Download Genetics of Viruses and Bacteria

Opening Activity
I. Jigsaw Beginning of Chapter 1
II. History of Discovery (Viruses)
III. Relative Size (Viruses)
IV. Viral Genome
V. Capsids and Envelopes
VI. What Viruses are, in general
VII. Lytic Cycle
VIII. Lysogenic Cycle
Genetics of Viruses and Bacteria
and DNA Cloning Applications
Chapters 18, 20
1. Tail fibers of
phage used
for attachment
to host
Lytic Cycle
Virulent Phages
3. Host DNA is
hydrolyzed and
destroyed
2. Injection of
genetic material
into host
5. Viral lysozymes
breakdown cell
wall, and cell lysis
occurs, releasing
new viruses.
4. Viral DNA replication, RNA
transcription and protein
translation occurs. Assembly
of viral particles begin
Lysogenic Cycle
Temperate Phages
6. Stress or other
factors cause prophage
to exit the host DNA and
start lytic cycle
1. Phage injects
its genetic
material into host
7. Lytic
cycle
proceeds
and ends
with phage
dispersal
2. Phage DNA circularizes
Lytic Cycle
Lysogenic Cycle
5. An entire
colony of
infected cells are
produced
4. Host reproduces
normally, and phage
DNA is copied in the
process
3. Phage DNA crosses over
and attaches with host DNA,
becoming a prophage
Retroviruses (RNA DNA)
video
RNA Viruses – higher rate of mutation
1. Virus enters cell and
delivers RNA and
reverse transcriptase
2. Reverse transcriptase
makes DNA from viral
RNA
3. DNA polymerase
copies 2nd strand of
4. Cross over btwn
Reverse trascriptase lacks DNA polymerase’s proofreading
viral DNA
viral and host DNA
mechanism
creates provirus
5. When provirus exits
(lysogenic cycle)
the host DNA, viral
transcription of RNA and
translation of viral
proteins begin for viral
assembly and release
Adaptability of Bacteria
5. DNA Plasmids with genes that increase a
4.
mutations
that
increase
fitness
are quickly and
bacterium’s
fitness
can
reproduce
independently
1.
2.
3.Any
Short
High
Sexual
Reproductive
generation
Reproduction
spans
Rate
by Conjugation
amplified
asexual
reproduction (binary fission)
transfer tobyother
bacterium
Bacteria have surface proteins that
The
recognize
uptakenaked
of foreign
DNADNA
fromfrom
closely
the related
surrounding
environment
species and transports them in.
How are new genes introduced to
bacteria?
Griffith’s Experiment with Pneumonia
and the accidental discovery of
Transformation
• Frederick
CONCLUSION:
Griffiths was a
bacteriologist studying
pneumonia
The smooth colonies
• He
discovered
types of
must
carry thetwo
disease!
bacteria:
– Smooth colonies
– Rough colonies
Griffith’s Experiment with Pneumonia
and the accidental discovery of
Transformation
• When heat was applied
to the deadly smooth
type…
• And injected into a
mouse…
• The mouse lived!
Griffith’s Experiment with Pneumonia
and the accidental discovery of
Transformation
• Griffith injected the heat-killed
type and the non-deadly rough
type of bacteria.
• The bacteria “transformed” itself
from the heated non-deadly type
to the deadly type.
Today we know…
• The DNA from the smooth colony was
taken up by the non-deadly rough colony
Transduction
• Phages (bacterial viruses) are vectors that
carry bacterial genes from one host to
another
Generalized Transduction
(virulent phage vectors)
2 types
Specialized Transduction
(temperate phage vectors)
Generalized
Transduction
Specialized
Transduction
When viral
genome is
excised from
prophase state,
it takes with it a
piece of host
bacterial DNA
Small piece of
bacterial DNA
is accidentally
assembled
inside a viral
capsid
Crossover
occurs
between new
transduced
DNA and new
host DNA
Conjugation
• Direct transfer of genetic material (usually plasmid DNA)
from two bacterial cells that are temporarily joined by a
sex pili.
• Plasmid genes are not required for survival, but they
tend to code for genes that increase fitness (ex. antibiotic resistance)
video
• The ability of a
bacterium to form
the sex-pili depends
on if they have the
“F-factor” gene
(fertility factor),
which is coded in
the bacterial DNA or
plasmid.
• F-factor bacterium
are considered
“male”
This information about bacteria
and viruses can be used in
biotechnology
to clone a gene
DNA Cloning:
Technique for making exact copies of DNA
2. Remove the gene of interest
from a cell (ex. gene for making
human growth hormone HGH)
1. Isolate plasmid
(cloning vector)
from bacteria
3. Insert gene of interest into
plasmid vector (create
recombinant DNA)
4. Return recombinant DNA
plasmid into bacteria by
transformation
5. Bacteria
multiplies,
plasmid replicates
6. Identify
bacteria of
interest and
remove
product
(HGH) from
bacteria
How do you create recombinant
DNA? (step 3)
• In nature, restriction enzymes
protect a cell by cutting out foreign
DNA that invades cells (ex. Cuts
out viral DNA from bacteria)
• Restriction Enzymes are used in
biotech. to cut a DNA cloning
vector and the desired genes in
specific locations. Creates “sticky
ends”
• Enzymes recognize specific DNA
sequences (4-8 nucleotides long)
= restriction site
How do you create recombinant
DNA? (step 3)
• Restriction enzymes cut plasmid and gene
of choice from DNA.
