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