Transcript Targeted Fluorescent Reporters: Additional slides

Chapter 18
Points of Emphasis
Know:
1.
all the bold-faced terms. You will be responsible for a good
portion of the chapter. I will emphasize HIV and gene
expression. The rest is probably up to you.
The difference between the lytic and lysogenic cycles and be
able to described them in an essay.
3.
The Genetics of Bacteria: know the basic concepts
4.
Control of Gene Expression or Operons: know very, very well.
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Figure 18.19 Regulation of a metabolic pathway
Feedback Inhibition
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Basic Components of an Operon
1. Operator
a. “Within” the promoter
b. Serves as a binding site for proteins to allow or disallow
transcription.
c. RNA polymerase may or may not be able to bind to the
promoter depending on the “state” of the operator
2. Promoter: where the RNA polymerase will bind
3. Operon: consists of the promoter, operator and the genes that are
transcribed as a unit, one right after another.
4. Regulatory Gene: produces a repressor protein
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Figure 18.20a The trp operon: regulated synthesis of repressible enzymes
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Figure 18.20b The trp operon: regulated synthesis of repressible enzymes (Layer 1)
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Figure 18.20b The trp operon: regulated synthesis of repressible enzymes (Layer 2)
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Figure 18.21a The lac operon: regulated synthesis of inducible enzymes
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Figure 18.21b The lac operon: regulated synthesis of inducible enzymes
Breaks down lactose into
glucose and galactose
Membrane protein that
transports lactose into the
cell
Unknown function
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Figure 18.22a Positive Regulation: cAMP receptor protein (CRP)
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Figure 18.22b Positive control: cAMP receptor protein
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Figure 18.1 Comparing the size of a virus, a bacterium, and a eukaryotic cell
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Figure 18.2 Viral structure
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Figure 18.3 A simplified viral reproductive cycle
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Figure 18.4 The lytic cycle of phage T4
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Figure 18.02x2 Phages
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Figure 18.5 The lysogenic and lytic reproductive cycles of phage , a temperate phage
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Figure 18.6 The reproductive cycle of an enveloped virus
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Figure 18.7 HIV, a retrovirus
gp41: binds the
glycoprotein to the
viral envelope
gp120: glycoprotein
p17: Matrix proteins
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Time, 2/16/98, pg 64
AIDS Notes I
When did AIDS begin?

A blood sample taken in 1959, frozen, and eventually
studied in the mid-1980s was discovered to contain HIV.
This is the oldest specimen of the AIDS virus and may help
explain how the virus made the leap from animals
(monkeys or chimpanzees) to humans.

It is thought the AIDS virus has been around since early
1950’s or even the 40’s.

This 1959 virus seems to be, genetically, at the root of
several other subtypes.

The theory is that AIDS started through contact with
infected monkeys in a remote area, maybe Kinshasa of
Central Africa, and spread through rest of population
through urbanization and mass inoculations.
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SJ Mercury 2/1/99
AIDS Notes II
When did AIDS begin?

Alabama researchers showed that the most prevalent form
of AIDS virus almost certainly passed to humans from a
subspecies of chimpanzees, Pan troglodytes troglodytes.

Chimpanzees can live with the virus without being ill

Univ. of Alabama, Birmingham researchers concluded the
virus passed to humans three separate times in a small
region of West Equatorial Africa about 50 years ago.

Transmission most likely occurred during the butchering of
animals, which were subsequently eaten.

Other sources of transmission: sootey mangabee monkey
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AIDS Notes III
 RNA core (retrovirus): about 2000 copies of the viral protein, p24, surround
two single strands of HIV RNA, each containing a copy of the virus’s 9
genes. Three of these genes, gag, pol and env make the new viral
proteins
 Viral envelope is the outermost membrane: consists of lipids and proteins.
One protein, called Envelope protein or Env protein protrudes from the
surface. Env protein consists of a cap made of glycoprotein (gp120) and
a stem, of gp41 molecules.
 protein capsid that envelopes the RNA
 infects the T cells, specifically the helper T cells, of the immune system which
stimulate cytotoxic T cells and B cells that act against HIV
 two membranes must fuse for the viral RNA to enter the HT cell: the viral
protein envelope and the cellular membrane.
 HT cells have at least two receptors for binding to HIV: CD4 receptor to
which the envelope binds and a CD26 that triggers a change in the CD4
receptor so the two membranes can fuse.
 When mouse cells were altered to express CD4 receptors but not CD26, 21
CD4
by itself would not allow fusion. “Neener. . . Neener. . . Neener!”
AIDS Notes III, cont’d
 How does CD4 normally function? It stabilizes the interaction of a HT cell
with another cell called an antigen-presenting cell (APC) and this
increases the stimulation of cytotoxic T cells and B cells in the immune
response.
HIV weakens this stability so less stimulation occurs.
 Why not does add CD4 “floaters” to the blood so HIV will bind to them all?
The CD4 floaters will bind to APC’s before HT cells do and lessen an
immune response.
 Why can’t the immune system fight off HIV?
Mutations occur too fast so few HIV molecules carry the antigen that
was recognized by the immune system, eliciting a response.
 Why are antibodies to AIDS virus found in fetus?
antibodies from the mother can cross the placenta. Antibodies have
evolved to cross the placenta so you can have an immune system when
you are young. So the infant can have the antibodies but not the virus.
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AIDS Notes III, cont’d
 What are these protease inhibitors that seem to be stopping the virus?
the virus makes a huge protein, I call it the “mother protein,” that
actually gets cut into several other functioning proteins.
Pro 1
Pro 2
Pro 3
Pro 4
Pro 5
Each protein is cleaved by a “protease.:
Pro 3
Pro 1
Pro 5
Pro 2
Pro 4
All now can help replicate HIV
A protease inhibitor prevents the cleaving of the “mother protein” into its subunits.
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Science News, Vol 160
AIDS Notes, cont’d
Rwandan patients show unusual HIV

Many Rwandans who have had HIV for many years
without getting AIDS appear to be harboring a rare
mutation.

Their T-cell count did not fall drastically during testing
years.

Rare mutation in the gene encoding a glycoprotein called
gp120 in the virus’ outer surface.

gp120 is involved in the binding of the virus to a T cell
during infection. No binding. . . No infection.
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AIDS Notes, cont’d
AIDS Vaccine

Shots in the Dark: The Wayward Search for an AIDS
Vaccine, ISBN 0-393-05027-0, 2001. Describes the dismal
attempt/funding for this effort.

Recent vaccine(AIDS-vax from VaxGen of Brisbane)
failure: 2546 injection drug abuses in Thailand were in this
3 year study. Those who received the vaccine were just as
likely to be infected as those who got dummy shots.
 15 years to develop
 large trial in Europe and North America was also
disappointing.
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AIDS Notes, cont’d
Three-Drug Therapy or a “Cocktail”

Combo of a protease inhibitor, reverse transcriptase inhibitor
(AZT) and a similar drug (3TC). This cocktail stops the
virus even if it does mutate. Its unlikely it would mutate at
all sites where all 3 drugs act.
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Chemical and Engineering
News, 10/27/2003, pg 10
AIDS Notes, cont’d
New Class of HIV Inhibitor, PA-457

Maturation Inhibitor: this drug acts by disrupting a late
stage in viral maturation.

HIV has a capsid protein that envelopes its RNA creating a
viral core structure.

This capsid protein is called “Gag protein.”

The treated viral particles develop defective core structures
and are noninfectious.

Preclinical studies indicate PA-457 is orally bioavailable and
works with approved HIV drugs.

Especially important for those infected individuals where
drug resistance has developed.
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