Download The Lytic Cycle

Biotechnology
• The use of living organisms or their components
to perform practical tasks.
• Ex: the use of bacteria to digest oil spills.
Restrictive Enzymes
• Cut DNA at specific nucleotide
sequences called “restriction sites”.
• Used to "cut and splice" DNA.
• Obtained from bacteria.
• Ex. EcoRI and Hind III
Plasmids
• Used extensively in Biotechnology
and Recombinant DNA.
• Serve as a “vehicle” for transporting
genes.
• Comment – other “vehicles” are used
in other methods
Steps for Plasmid Use
1. Get the DNA for the trait.
2. Insert DNA into the plasmid.
3. Bacterial Transformation.
4. Identification of the new
trait.
Insertion
• Placing foreign DNA into a plasmid.
• Open plasmid with enzymes to create “sticky
ends”.
• Splice the new DNA and plasmid together.
Transformation
• Placing the plasmid into a bacterial cell.
Methods
• Temperature shock & salt treatment
• Electric current
• Injection
Identification
• Screening the altered cells for the desired gene.
• Ex: Antibiotic sensitivity or the expression of a
“new” trait (color, glowing etc.).
Example Applications
1. Insulin
2. Human Growth Hormone
3. Other Proteins
Comment
• Gene can’t be above a certain size
(12 kb) or a plasmid won’t work.
• mRNA must not need splicing to
remove introns.
DNA Sources
1. Organism - use a section of their chromosome.
2. cDNA - Complementary DNA - created copy of
DNA from the mRNA transcript to avoid introns.
Uses reverse transcriptase.
PCR
• Polymerase Chain Reaction
• Method for making many copies of a specific
segment of DNA.
• Also called “DNA Amplification”.
PCR - Method
1. Separate strands by heating (denature the
DNA).
2. Cool slightly.
3. Build new strand from primers and nucleotides.
4. Repeat.
Importance - PCR
• Can amplify any DNA with as little as one
original copy.
• Very useful in a variety of techniques and tests.
Chapter 19
Viruses
PowerPoint® Lecture Presentations for
Biology
Eighth Edition
Neil Campbell and Jane Reece
Lectures by Chris Romero, updated by Erin Barley with contributions from Joan Sharp
Copyright © 2008 Pearson Education, Inc., publishing as Pearson Benjamin Cummings
Overview: A Borrowed Life
• Viruses called bacteriophages can infect and
set in motion a genetic takeover of bacteria,
such as Escherichia coli
• Viruses lead “a kind of borrowed life” between
life-forms and chemicals
• The origins of molecular biology lie in early
studies of viruses that infect bacteria
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Fig. 19-1
0.5 µm
Structure of Viruses
• Viruses are not cells
• Viruses are very small infectious particles
consisting of nucleic acid enclosed in a protein
coat and, in some cases, a membranous
envelope
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Viral Genomes
• Viral genomes may consist of either
– Double- or single-stranded DNA, or
– Double- or single-stranded RNA
• Depending on its type of nucleic acid, a virus is
called a DNA virus or an RNA virus
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Capsids and Envelopes
• A capsid is the protein shell that encloses the
viral genome
• Capsids are built from protein subunits called
capsomeres
• A capsid can have various structures
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Fig. 19-3
RNA
DNA
Capsomere
Membranous
envelope
RNA
Head
DNA
Capsid
Tail
sheath
Capsomere
of capsid
Glycoproteins
Glycoprotein
18  250 nm
70–90 nm (diameter) 80–200 nm (diameter)
20 nm
50 nm
(a) Tobacco mosaic (b) Adenoviruses
virus
50 nm
Tail
fiber
80  225 nm
50 nm
(c) Influenza viruses (d) Bacteriophage T4
• Some viruses have membranous envelopes
that help them infect hosts
• These viral envelopes surround the capsids of
influenza viruses and many other viruses found
in animals
• Viral envelopes, which are derived from the
host cell’s membrane, contain a combination of
viral and host cell molecules
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
• Bacteriophages, also called phages, are
viruses that infect bacteria
• They have the most complex capsids found
among viruses
• Phages have an elongated capsid head that
encloses their DNA
• A protein tail piece attaches the phage to the
host and injects the phage DNA inside
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
General Features of Viral Reproductive Cycles
• Once a viral genome has entered a cell, the
cell begins to manufacture viral proteins
• The virus makes use of host enzymes,
ribosomes, tRNAs, amino acids, ATP, and
other molecules