• Sticky ends of both the gene of choice and
the DNA plasmid vector match. Base
pairing occurs.
• DNA ligase covalently seals 5’ end and 3’
end of the cut strands together
Examples of Restriction
Enzymes
• EcoR1
TTAA
AATT
• Bam1
CTAG
GATC
• HaeII
CC GG
GG CC
How do you identify cell
clones carrying genes of
interest? (step 7)
Method One: Antibioitic Resistance
• Cloning vector (plasmid) usually has a gene for
antibiotic resistance. (ex. Ampicillin resistance)
• Bacteria grows on a petri dish with ampicillin in
it.
• Bacteria w/o the vector will not have resistance
and will die, leaving only the desired bacteria
with the vector on the plate.
• Product then can be removed and isolated from
the cell clones.
Method Two: Phenotypic Color
• If the product has a specific color, isolation by
color.
Method 3: Nucleic Acid Probe
Isolation by locating the gene instead of the product.
1. Transfer cells
onto a filter then
denature the
DNA so the
bases are
exposed
2. Create a
radioactively labeled
DNA probe that has
base-pairs
complementary to the
desired gene
3. Develop the film
4. Compare film to original plate to
identify bacterial cells with the
desired gene
Are there problems with
combining eukaryotic genes into
prokaryotic plasmids?
Problem #1
Eukaryotic DNA have introns that
prokaryotic DNA does not.
Prokaryotic cells are not equipped
to cut out the introns to make
functional mRNA.
Solution?
Create “cDNA” or DNA
without introns
Intron and Exon in Eukaryotic Cells
exon
promotor
3’
exon
intron
exon
intron
DNA
5’
stop codon
start codon
Transcription
5’
3’ mRNA
Processing
cap
poly A
tail
Splicing
Intron deleted
Take place in nucleus
mature mRNA
To cytoplasm
cDNA Is Reverse Transcribed from mRNA
5’
Reverse
transcription
3’
mature mRNA
RNA
hydrolysis
3’
5’
poly A tail
TTTT
5’
5’
DNA
3’
3’
polymerase
5’
3’
Target Genes Carried by Plasmid
Target Genes
Restriction
Enzyme
DNA Recombination
Target Gene
Recombination
Chromosomal cDNA
Restriction
Enzyme
Transformation
Host Cells
Recombinant
Plasmid
Transformation
1 plasmid
1 cell
Juang RH (2004) BCbasics
Problem #2
Eukaryotic DNA inserted into a plasmid
does not have a prokaryotic promoter for
bacterial RNA polymerase to bind and
transcribe
Solution?
Insert an “expression vector” or a prokaryotic
promoter, just in front of the area where the
eukaryotic gene will be inserted into the
plasmid for transcription to occur.
Problem #3
Overall, there can be eukaryotic
and prokaryotic incompatibility
Solution?
Use eukaryotic yeast instead of bacteria
Yeast offer the same advantages of bacteria.
1) Easy to grow
2) Also have plasmids (rare among eukaryotes)
But more cool Biotechnology
methods awaits…
Phosphate groups of nucleotides have a - charge
5’
5’
2-
1
PO4
1
2
3’
OH
O
O-P=O
O
3
5’
4
2-
PO4
Phosphodiester bond
2
3’ OH
5
6
3’
Charge on a DNA Double Helix
3’
5’
1
2
3
4
5
Large groove
6
Small groove
7
8
9
10
1 Twist = 10.5 bp
5’
3’
Gel Electrophoresis
• Gel Electrophoresis: technique uses the
difference in electrical charge to separate
polymers (DNA, RNA, protein) on the
basis of size
Let’s see a model of how gel electrophoresis
works
DNA Electrophoresis analysis after endonuclease
(restriction enzyme) digestion
Restriction
enzymes
C
A
B A+B L
A
B
10 kb
A 8 kb
2 kb
B 7 kb
3 kb
A 5 kb
+ 3 kb
B 2 kb
Juang RH (2004) BCbasics
What is RFLP?
An RFLP is a sequence of DNA that has a
restriction site on each end with a "target"
sequence in between
A target sequence is any segment of DNA that can bind
to a radioactive probe by forming complementary base
pairs. The target sequence then can be detected by a
southern blot analysis.
Purpose of RFLP Analysis?
• Trace a sequence of genetic markers in
families
• Diagnose disease
• Prepare DNA fingerprints for forensics
• Compare genomes of different species
• Find mutations
• Paternity tests
Southern Blot Analysis for Paternity
Mother
Child
?
Answer:
B!
?
Wells B and D
represent
possible
fathers
Based on
this RFLP
analysis,
who’s the
dad?
Other Biotech Methods: PCR
• Used when DNA is rare or
impure
• Quick amplification of
DNA (Billions made in a
few hours)
• Use DNA pol. (from Taq
bacteria) to copy strands.
• Use synthetic DNA
primers for DNA pol. to
extend from