Download Chapters 18, 19, 20, 27) Virus, bacteria, gene expression

Chapter 19: Viruses
Structure of Viruses
Virus = DNA/RNA + capsid
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Capsid – protein coat
Capsomeres- small units that make up the capsid
Viral envelope –
- Are derived from membranes of host cells: as a virus is brought into a cell, it brings part of
the host cell membrane in through endocytosis
- May cloak the capsids of viruses found in animals
 Viral genomes may be single or double stranded DNA or single or double stranded RNA.
- Viral genes are usually contained on a single linear or circular nucleic acid molecule
 May be rod-shaped (helical viruses), polyhedral (icosahedral viruses), or more complex in shape
 Some viruses also contain a few viral enzymes.
 Viruses are specific to organisms and tissues
- Colds/flus = different strains and mutations
 Ways of entering the cell = Inject genome, use endocytosis or budding (=passes through
membrane without breaking it)
Bacteriophage – viruses that infect bacteria only
- Complex capsids are found among phages
- Inject viral DNA into host bacteria cell
- Use bacteria cell to produce viral DNA proteins (capsid and capsomeres)
- Cell gets packed with self assembled virus particles
- The cell lyses and virus particles are released to infect other cells
*Same process in all living organisms  evolution, continuity and change
- Use bacteria for DNA replication and protein synthesis with transcription and translation
Frederick Griffith – tested mice – mixed nonpathogenic and pathogenic bacteria  killed mice
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Nonpathogenic bacteria were transformed into pathogenic bacteria
Transforming factor = DNA
Caused inactive DNA of dead bacterial cell leaking from one cell into another live bacterial and
becoming active
Zone of inhibition:
penicillin (protein)
zone of inhibition:
-chemicals diffuse out of the penicillin and prevents
bacteria from growing their cell walls
1) Transformation: nonliving cell’s DNA is picked up by a living cell
- Avery, McCarty, and McCleod – discovered it was DNA
- Alexander Flemming – discovered penicillin (antibiotic)
2) Transduction: virus injects DNA into host cell and takes over its machinery to produce viral
DNA and proteins
- Plasmids act as resistance factors
- Example = E.coli (Eschercia (genus) coli (species) )
3) Conjugation: living bacteria cells transmit DNA between each other; called bacteria sex
- A conjugation bridge
- Contributes to variation
Reproduction of Virus
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Viruses are obligate intracellular parasites that lack metabolic enzymes and other equipment
needed to reproduce
Each virus type has a limited host range due to proteins on the outside of the virus that
recognize only specific receptor molecules on the host cell surface
General Features of Viral Reproductive Cells
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Once the viral genome enters the host cell, the cell’s enzymes, nucleotides, amino acids,
ribosomes, ATP, and other resources are used to replicate the viral genome and produce
capside proteins
- The virus basically takes over the cell and turns it into a virus factory
- DNA viruses use host DNA polymerases to copy their genome, whereas RNA viruses use
virus-encoded polymerases for replicating their RNA genome
After replication, viral nucleic acid and capside proteins spontaneously assemble to form new
viruses within the host cell, a process called “self-assembly”
Hundreds or thousands of newly formed virus particles are released, often destroying the host
cell in the process
Reproductive Cycles of Phages
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There are 2 different types of viral reproductive cycles:
Lytic cycle
Lytic cycle = a reproductive cycle of a phage that culminates in lysis of the host cell and release
of newly produced phages
- The virus attaches to the cell by sticking to a receptor site on the surface of a cell
- The virus injects its DNA into the host cell, leaving behind the empty capside
- The cell begins to transcribe and translate phage genes, one of which codes for an enzyme
that chops up host cell DNA.
- Virus parts assemble: Capsid proteins are made and assembled into phage tails, tail fibers,
heads
- Virus particles are released from the cell, damaging its cell wall and often lysing the cell
Virulent = very aggressive infection
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2.
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Lowering viral infection in bacteria:
1. Mutations that change their receptor sides
2. Restriction enzymes that chop up foreign viral DNA once it enters the cell
Lysogenic cycle
Lysogenic cycle = a virus reproduces its genome without killings its host by incorporating itself
into the DNA of the host cell but doesn’t express itself
Temperate phages can reproduce by the lytic and lysogenic cycles.