Animation: Simplified Viral Reproductive Cycle
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Fig. 19-4
VIRUS
1 Entry and
DNA
uncoating
Capsid
3 Transcription
and manufacture
of capsid proteins
2 Replication
HOST CELL
Viral DNA
mRNA
Viral DNA
Capsid
proteins
4 Self-assembly of
new virus particles
and their exit from
the cell
Reproductive Cycles of Phages
• Phages are the best understood of all viruses
• Phages have two reproductive mechanisms:
the lytic cycle and the lysogenic cycle
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
The Lytic Cycle
• The lytic cycle is a phage reproductive cycle
that culminates in the death of the host cell
• The lytic cycle 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
Animation: Phage T4 Lytic Cycle
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Fig. 19-5-5
1 Attachment
2 Entry of phage
5 Release
DNA and
degradation of
host DNA
Phage assembly
4 Assembly
3 Synthesis of viral
genomes and
proteins
Head
Tail Tail fibers
The Lysogenic Cycle
• The lysogenic cycle replicates the phage
genome without destroying the host
• The viral DNA molecule is incorporated 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 the copies to daughter cells
Animation: Phage Lambda Lysogenic and Lytic Cycles
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
• An environmental signal can trigger the virus
genome to exit the bacterial chromosome and
switch to the lytic mode
• Phages that use both the lytic and lysogenic
cycles are called temperate phages
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Fig. 19-6
Phage
DNA
Daughter cell
with prophage
The phage injects its DNA.
Cell divisions
produce
population of
bacteria infected
with the prophage.
Phage DNA
circularizes.
Phage
Bacterial
chromosome
Occasionally, a prophage
exits the bacterial
chromosome,
initiating a lytic cycle.
Lytic cycle
Lysogenic cycle
The bacterium reproduces,
copying the prophage and
transmitting it to daughter cells.
The cell lyses, releasing phages.
Lytic cycle
is induced
or
New phage DNA and proteins
are synthesized and
assembled into phages.
Lysogenic cycle
is entered
Prophage
Phage DNA integrates into
the bacterial chromosome,
becoming a prophage.
Fig. 19-UN1
Phage
DNA
The phage attaches to a
host cell and injects its DNA
Bacterial
chromosome
Lytic cycle
• Virulent or temperate phage
• Destruction of host DNA
• Production of new phages
• Lysis of host cell causes release
of progeny phages
Prophage
Lysogenic cycle
• Temperate phage only
• Genome integrates into bacterial
chromosome as prophage, which
(1) is replicated and passed on to
daughter cells and
(2) can be induced to leave the
chromosome and initiate a lytic cycle
Reproductive Cycles of Animal Viruses
• There are two key variables used to classify
viruses that infect animals:
– DNA or RNA?
– Single-stranded or double-stranded?
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Table 19-1a
Table 19-1b
Viral Envelopes
• Many viruses that infect animals have a
membranous envelope
• Viral glycoproteins on the envelope bind to
specific receptor molecules on the surface of a
host cell
• Some viral envelopes are formed from the host
cell’s plasma membrane as the viral capsids
exit
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Fig. 19-7
Capsid and viral genome
enter the cell
Capsid
RNA
HOST CELL
Envelope (with
glycoproteins)
Viral genome (RNA)
Template
mRNA
Capsid
proteins
ER
Glycoproteins
Copy of
genome (RNA)
New virus
RNA as Viral Genetic Material
• The broadest variety of RNA genomes is found
in viruses that infect animals
• Retroviruses use reverse transcriptase to
copy their RNA genome into DNA
• HIV (human immunodeficiency virus) is the
retrovirus that causes AIDS (acquired
immunodeficiency syndrome)
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Fig. 19-8
Glycoprotein
Viral envelope
Capsid
Reverse
transcriptase
HIV
RNA (two
identical
strands)
HIV
Membrane of
white blood cell
HOST CELL
Reverse
transcriptase
Viral RNA
RNA-DNA
hybrid
0.25 µm
DNA
HIV entering a cell
NUCLEUS
Provirus
Chromosomal
DNA
RNA genome
for the
next viral
generation
New virus
New HIV leaving a cell
mRNA
Fig. 19-8a
Glycoprotein
Viral envelope
Capsid
Reverse
transcriptase
RNA (two
identical
strands)
HOST CELL
HIV
Reverse
transcriptase
Viral RNA
RNA-DNA
hybrid
DNA
NUCLEUS
Provirus
Chromosomal
DNA
RNA genome
for the
next viral
generation
New virus
mRNA
Chapter 18
Regulation of Gene Expression
Regulation of Gene Expression
Important for cellular control and
differentiation.
