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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