 Temperate phage – a virus that stays in your body and can go from active to inactive to
active
When the phage injects its DNA into a cell, it can begin a lytic cycle, or its DNA may be
incorporated into the host cell’s chromosomes and begin a lysogenic cycle as prophage.
 Prophage/ provirus – unexpressed bacteria / unexpressed virus
Most of the genes of the inserted phage genome are repressed by a protein coded for by a
prophage gene. Reproduction of the host cell replicates the phage DNA along with the bacterial
DNA. the prophage may exit the bacterial chromosome, usually in response to environmental
stimuli and start the lytic cycle.
Expression of prophage genes may cause a change in the bacterial phenotype. Several diseasecausing bacteria would be harmless except for the expression of prophage genes that code for
toxins.
Retroviruses
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Retrovirus – contains RNA (not DNA)
Retroviruses convert viral RNA into DNA with the viral enzyme reverse transcriptase
The viral DNA is then integrated into a chromosome, where it is transcribed by the host cell into
viral RNA which acts both as a new viral genome and as mRNA for viral proteins
HIV (human immunodeficiency virus) is a retrovirus that causes AIDS (acquired
immunodeficiency syndrome)
The integrated viral DNA remains as a provirus (=unexpressed virus) within the host cell DNA
New viruses, assembled with 2 copies of both the RNA genome and reverse transcriptase, bud
off covered in host cell plasma membrane studded with viral glycoproteins
Viral Diseases in Animals
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The symptoms of a viral infection may be caused by viral-programmed toxins produced by
infected cells, cells killed or damaged by the virus, or the body’s defense mechanisms fighting
the infection.
Vaccines are variants or derivatives of pathogens that induce the immune system to react
against the actual disease agent.
- Vaccines – stimulates body’s immune system and shortens the lag time for antibodies to
find antigens
- Can be made from a protein coat or an attenuated virus (=dead/inactive form of the virus)
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Unlike bacteria, viruses use the host’s cellular machinery to replicate, and few drugs have been
found to treat or cure viral infections. Some antiviral drugs resemble nucleosides and interfere
with viral nucleic acid synthesis.
Emerging Viruses
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Emerging viruses: new viruses that appear and disappear
The emergence of “new” viral diseases may be linked to the mutation of an existing virus (as in
influenza viruses), the dissemination of an existing virus to a more widespread population (as in
HIV), or the spread from one host species to another (as in SARs). Many new human diseases
are estimated to originate by the third mechanism.
A general or local outbreak of a disease is called an epidemic. Flue epidemics may be caused by
new strains of viruses to which people have little immunity.
Pandemics are global outbreaks.
A recombinant virus may be highly pathogenic
Viroids and Prions: The Simplest Infectious Agents
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Viroids are very small infectious molecules of circular RNA that can replicate in host plant cells,
causing abnormal development and stunted growth.
Prions are protein infectious agents that may be linked to several degenerative brain diseases
such as mad cow disease and Creutzfeldt-Jacob disease in humans.
Prions apparently spread disease by converting normal cellular proteins into the defective form
of the protein.
Bacterial Diseases
vs.
Bacteria shapes: rod, spherical, spiral
Treat with antibiotics
Salmonella
Cholera
Legionnaire’s disease
Stomach ulcers
Syphilis
Gonorrhea
Lyme disease
Botulism
Strep/staph infection
Black death (bubonic plague)
E.coli
Viral Diseases
Viruses: variety of shapes (icosahedrons, rod)
Treat with vaccines or antiviral drugs
Warts
Cold sores
Chicken pox
Small pox
Polio
Rhinovirus
Hepatitis
SARS
Influenza
Measles
Mumps
Rabies
HIV
Ebola
Defenses:
Antigens: foreign agents
- Take time to infect the body’s cells
B-CELLS 
Antibodies: Y-shaped proteins that tag and secure foreign particles for the immune
system to fend against and get rid of them
- Look for and recognize matching virus spikes
- Virus spikes: can mutate so the virus particles are not recognized by the immune
system
T-CELLS 
Natural killer cells:
- HIV – kills T cells
- Ebola – high virulence and causes “bleeding out” in a few days
mRNA splicing: exons joined, introns out
- Gives you specific proteins to recognize antigens
Chapter 27: Bacteria and Archaea
Structure and Function
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3 shapes: spherical (cocci), rod-shaped (bacilli), spiral
Cell-Surface Structure
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Structure:
- Nucleoid= single loop of DNA
- Plasmids= extra loops of DNA that resist antibiotics
- Cell wall= made of peptidoglycan; can have an outer membrane that makes it more
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resistant  maintain cell shape and prevent the cell from bursting in a hypotonic
surrounding
 Gram positive = bacteria with walls containing just peptidoglycan
 Gram negative = bacteria with more complex walls like an outer membrane
Plasma membrane= phospholipid bilayer imbedded with proteins
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Capsule= sticky polysaccharide coating that makes bacteria more resistant to attack by a
host’s immune system and serves as a glue for adhering to a subrate or other prokaryotes
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Fimbriae/sex pili = allow bacteria to stick to their substrate or to other individuals/ allow
bacteria to exchange DNA
Motility
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Many prokaryotes are equipped with flagella, either scattered over the cell surface or
concentrated at one or both ends of the cell. These flagella differ from eukaryotic flagella in
their lack of a plasma membrane cover, structure, and function.
