Agenda 11/28/11
• Do DNA modeling – get through all the building
parts and then each person answers question
sheet (questions can be done for homework)
• I check Ch. 17 & 18 Notes and SQ while you do
this
• 5 min left- break down and put away except I
keep one good group for demo tomorrow
• Homework –
Finish activity questions tonight!!!
Quiz Fri on Ch. 16 and 17
Ch. 19 Notes and SQ due next Monday
Me- pour plates for transformation
Agenda
11/29/11
Start with next slide as Intro
•
• Ch. 16 and 17 highlights with guided notes – focus on pics and
especially new material, use models to demo and go over
yesterday’s answers between 16&17 – will likely discuss mutations
another day
• Ch. 17 is one of the top 5 chapters you must
know to perform well on the AP exam!!!
• Know these: !!!!
– DNA DNA=
– DNA RNA =
– RNA  protein =
Homework –
•Quiz Fri on Ch. 16, 17
•Ch. 19 Notes and SQ due next Monday
•Read Transformation Lab 6A and do prelab worksheet to
discuss
tomorrow!!!
Me-streak starter
plates at lunch – 2 per class
• Transcription,
RNA
processing, and
translation are
the processes
that link DNA
sequences to
the synthesis of
a specific
polypeptide
chain.
Fig. 17.25
•A gene is a region of DNA whose final product is either a
polypeptide or an RNA molecule.
Agenda 11/30/11
• Finish guided notes from yesterday
• Discuss plasmids and bacterial genomes
• Intro Transformation lab (go over prelab
worksheet) and assign roles for tomorrow
Homework –
Quiz Fri on Ch. 16, 17
•Ch. 19 Notes and SQ due next Monday
•Go over 6A in manual and quickguide and
know procedure well, and study for quiz!
Me- set up lab group stuff
• Transcription,
RNA
processing, and
translation are
the processes
that link DNA
sequences to
the synthesis of
a specific
polypeptide
chain.
Fig. 17.25
•A gene is a region of DNA whose final product is either a
polypeptide or an RNA molecule.
• A tRNA molecule consists of a strand of
about 80 nucleotides that folds back on
itself to form a three-dimensional structure.
• 45 tRNA’s exist (not 61) because of
wobble
Fig. 17.13
• Recent advances in our understanding of
the structure of the ribosome strongly
supports the hypothesis that rRNA, not
protein, carries out the ribosome’s
functions.
– RNA is the main constituent at the inter-face between
the two subunits and of the A and P sites.
– rRNA is the catalyst for
peptide bond formation
Fig. 17.16
• Translation can be divided into three
stages:
initiation
elongation
termination
• All three phase require protein “factors”
that aid in the translation process.
• Both initiation and chain elongation require
energy provided by the hydrolysis of
GTP.
• Initiation brings together mRNA, a tRNA
with the first amino acid, and the two
ribosomal subunits.
– First, a small ribosomal subunit binds with mRNA and a
special initiator tRNA, which carries methionine and
attaches to the start codon.
– Initiation factors bring in the large subunit such that the
initiator tRNA occupies the P site.
Fig. 17.17
Elongation – 3 steps that continue codon by
codon to add amino acids until the
polypeptide chain is completed.
Fig. 17.18
• Termination occurs when one of the three
stop codons reaches the A site.
• A release factor binds to the stop codon
and hydrolyzes the bond between the
polypeptide and its tRNA in the P site.
• This frees the polypeptide and the
translation complex disassembles.
Fig. 17.19
• Typically a single mRNA is used to make many
copies of a polypeptide simultaneously.
• Multiple ribosomes, polyribosomes, may trail
along the same mRNA.
• A ribosome requires less than a minute to translate
an average-sized mRNA into a polypeptide.
Fig. 17.20
• While bound and free ribosomes are identical in
structure, their location depends on the type of
protein that they are synthesizing.
• Translation in all ribosomes begins in the cytosol,
but a polypeptide destined for the endomembrane
system or for export has a specific signal peptide
region at or near the leading end.
