opening activity
DESCRIPTION
Opening Activity. Jigsaw Beginning of Chapter 1 History of Discovery (Viruses) Relative Size (Viruses) Viral Genome Capsids and Envelopes What Viruses are, in general Lytic Cycle Lysogenic Cycle. Genetics of Viruses and Bacteria and DNA Cloning Applications Chapters 18, 20. - PowerPoint PPT PresentationTRANSCRIPT
Opening Activity
I. Jigsaw Beginning of Chapter 1II. History of Discovery (Viruses)III. Relative Size (Viruses)IV. Viral GenomeV. Capsids and EnvelopesVI. What Viruses are, in generalVII. Lytic CycleVIII. Lysogenic Cycle
Genetics of Viruses and Bacteria and DNA Cloning Applications
Chapters 18, 20
Lytic CycleVirulent Phages1. Tail fibers
of phage used for attachment to host
2. Injection of genetic material into host
3. Host DNA is hydrolyzed and destroyed
4. Viral DNA replication, RNA transcription and protein translation occurs. Assembly of viral particles begin
5. Viral lysozymes breakdown cell wall, and cell lysis occurs, releasing new viruses.
Lysogenic CycleTemperate Phages
1. Phage injects its genetic material into host
2. Phage DNA circularizes
3. Phage DNA crosses over and attaches with host DNA, becoming a prophage
4. Host reproduces normally, and phage DNA is copied in the process
5. An entire colony of infected cells are produced
6. Stress or other factors cause prophage to exit the host DNA and start lytic cycle
Lytic Cycle Lysogenic Cycle
7. Lytic cycle proceeds and ends with phage dispersal
Retroviruses (RNA DNA)
RNA Viruses – higher rate of mutation
Reverse trascriptase lacks DNA polymerase’s proofreading mechanism
1. Virus enters cell and delivers RNA and reverse transcriptase
2. Reverse transcriptase makes DNA from viral RNA3. DNA polymerase
copies 2nd strand of viral DNA
4. Cross over btwn viral and host DNA creates provirus (lysogenic cycle)
5. When provirus exits the host DNA, viral transcription of RNA and translation of viral proteins begin for viral assembly and release
video
Adaptability of Bacteria1. Short generation spans2. High Reproductive Rate3. Sexual Reproduction by Conjugation4. Any mutations that increase fitness are quickly amplified by asexual reproduction (binary fission)
5. DNA Plasmids with genes that increase a bacterium’s fitness can reproduce independently and transfer to other bacterium
How are new genes introduced to bacteria?
The uptake of foreign DNA from the surrounding environment
Bacteria have surface proteins that recognize naked DNA from closely related species and transports them in.
Griffith’s Experiment with Pneumonia and the accidental discovery of Transformation
• Frederick Griffiths was a bacteriologist studying pneumonia
• He discovered two types of bacteria:– Smooth colonies– Rough colonies
CONCLUSION:
The smooth colonies must carry
the disease!
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 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.
Griffith’s Experiment with Pneumonia and the accidental discovery of Transformation
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
2 types
Generalized Transduction (virulent phage vectors)
Specialized Transduction (temperate phage vectors)
Generalized Transduction
Specialized Transduction
Small piece of bacterial DNA is accidentally assembled inside a viral capsid
When viral genome is excised from prophase state, it takes with it a piece of host bacterial DNA
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. anti-biotic 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
biotechnologyto clone a gene
DNA Cloning:Technique for making exact copies of DNA
1. Isolate plasmid (cloning vector) from bacteria
2. Remove the gene of interest from a cell (ex. gene for making human growth hormone HGH)
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 TTAAAATT
• Bam1 CTAGGATC
• HaeII CC GGGG 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 #1Eukaryotic 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
mRNA
DNA
5’ 3’
cappoly A
tail
exon exonexonintron intron
mature mRNA
Processing
Transcription
Splicing
promotor3’ 5’
Take place in nucleus
start codon stop codon
To cytoplasm
Intron deleted
cDNA Is Reverse Transcribed from mRNA
mature mRNApoly A tail
5’ 3’
TTTTReverse transcription 3’ 5’
3’5’ 3’
DNA polymerase
RNA hydrolysis
5’
3’ 5’
Target Genes Carried by Plasmid
1 plasmid1 cellRecombinant
PlasmidTransformation
Target GeneRecombination
Restriction
Enzyme
Restriction
Enzyme
Chrom
osomal
cDN
ATarget Genes
DNA Recombination
TransformationHost Cells
Juang RH (2004) BCbasics
Problem #2Eukaryotic 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 #3Overall, there can be eukaryotic and prokaryotic incompatibility
Solution?Use eukaryotic yeast instead of bacteria
Yeast offer the same advantages of bacteria.1) Easy to grow2) Also have plasmids (rare among eukaryotes)
But more cool Biotechnology methods awaits…
Phosphate groups of nucleotides have a - charge
OH
PO4
2-
Phosphodiester bond
O-P=O-
O
O
5’
3’
PO4
2-
OH3’
5’
1
23’
5’
1
2
3
4
5
6
Large grooveSmall groove
1 Twist = 10.5 bp
1
2
3
4
5
6
7
8
9
10
Charge on a D
NA
Double H
elix
3’
5’
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
A B 10 kb
8 kb2 kb A
7 kb3 kb B
5 kb3 kb2 kb
A+B
C A B A+B L
Restriction enzymes
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 PaternityMother Child
Wells B and D represent possible fathers
? ?
Based on this RFLP analysis, who’s the dad?
Answer:
B!
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