Many motile prokaryotes exhibit taxis, an oriented movement in response to chemical, light, or
other stimuli.
Reproduction and Adaptation
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Prokaryotes have a huge reproductive potential. The growth of colonies usually stops due to the
exhaustion of nutrients or the toxic accumulation of wastes
Some bacteria produce endospores, which are tough-walled dormant cells formed in response
to a a lack of nutrients
Due to short generation times, favorable mutations are rapidly expanded, increasing adaptive
evolution to environmental changes
Prokaryotes
vs.
Bacteria
Eukaryotes
Protists, fungi, plants, animals
Archea Eubacteria
or
“extremophiles”
Genetic variation
1) Rapid reproduction
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Prokaryotes reproduce very rapidly and have large population sizes
2) Mutation
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Mutations, although statistically rare, produce a great deal of genetic diversity because the
mutated cells reproduce so fast
3) Genetic recombination
1. Transformation= nonliving cell’s DNA from the environment or plasmid is picked up by a
living one
- The foreign DNA is integrated into the bacterial chromosome by an exchange of
homologous DNA segments.
- Many bacteria have surface proteins specialized for the uptake of DNA from closely related
species.
2. Transduction = exchange between bacteria and bacteriophage/virus
- A random piece of host DNA may be accidently packaged within a phage, introduced into a
new bacterium, and replace the homologous region of a bacterial chromosome
3. Conjugation = transfer of genetic material (often plasmids) between living bacteria cells;
called bacteria sex
- Sex pili form the conjugation bridge for plasmid exchange
- F- factor is required to produce sex pili and donate DNA. Bacterial cells containing the F
factor on the F plasmid are called F+ cells
- During conjugation, a single strand of the plasmid DNA is transferred through a mating
bridge to the recipient cell
Growth Curve for Bacteria
Number of
bacteria
Bacteria start dying because
they’ve used up their resources
Best for transformation
experiment
Time (days)
The Harmful and Beneficial Impacts of Prokaryotes
Pathogenic Prokaryotes
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About half of all human diseases are caused by pathogenic prokaryotes
Pathogens most commonly cause disease by producing toxins.
Exotoxins are proteins secreted by prokaryotes that cause such diseases as botulism and
cholera.
Endotoxins are lipopolysaccharides released from the outer membrane of gram negative
bacteria, and they cause diseases like typhoid fever
Improved sanitation and development of antibiotics have decreased the incidence of bacterial
disease in developed countries.
The evolution of antibiotic-resistant strains of pathogenic bacteria, however, poses a serious
health threat.
R plasmids carry genes that code for various mechanisms of resistance to antibiotics. R plasmids
also have genes coding for sex pili and are transferred to nonresistant cells during conjugation,
creating the medical problem of antibiotic-resistant pathogens
Horizontal gene transfer also spreads genes connected with virulence
Some pathogenic prokaryotes are potential bioterrorism weapons
Prokaryotes in Research and Technology
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Bioremediation is the use of organisms to remove environmental pollutants.
Prokaryotes are used to treat sewage and clean up oil spills
They are used in the mining industry to recover metals from ores.