– This consists of a sequence of about 20 amino acids.
• A signal recognition particle (SRP) binds to the
signal peptide and attaches it and its ribosome to a
receptor protein in the ER membrane.
– The SRP consists of a protein-RNA complex.
Fig. 17.21
• The diverse
functions of
RNA range
from structural
to
informational
to catalytic.
• COMPARING EUK & PROK
TRANSCRIPTION & TRANSLATION
• E – Compartmentalized with Transcription in nucleus,
Translation in Cytoplasm and extensive RNA processing in
between, also complicated mechanisms for targeting
proteins to the appropriate organelle.
• P- no nuclei so transcription and translation can occur
simultaneously - Ribosomes attach to the leading end of a
mRNA molecule while transcription is still in progress,
protein diffuses to where needed
Fig. 17.2a
INITIATION AND ELONGATION
E - The promoter also includes a binding site for RNA
polymerase several dozen nucleotides upstream of
the start point.
P - In prokaryotes, RNA polymerase can recognize and
bind directly to the promoter region.
E - eukaryotes have three RNA polymerases (I,
II, and III) in their nuclei.
– RNA polymerase II is used for mRNA synthesis.
P - Bacteria have a single type of RNA
polymerase that synthesizes all RNA molecules.
• STEP 3 - TERMINATION
– E - RNA polymerase continues for hundreds of
nucleotides past the terminator sequence,
AAUAAA.
– P - RNA polymerase stops transcription right at
the end of the terminator.
• Both the RNA and DNA are then released.
RIBOSOMES
• While very similar in structure and
function, prokaryotic and eukaryotic
ribosomes have enough differences that
certain antibiotic drugs (like tetracycline)
can paralyze prokaryotic ribosomes
without inhibiting eukaryotic ribosomes.
• More on Bacteria…
1) Let’s draw a bacteria cell with nucleoid
and plasmid
2) How do bacterial cells divide?
• Bacterial cells
divide by binary
fission.
• This is preceded
by replication of
the bacterial
chromosome
from a single
origin of
replication.
Fig. 18.11
• Bacteria proliferate very rapidly
- In a lab, one cell divides after 20 min
producing a colony of 107 to 108 bacteria in
as little as 12 hours.
– In the human colon, E. coli reproduces rapidly
enough to replace the 2 x 1010 bacteria lost
each day in feces.
• Through binary fission, most of the bacteria
in a colony are genetically identical to the
parent cell.
• New mutations, though individually rare,
can have a significant impact on genetic
diversity when reproductive rates are very
high because of short generation spans.
• Individual bacteria that are genetically well
equipped for the local environment clone
themselves more prolifically than do less fit
individuals.
• In contrast, organisms with slower
reproduction rates (like humans) create
most genetic variation not by novel alleles
produced through mutation, but by sexual
recombination of existing alleles.
2. Genetic recombination
produces new bacterial strains
• In addition to mutations, genetic
recombination generates diversity within
bacterial populations.
• Recombination occurs through three
processes:
transformation
transduction
conjugation
• Transformation is the alteration of a
bacterial cell’s genotype by the uptake of
naked, foreign DNA from the surrounding
environment.
– For example, harmless Streptococcus pneumoniae
bacteria can be transformed to pneumonia-causing cells.
(Remember Griffith’s experiments?)
– This occurs when a live nonpathogenic cell takes up a
piece of DNA with allele for pathogenicity from dead,
broken-open pathogenic cells.
– The foreign allele replaces the native allele & resulting
cell is now recombinant with DNA derived from two
different cells.
• Transduction
• Both generalized and specialized transduction use
phage as a vector to transfer genes between
bacteria.
Fig. 18.13
• Conjugation = “bacterial sex”
• One cell (“male”) donates DNA and its
“mate” (“female”) receives the genes.
• A sex pilus from the male initially joins the
two cells and creates a cytoplasmic
bridge between cells.
• “Maleness”, the ability to form
a sex pilus and donate DNA,
results from an F factor as a
section of the bacterial
chromosome or as a plasmid.