Understanding “expression” is a “hot” area
in Biology.
General Mechanisms
1. Regulate Gene Expression
2. Regulate Protein Activity
Operon Structure
1. Regulatory Gene
2. Operon Area
Gene Structures
Regulatory Gene
Makes Repressor Protein which may bind to the operator.
Repressor protein blocks transcription.
Promoter
Attachment sequence on the DNA for RNA polymerase to start
transcription.
Operator
The "Switch”, binding site for Repressor Protein.
If blocked, will not permit RNA polymerase to pass, preventing
transcription.
Gene Structures
Structural Genes
Make the enzymes for the metabolic pathway.
Lac Operon
For digesting Lactose.
Inducible Operon - only works (on) when the substrate (lactose) is
present.
If no Lactose
Repressor binds to operator.
Operon is "off”, no transcription,
no enzymes made
If Lactose is absent
If Lactose is present
Repressor binds to Lactose instead of operator.
Operon is "on”, transcription occurs, enzymes are made.
If Lactose is present
Enzymes
Digest Lactose.
When enough Lactose is digested, the Repressor can bind to the
operator and switch the Operon "off”.
Net Result
The cell only makes the Lactose digestive enzymes when the substrate
is present, saving time and energy.
Animation
http://www.biostudio.com/d_%20Lac%20Operon.htm
trp Operon
Makes Tryptophan.
Repressible Operon.
If no Tryptophan
Repressor protein is inactive,
“Normal” state for the cell.
Operon "on” Tryptophan made.
Tryptophan absent
If Tryptophan present
Repressor protein is active,
no enzymes.
Result - no Tryptophan made.
Operon "off”, no transcription,
If Tryptophan present
Repressible Operons
Are examples of Feedback Inhibition.
Result - keeps the substrate at a constant level.
DNA Fingerprinting
Forensic science with DNA
Cut DNA
Restriction Enzyme
– Cuts DNA at specific site
– Found in bacteria as defense against viruses
Cut DNA
Recall we each
have unique
sequence
Sort DNA by size
Load DNA into agarose gel
Apply voltage
– DNA Negative –
which way will go?
Sort DNA by size
Small pieces can move faster, so they move farther
Pouring a gel
Loading the gel
Using a micropipette, load DNA into each well
Use clean tip for each sample
Don’t poke too deep in the gel and puncture the well
Ideally the DNA is
released in the
middle of the well
What we will do
Restriction enzyme
makes different
sized pieces
Voltage will separate
pieces by size
Individual
One
Individual
Two
Individual
One
Individual
Two
Uses of DNA fingerprinting
Genetic disease testing
Paternity cases
Forensic cases
Identity cases
Diagnose inherited disorders
Determine if individual has
genetic makeup to develop
genetic disease
Develop cures
Find DNA sequences shared by individuals with genetic disease
Locates gene responsible
Can determine what does wrong, how to fix
Determine paternity
Prove child belongs to parents
Determine if man is father
Determine paternity
Recall child gets half
genetic material from
mom, half from dad
Each child fragment should
match one of their
parents
Paternity
Which male
might be
the father?
A
B
C
D
E
A mother
B male 1
C male 2
D child
E standards
Determine paternity
Mary is mother.
Bob or Larry might be the father.
Who is the father?
Explain where EACH
child band came from.
Forensics
Link suspects to biological
evidence
– Blood
– Semen
– Hair
Forensics
Look for a match between suspects and evidence
Shows evidence came
from the individual
Does this always
mean someone is
guilty?
Sample
Who’s blood was on the victim’s clothes?
Personal identification
DNA samples collected from family, military personal
Used to identify casualties