They have been engineered to make vitamins (vitamin K), antibiotics, and other products
Chapter 18: Regulating Gene Expression
Feedback inhibition
Bacteria can respond to short-term environmental fluctuations by regulating enzyme activity
through feedback inhibition
 Feedback inhibition is when the product inhibits an enzyme earlier in the process
1) Prevents overproduction of a product
2) Energy conservation: turns the process on or off to prevent waste
Eukaryotic chromosomes are more complex than prokaryotes
- It’s easier to understand prokaryotic gene expression because their DNA is in a single loop, not
strands like eukaryotes
Prokaryotic Gene Expression
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Genes can be under coordinated control by a single on-off switch
An operator is a segment of DNA within or near the promoter that controls the access of RNA
polymerase to the genes
An operon is the entire stretch of DNA that includes the group of structural genes that will make
a protein, the operator, and the promoter
The operon can be switched off by a protein repressor
Repressor: protein that binds to a specific operator, blocking attachment of RNA polymerase
and thus turning an operon “off”  Prevents gene transcription
Repressors are produced by a separate regulatory gene
Regulatory genes code for repressor proteins. These allosteric proteins, which may assume
active or inactive shapes, are usually produced at a slow but continuous rate.
The activity of the repressor protein may be determined by the presence or absence of a
corepressor.
2 types of operons:
1) Lac operon
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RNA polymerase: makes a complementary transcript of DNA and always binds to the promoter
Operator: where the repressor will bind
If the repressor binds  off switch
If the repressor doesn’t bind  on switch
2) Trp operon
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Tryptophan absent  repressor inactive  gene is transcribed
Tryptophan is the corepressor that binds to the repressor protein, changing it into its active
shape, which has a high affinity for the operator and switches off the trp operon
If Tryptophan is absent: repressor proteins aren’t bound with try  the operator is no longer
repressed  RNA polymerase attaches to the promoter  mRNA for the enzymes needed to
tryptophan synthesis is produced
Differential Gene Expression
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Almost all the cells in an organism are genetically identical
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Differences between cell types result from differential gene expression: the expression of
different genes by cells with the same genome
Basically: not every cell of every organism expresses every gene at the same time
Only a small portion of a cell’s DNA codes for proteins; some codes for RNA; and most is
noncoding DNA
Errors in gene expression can lead to diseases like cancer
Gene expression is regulated at many stages
Example: embryonic development
It is most commonly regulated at transcription
Regulation of Chromatin Structure
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DNA packing and the location of a gene’s promoter relative to nucleosomes may help control
which genes are available for transcription
Genes within highly packed heterochromatin are not usually expressed
Heterochromatin is tightly wound up while euchromatin is long and skinny
Chemical modifications to histones and DNA of chromatin influence both chromatin structure
and gene expression
These modifications can either allow or stop transcription:
1) Acetylation – CH2CH3  promotes initiation of transcription
- Loosens attachment of DNA to proteins
2) Methylation – CH3  stops transcription
- Condenses and tightens DNA around proteins
- It “silences” gene expression
According to the histone code hypothesis, the specific patterns of modifications determine
chromatin shape and influence transcription
DNA methylation
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In a process called DNA methylation, methyl groups are added to DNA bases (usually cytosine)
Genes are more heavily methylated in cells in which they are not expressed.
Some proteins that bind to methylated DNA also interact with histone deacetylation enzymes,
reinforcing the transcription repression.
DNA methylation may reinforce gene regulatory decisions of early development as enzymes act
during DNA replication to methylate daughter DNA strands and pass on methylation records.
Such methylation patterns account for genomic imprinting in mammals in which either
maternal or paternal allele of certain genes is permanently regulated.
Epigenetics
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Epigenetics means “above the genome”
It is a second “code” that “sits on top” of our regular DNA and controls how are genes
are expressed by turning them on and off with altered consequences
This 2nd code consists of gene regulators that turn genes on or off. (e.g. methylation turns genes
off)
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Epigenic inheritance is the transmission of traits by mechanisms that do not involve changes in
DNA sequences, such as chromatin modifications that affect gene expression in future
generations of cells.
Regulation of Transcription Initiation
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A typical gene consists of a promoter sequence, where a transcription initiation complex that
includes RNA polymerase II attaches, and a sequence of introns interspersed among the coding
exons.