Fig. 18.14
• Plasmids,
– small, circular, self-replicating DNA molecules.
– usually have only a few genes that are not
required for normal survival and reproduction.
– generally, benefit the bacterial cell by providing
genes that are advantageous in stressful
conditions.
• The F plasmid facilitates genetic recombination when
environmental conditions no longer favor existing strains.
• R plasmid has genes for antibiotic resistance
• Episomes, like the F plasmid, can undergo
reversible incorporation into the cell’s
chromosome.
– Temperate viruses also qualify as episomes.
• The F factor or its F plasmid consists of
about 25 genes, most required for the
production of sex pili.
– Cells with either the F factor or the F plasmid are
called
F+ and they pass this condition to their offspring.
– Cells lacking either form of the F factor, are
called F-, and they function as DNA recipients.
• When an F+ and F- cell meet, the F+ cell passes a
copy of the F plasmid to the F- cell, converting it.
Fig. 18.15a
• The plasmid form of the F factor can become
integrated into the bacterial chromosome.
• The resulting Hfr cell (high frequency of
recombination) functions as a male during
conjugation.
Fig. 18.15b
• In the 1950s, Japanese physicians began to
notice that some bacterial strains had
evolved antibiotic resistance.
– The genes conferring resistance are carried by
plasmids, specifically the R plasmid (R for
resistance).
– Some of these genes code for enzymes that
specifically destroy certain antibiotics, like
tetracycline or ampicillin.
• When a bacterial population is exposed to
an antibiotic, individuals with the R plasmid
will survive and increase in the overall
population.
• Because R plasmids also have genes that
encode for sex pili, they can be transferred
from one cell to another by conjugation.
• A transposon is a piece of DNA that can
move from one location to another in a cell’s
genome.
• Transposon movement occurs as a type of
recombination between the transposon and
another DNA site, a target site.
– In bacteria, the target site may be within the
chromosome, from a plasmid to chromosome (or
vice versa), or between plasmids.
• Transposons can bring multiple copies for
antibiotic resistance into a single R plasmid
by moving genes to that location from
different plasmids.
– This explains why some R plasmids convey
resistance to many antibiotics.
• The transposase
enzyme recognizes the
inverted repeats as the
edges of the
transposon.
• Transposase cuts the
transposon from its
initial site and inserts it
into the target site.
• The simplest bacterial
transposon, an insertion
sequence, consists only
of the transposase gene
Fig. 18.17
• Composite transposons (complex
transposons) include extra genes
sandwiched between two insertion
sequences.
– It is as though two insertion sequences
happened to land relatively close together and
now travel together, along with all the DNA
between them, as a single transposon.
Fig. 18.18
• While insertion sequences may not benefit
bacteria in any specific way, composite
transposons may help bacteria adapt to new
environments.
– For example, repeated movements of resistance
genes by composite transposition may
concentrate several genes for antibiotic
resistance onto a single R plasmid.
– In an antibiotic-rich environment, natural
selection favors bacterial clones that have built
up composite R plasmids through a series of
transpositions.
Describe what you see.
What do you think causes the differences
between these two adult dogs?
All dogs are descendants of wolves, in fact, dogs and
wolves are almost indistinguishable genetically. But if that’s
the case, how do we get dogs as different as Chihuahuas
and Great Danes?
Describe what you see (1)
Describe what you see (2)
• These two-day-old
zebrafish (Danio rerio)
embryos are expressing
a gene for Green
Fluorescent Protein
(GFP) in cells lining their
circulatory system. This
causes the embryo's
circulatory system to
glow green when
exposed to light of a
certain wavelength.
• How would this be useful
to scientists?
• GFP is a visual marker
• Study of biological processes
(example: synthesis of proteins)
• Localization and regulation of gene expression
Links to
Real-world
• Cell movement
• Cell fate during development
• Formation of different organs
• Screenable marker to identify transgenic
organisms
How are FP’s used by scientists?
