter

1Solutioncontainingprobe

Treat cellson filter toseparateDNA strands

2 Add probeto filter

3 ProbeDNA

Gene ofinterest

Single-strandedDNA from cell

Hydrogen-bonding

Autoradiography4

Developed filmColonies of livingcells containinggene of interest

Compare autoradiographwith master plate

5

Master plate

Filterpaper


• A labeled probe can reveal patterns of gene expression in different kinds of cells

• This technique may revolutionize the diagnosis and treatment of cancer

12.9 Connection: DNA microarrays test for the expression of many genes at once

Figure 12.9

cDNA

DNA of gene

DNAmicroarray,actual size(6,400 genes)

Documents

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings DNA technology has many useful applications –The Human Genome Project –The production