After transcription, RNA processing of the pre-mRNA removes the introns and adds a 5’ cap and
a poly-A tail at the 3’ end.
Control elements are noncoding sequences to which transcription factors bind that help
regulate transcription.
mRNA processing
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Alternative mRNA splicing may produce different mRNA molecules when regulatory proteins
control which segments of the primary transcript are chosen as introns and which are chosen as
exons.
Introns are taken out, exons are joined together by ligase
Provides diversity and variation in terms of proteins
mRNA Degradation
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In contrast to prokaryotic mRNA that is degraded after a few minutes, eukaryotic mRNA can last
hours or even weeks.
The hydrolysis of mRNA by nucleases is usually preceded by the enzymatic shortening of the
poly-A tail and removal of the 5’ cap.
Poly A tail and methyl-guanine cap help to prevent mRNA degradation
Initiation of Translation
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Translation may begin when enzymes add more A nucleotides to poly-A tails or when translation
initiation factors are activated following fertilization
The translation of eukaryotic mRNA can be delayed by the binding of regulatory proteins to the
5’ UTR (untranslated region) that prevent ribosome binding
A great deal of mRNA is synthesized and stored in egg cells
Protein Processing and Degradation
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Following translation, polypeptides are often cleaved or chemical groups added to yield an
active protein. Some proteins must be transported to target locations.
Selective degredation of proteins may serve as a control mechanism in the cell.
Molecules of the protein ubiquitin are added to mark proteins for destruction
Proteasomes recognize and degrade the marked proteins
Cancer results from genetic changes that affect cell control
Types of Genes Associated with Cancer
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Chemical carcinogens, X-rays, or certain viruses most often cause the changes in the genes that
regulate cell growth and division that lead to cancer.
Viruses that can cause cancer in animals are called tumor viruses.
Mutations in tumor-suppressor genes can contribute to the onset of cancer when they result in
a decrease in the activity of proteins that prevent uncontrolled cell growth
Chapter 20: Biotechnology
The DNA Toolbox
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DNA technology began with techniques for making recombinant DNA, which combines
nucleotide sequences from different organisms or species into the same DNA molecule.
Genetic engineering, the direct manipulation of genetic material for practical purposes, has
begun a revolution in biotechnology.
Biotechnology is the use of living organisms or their components to manufacture desirable
produces, and new advances have resulted in hundreds of new products and applications.
Stanley Cohen and Herbert Boyer (1980s)- demonstrated that the gene for frog ribosomal RNA
could be transferred into bacterial cells and expressed by them
DNA Cloning
An Overview of DNA Cloning
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The “gene of interest” is the gene being placed into the bacterial/eukaryotic cell
Plasmids of bacterial cells can be used to clone DNA
Recombinant DNA can be made by inserting foreign DNA into plasmids.
These plasmids are put back into bacterial cells where they will replicate as the recombinant
bacteria reproduce to form clones of identical cells.
2 purposes of gene cloning:
1) Provides multiple copies of a particular gene
2) Produces a protein product
Using Restriction Enzymes to Make Recombinant DNA
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Restriction enzymes “cut” DNA at specific restriction sites, short nucleotide sequences.
- Common restriction enzymes: EcoRI, HindIII, BamHI
- In bacteria, restriction enzymes defend against viruses by cutting up the foreign DNA
- The cell protects its own DNA from restriction by methylating nucleotide bases within its
own restriction sequences.
Restriction enzymes cut the sugar-phosphate backbone of DNA in specific staggered patterns,
forming sticky ends on both sides of the resulting restriction fragment
DNA from different sources can be combined in the laboratory when the DNA is cut by the same
restriction enzyme  it needs to be cut by the same restriction enzyme so that sticky ends are
complementary and can form base pairs
DNA ligase is used to seal the strands together.
Cloning a Eukaryotic Gene in a Bacterial Plasmid
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Cloning vectors are DNA molecules that can move foreign DNA into a cell and replicate there.
Recombinant plasmids returned to bacterial cells will replicate the foreign DNA as the bacteria
reproduce.