•
Researcher wants to study
a protein of interest to find
out what it does, or where
this protein is expressed in
the cell/tissue/organism
•
But how does the
researcher see the protein
and find out where it is
expressed if most proteins
are colorless and can’t be
distinguished from the
“soup” of proteins in the
cell?
http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP4.htm
How are FP’s used by scientists?
•
The FP gene is inserted
into the plasmid right after
the gene for the protein,
before the stop codon.
•
The protein of interest AND
the FP are translated at the
ribosome together.
•
The FP can be seen and
measured, even though the
protein of interest cannot
be seen. Anytime the
protein of interest is made
in the cell, the FP will also
be made.
http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP4.htm
Cellular organelles targeted with FPs
Human cell stained with two different
fluorescent proteins to visulalize
cytoskeletal components. Transfected
with GFP-tubulin / mCherry actin
(Ben Giepmans)
C Elegans transfected with GFP
tubulin construct (Susan Kline)
What is Transformation?
• Uptake of
foreign DNA,
often a
circular
plasmid
Allow bacteria to grow for 1-3
days on plate with ampicillin.
Bacteria now express cloned fluorescent protein
(transcription of gene and translation of mRNA to
protein at ribosomes).
GFP
Beta-lactamase
Ampicillin
Resistance
What is a plasmid?
•
A small circular piece of DNA
that replicates separately from
the main bacterial chromosome
•
Originated in bacteria to allow
survival in specific
environmental conditions
•
May express antibiotic
resistance gene or be modified
in the lab to express proteins of
interest
How are plasmids engineered?
DNA Plasmid Vector
Host DNA fragments
(i.e. coral or jellyfish
FP coding DNA)
Ligate (paste) fragments
into cut DNA vector
Cut genomic
DNA into
fragments
+
Cut plasmids
open with
restriction
enzymes
End result: Plasmid
containing FP gene
Transformation procedure
1. Suspend bacterial colonies in cold CaCl2
2. Add plasmid DNA
3. Place tubes on ice for 10 min
4. Heat-shock at 42°C for 45 seconds &
place on ice again for 2 min
5. Plate out bacteria
PLEASE READ AND FOLLOW LAB
INSTRUCTIONS CAREFULLY!
Why calcium chloride?
Ca++
Ca++
O P O
O
• Transformation solution = CaCl2
• Positive charge of Ca++ ions shields
negative charge of DNA
phosphates so the plasmid DNA
can more easily move through the
cell membrane
O
CH2
Base
O
Sugar
O
Ca++
O P O
Base
O
CH2
O
Sugar
OH
Why ice and heat?
•
Incubate on ice slows fluid
cell membrane
•
Heat-shock increases
permeability of membranes
Why Ampicillin?
• Ampicillin inhibits cell growth. Only cells that can deactivate the
ampicillin around them will grow.
• Ampicillin resistance is tied to (expressed with) the fluorescent
protein gene
• The ampicillin is the selection mechanism that allows only the
transformed bacteria to grow on the plate
Gene
Expression
• Beta Lactamase
– Ampicillin resistance
• Green Fluorescent
Protein (GFP)
– Aequorea victoria
jellyfish gene
• araC regulator
protein
– Regulates GFP
transcription
Scanning electron micrograph
of supercoiled plasmid
Grow? Glow?
• Follow protocol
• On which plates will
colonies grow?
• Which colonies will
glow?
Laboratory Quick Guide
Agenda 12/1/11
• Transformation lab – 6A
Homework –
•Ch. 19 Notes and SQ due next Monday
•Do any analysis questions for transformation lab
that you can
•Study for quiz tomorrow on Ch. 16, 17
Agenda 12/2/11
• Quiz Ch. 16, 17 and correct
• Analyze Transformation data and wrap up/ discuss
Analysis
• If extra time, could do mutation slides (Ch. 17, slides 7482) – otherwise will do it next Tuesday
Homework –
• Ch. 19 Notes and SQ due next Monday, short
Transformation quiz so be comfortable with lab
• Mutation practice due next Tuesday