Opening Activity

1. 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

2. Genetics of Viruses and Bacteria and DNA Cloning ApplicationsChapters 18, 20

3. Lytic CycleVirulent Phages 1. Tail fibers of phage used for attachment to host 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

4. Lysogenic CycleTemperate 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 5. An entire colony of infected cells are produced 7. Lytic cycle proceeds and ends with phage dispersal 4. Host reproduces normally, and phage DNA is copied in the process 2. Phage DNA circularizes Lytic Cycle Lysogenic Cycle 3. Phage DNA crosses over and attaches with host DNA, becoming a prophage

5. Retroviruses (RNA DNA) video 1. Virus enters cell and delivers RNA and reverse transcriptase 2. Reverse transcriptase makes DNA from viral RNA RNA Viruses – higher rate of mutation Reverse trascriptase lacks DNA polymerase’s proofreading mechanism 3. 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

6. Adaptability of Bacteria 5. DNA Plasmids with genes that increase a bacterium’s fitness can reproduce independently and transfer to other bacterium 4. Any mutations that increase fitness are quickly amplified by asexual reproduction (binary fission) 1. Short generation spans 2. High Reproductive Rate 3. Sexual Reproduction by Conjugation

7. How are new genes introduced to bacteria? Bacteria have surface proteins that recognize naked DNA from closely related species and transports them in. The uptake of foreign DNA from the surrounding environment

8. 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!

9. 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

10. 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.

11. Today we know… • The DNA from the smooth colony was taken up by the non-deadly rough colony

12. 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)

13. 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

14. 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

15. 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”

16. This information about bacteria and viruses can be used in biotechnologyto clone a gene

17. 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 6. Identify bacteria of interest and remove product (HGH) from bacteria 5. Bacteria multiplies, plasmid replicates

18. 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

19. 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

20. Examples of Restriction Enzymes • EcoR1 TTAA AATT • Bam1 CTAG GATC • HaeII CC GG GG CC

21. How do you identify cell clones carrying genes of interest? (step 7)

22. 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.

23. Method 3: Nucleic Acid ProbeIsolation by locating the gene instead of the product. 2. Create a radioactively labeled DNA probe that has base-pairs complementary to the desired gene 1. Transfer cells onto a filter then denature the DNA so the bases are exposed 3. Develop the film 4. Compare film to original plate to identify bacterial cells with the desired gene

24. Are there problems with combining eukaryotic genes into prokaryotic plasmids?

25. 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

26. Intron and Exon in Eukaryotic Cells exon exon exon intron intron 5’ 3’ 5’ 3’ DNA promotor stop codon start codon Transcription mRNA Processing cap poly A tail Splicing Intron deleted mature mRNA To cytoplasm Take place in nucleus

27. cDNA Is Reverse Transcribed from mRNA 5’ 3’ 3’ 5’ 5’ mature mRNA poly A tail Reverse transcription TTTT 3’ 5’ RNA hydrolysis DNA polymerase 3’ 3’ 5’

28. Target Genes Carried by Plasmid Target Genes Restriction Enzyme Restriction Enzyme Chromosomal cDNA DNA Recombination Target Gene Recombination Transformation 1 plasmid 1 cell Host Cells Recombinant Plasmid Transformation Juang RH (2004) BCbasics

29. 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.

30. Problem #3Overall, there can be eukaryotic and prokaryotic incompatibility Solution? Use eukaryotic yeast instead of bacteria Yeast offer the same advantages of bacteria. • Easy to grow • Also have plasmids (rare among eukaryotes)

31. But more cool Biotechnology methods awaits…

32. Phosphate groups of nucleotides have a - charge 1 2 3 2- 2- 5’ O 4 PO4 PO4 - O-P=O 5 O 6 3’ OH 5’ 5’ 1 3’ OH 2 Phosphodiester bond 3’

33. Charge on a DNA Double Helix 3’ 5’ 1 2 3 4 5 6 7 8 9 10 5’ 3’ Large groove Small groove 1 Twist = 10.5 bp

34. 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

35. DNA Electrophoresis analysis after endonuclease (restriction enzyme) digestion 8 kb 2 kb A 7 kb 3 kb B 5 kb 3 kb 2 kb A + B Restriction enzymes A B 10 kb C A B A+B L Juang RH (2004) BCbasics

36. 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.

37. 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

38. Southern Blot Analysis for Paternity Child Mother Wells B and D represent possible fathers ? ? Answer: B! Based on this RFLP analysis, who’s the dad?

39. 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