Producing Clones of Cells Carrying Recombinant Plasmids
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The plasmid method of gene cloning involves treating plasmids containing an antibiotic-resistant
gene with a restriction enzyme that cuts the DNA ring at a single restriction site and disrupts a
gene
The clipped plasmids are mixed with the DNA containing the gene of interest, which has been
treated with the same restriction enzyme
- The sticky ends of the cut plasmids are complementary to the sticky ends of the DNA with
the gene of interest
- The sticky ends form hydrogen bonds with each other, and DNA ligase seals the
recombinant molecules
The challenge is getting recombinant plasmid back into the bacteria.
There are different processes for inserting the recombinant plasmid:
1. Electrophoration – an electric pulse briefly opens holes in the plasma membrane
through which DNA can enter
2. Gene gun – injection of plasmids into cells using microscopically thin needles
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3. Bacterial transformation – the bacteria cell’s DNA picks up the DNA of the
recombinant plasmid
*Competent cells are likely to take up a plasmid (18–20% chance)
For example: E.coli (control-not transformed) vs. E.coli with plasmid (hopefully transformed)
-The plasmid has an ampicillin resistance factor
-If placed in ampicillin (an antibiotic):
*E.coli cells will not grow  the ampicillin kills them
*E.coli cells that have been transformed with recombinant plasmids will GROW
 they will not be killed because the plasmid has an amp resistance factor
After it is proved that E.coli cells have been transformed, scientists take the transformed cells
and grow them in a culture flask.
- Fermentation tanks are usually glass, metal or plastic tanks used in the pharmaceutical
industry for the growth of specialized pure cultures of bacteria, fungi and yeast, and the
production of enzymes and drugs
Storing Cloned Genes in DNA Libraries
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An organism’s DNA can be cut into thousands of pieces with restriction enzymes and inserted
into plasmids.
A genomic library is the collection of the thousands of clones of bacteria that contain
recombinant plasmids.
Bacteriophages are also used as vectors for creating genomic libraries.
- DNA fragments are spliced into phage DNA, which is packaged into capsids and used to
infect bacteria. The production of new phage clones the foreign DNA.
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Cloning vectors also include bacterial artificial chromosomes (BACs) which are large plasmids
that carry longer inserts that standard plasmid or phage vectors.
BAC clones are often stored in multiwelled plastic plates.
A partial genomic library can be produced using the mRNA molecules isolated from a cell. Using
reverse transcriptase from retroviruses, mRNA is used as a template to produce complementary
DNA (cDNA)
Restriction sites are added to the ends and the cDNA is inserted into vector DNA, which is used
to create a cDNA library.
Screening a Library for Clones Carrying a Gene of Interest
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A genomic library can consist of thousands of bacterial colonies, so how do you find the specific
gene?
The colonies with recombinant plasmids may be tested to find the ones that contain the gene of
interest by nucleic acid hybridization, using a nucleic acid probe that has complementary
sequences to segments of the gene.
Nucleic acid probes identify a specific gene.
- They have complementary sequences to segments of the gene, and this complementary
molecule is labeled with a radioactive isotope or fluorescent dye.
- It’s called a probe because it can be used to find the gene.
To find the bacterial clone that holds the gene:
- DNA is obtained from each colony of bacteria and treated to separate the DNA strands.
- The probe is then mixed with the DNA strands and it only bonds with the recombinant DNA
with a complementary base sequence.
Once you have figured out which bacterial colony in the library contains the gene of interest,
you can grow these bacteria in larger amounts.
Polymerase Chain Reaction (PCR)
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The polymerase chain reaction (PCR) amplifies DNA by taking a small segment and copying it,
increasing the amount of DNA
- It can produce billions of copies of a section of DNA in only a few hours.
PCR is often used prior to cloning a gene in cells.
- By amplifying the gene prior to cloning, the later task of identifying clones carrying the
desired gene is simplified.
Even though PCR produces so many copies so fast, gene cloning is a necessary process.
- There is a limit to the number of accurate copies that can be made due to the accumulation
of relatively rare copying errors.
- Large quantities of a gene are better prepared by DNA cloning in cells.
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
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Differences between prokaryotic and eukaryotic mechanisms for gene expression can be
overcome by using an expression vector
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An expression vector is a cloning vector that has an active prokaryotic promoter just
upstream from the eukaryotic gene insertion site.
Using a cDNA gene removes the problem of long introns, especially since bacteria don’t have
RNA-splicing machinery
Eukaryotic Cloning and Expression Systems
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Yeast cells are eukaryotic hosts used to clone and express eukaryotic genes
- They are easy to grow and have plasmids that serve as vectors.
Yeast artificial chromosomes (YACs) carry foreign DNA and contain an origin for DNA
replication, a centromere, and 2 telomeres
- These vectors behave normally in mitosis and are able to clone large pieces of DNA
Plant and animal cells in culture can serve as hosts and may be necessary when a protein must
be modified following translation
DNA Technology
Gel electrophoresis
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Many of the methods for analyzing and comparing DNA involve gel electrophoresis, a technique
that separates nucleic acids and proteins on the basis of their SIZE and electrical CHARGE
Gel electrophoresis is used to separate fragments of DNA according to their size
Due to the negative charge of their phosphate groups, DNA molecules are negative.
As a result, when it is placed in the electric field of the gel, the DNA will migrate toward the
positive pole.
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The process:
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The gel used in gel electrophoresis is called agarose. This is very similar to the agar used in
microbiology and is like Jello. When the agarose is poured into the tray, a device called a “comb”
is in place. When the agarose gels, or solidifies, the comb is removed, leaving small holes or
“wells” in the gel. Different sized fragments of DNA are loaded into wells with capillary tubes.
The electric current is turned on and the DNA begins to move to the positive end.
The rate depends on SIZE:
-The smaller the DNA  the faster and further it will move
-The longer the DNA  the slower and less far it will move
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Restriction Analysis
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Restriction fragment analysis is the process used to produce DNA fingerprints.
Another name for this is RFLP (=Restriction Fragment Length Polymorphisms)
In the process of DNA fingerprinting:
1. A small sample of human DNA is cut with a particular restriction enzyme
2. The resulting restriction fragments are separated by gel electrophoresis
 This produces a series of characteristic patterns of bands (called a DNA FINGERRPINT) that show
the lengths of the fragments.
- A unique pattern of bands (=DNA FINGERPRINT) can be established for each individual in the
world.
20.3 – Cloning organisms may lead to production of stem cells
I don’t know how much detail we have to know, so just read over it.
20.4 – The practical applications of DNA technology
Medical Applications
Diagnosis of Diseases
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DNA technology is identifying genes responsible for genetic diseases; it is hoped this will lead to
new ways to diagnose, treat, and even prevent those disorders.
DNA microarray assays allow comparisons of gene expression in healthy and diseased tissues.
Even if a gene hasn’t yet been cloned, a disease allele may be diagnosed when closely linked
with a genetic marker, a sequence of variation, or polymorphism, found among individuals
- Single nucleotide polymorphisms (SNPs) are variations that occur in at least 1% of humans.
- When such variations change a restriction site, they result in restriction fragment length
polymorphisms (RFLPs).
Human Gene Therapy
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Gene therapy may provide the means for correcting genetic disorders in individuals by replacing
or supplementing defective genes.
New genes would be introduced into somatic cells of the affected tissue. For the correction to
be permanent, the cells must be actively reproducing within the body, like bone marrow cells.
Pharmaceutical Products
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Many pharmaceutical proteins are produced using biotechnology.
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Cancer: New approaches to cancer treatments include genetically engineered proteins that
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block a protein necessary for a tumor cell’s survival.
Vaccines: Recombinant DNA techniques can be used to make vaccines that consist of surface
molecules on pathogens, or to modify the genome of a pathogen as to attenuate it (make it
nonpathogenic) so it can be used as a vaccine
Forensic Evidence and Genetic Profiles
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Criminal cases: RFLP analysis has been used in criminal cases to compare the genetic profile of
a suspect to that of crime DNA samples.
Environmental Cleanup
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Mining: Genetically engineered microorganisms that are able to extract heavy metals, such as
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copper, may be important in mining and cleaning up mining waste.
Fuel: Biofuels use microorganisms to ferment plant sugars to “bioethanol” or plant oils to
“biodiesel”
Agricultural Applications
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Animal husbandry: DNA technology allows scientists to produce transgenic animals which
speed up the selective breeding process. Transgenic animals (“Pharm” animals) are produced by
injecting a foreign gene into egg cells fertilized in vitro (=in a test tube). The eggs are then
transplanted into surrogate mothers.