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Do Now

Do Now .

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Do Now

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  1. Do Now • We have been studying patterns of inheritance for the last several weeks. We have used such terminology as traits and genes, but have not specifically targeted the mechanism behind the transfer of those traits and genes from generation to generation. Identify the material that provides the instructions for ALL of your traits and design an experiment to prove that this material is in fact the “stuff” of what you are made.

  2. DNA! The stuff of Life

  3. The beginning: Chromosomes related to phenotype 1908 | 1933 • T.H. Morgan • worked with Drosophila • associated phenotype with specific chromosome • white-eyed male had specific X chromosome

  4. Morgan’s conclusions • genes on chromosomes • BIG QUESTION – The Gene Wars: Is protein orDNA that are the genes? • Why all the fuss???

  5. Frederick Griffith 1928 Observe the following data – explain what is going on

  6. Conclusion: heat-killed, virulent bacteria must have released genetic material  transferred to R cells • Transformation – DNA from dead cells cut into fragments & exits cell → healthy cells pick up free floating DNA and integrate chromosomes via recombination

  7. Avery, McCarty & MacLeod - 1944 Oswald Avery Colin MacLeod Maclyn McCarty

  8. Avery, McCarty & MacLeod • purified DNA & proteins separately from Streptococcus pneumonia bacteria • Experimental Question: which will transform non-pathogenic bacteria? • 1. injected protein into bacteria • Mice lived! • 2. injected DNA into bacteria • transformed bacteria • Mice died!

  9. Hershey & Chase 1952 | 1969 Martha Chase Alfred Hershey

  10. Protein coat labeled with 35S DNA labeled with 32P T2 bacteriophages are labeled with radioactive isotopes S vs. P Which radioactive marker is found inside the cell? bacteriophages infect bacterial cells This will be the molecule containing genetic info! bacterial cells are agitated to remove viral protein coats 32P radioactivity foundin the bacterial cells 35S radioactivity found in the medium

  11. Watson and Crick 1952

  12. Rosalind Franklin & Maurice Wilkins

  13. 1947 Chargaff • DNA composition: “Chargaff’s rules” • varies from species to species • all 4 bases not in equal quantity • bases present in characteristic ratio • humans: A = 30.9% T = 29.4% G = 19.9% C = 19.8% RulesA = T C = G

  14. Review with Hank!

  15. The Cell Cycle

  16. DNA Replication (Hank Video) • base pairing suggests that each side can serve as a template for a new strand * DNA Replication Machinery – 1:45 “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” —Watson & Crick

  17. A little more on DNA Replication… • Prokaryotes: DNA usually circular(1 fork) • Eukaryotes: DNA linear(many forks)

  18. Telomeric Replication • Bacterial DNA is circular - Animation • Eukaryotic DNA is linear • Can you think of any problems this may pose in the successful completion of replication? Animation

  19. A scientist is using an ampicillin-sensitive strain of bacteria that cannot use lactose because it has a nonfunctional gene in the lac operon. She has two plasmids. One contains a functional copy of the affected gene of the lac operon, and the other contains the gene for ampicillin resistance. Using restriction enzymes and DNA ligase, she forms a recombinant plasmid containing both genes. She then adds a high concentration of the plasmid to a tube of the bacteria in a medium for bacterial growth that contains glucose as the only energy source. This tube (+) and a control tube (-) with similar bacteria but no plasmid are both incubated under the appropriate conditions for growth and plasmid uptake. The scientist then spreads a sample of each bacterial culture (+ and -) on each of the three types of plates indicated below.

  20. If no new mutations occur, it would be most reasonable to expect bacterial growth on which of the following plates? • a. 1 and 2 only • b. 3 and 4 only • c. 5 and 6 only • d. 4, 5, and 6 only • e. 1, 2, 3, and 4 only

  21. If the scientist had forgotten to use DNA ligase during the preparation of the recombinant plasmid, bacterial growth would most likely have occurred on which of the following? • a. 1 and 2 only • b. 1 and 4 only • c. 4 and 5 only • d. 1, 2, and 3 only • e. 4, 5, and 6 only

  22. From Gene to Protein sections of DNA that code for • How does DNA code for cells & bodies? DNA proteins cells bodies

  23. The “Central Dogma” • Flow of genetic information in a cell • DNA to proteins? transcription translation RNA DNA protein trait replication

  24. A B C D E disease disease disease disease Metabolic Pathways… • suggest genes code for enzymes • Disruptions in pathways result in • lack of an enzyme • disease • variation of phenotype metabolic pathway

  25. Transcription in short… • Make a model! • Steps • Structures

  26. RNA review • ribose sugar • N-bases • uracil instead of thymine • U : A • C : G • single stranded • many RNAs • mRNA, tRNA, rRNA, siRNA… transcription DNA RNA

  27. Making mRNA • transcribed DNA strand = template strand • untranscribed DNA strand = coding strand • synthesize complementary RNA strand • transcription bubble • Enzymes involved • RNA polymerase • Helicase coding strand 3 A G C A T C G T 5 A G A A A C G T T T T C A T G A C T DNA 3 C T A A 5 T G G C A U C G U T C 3 G T A G C A mRNA template strand RNA polymerase 5 build RNA 53

  28. RNA polymerases • 3 types of RNA polymerases • 1.RNA polymerase 1 • transcribe rRNA genes ONLY • makes ribosomes • 2. RNA polymerase 2 • transcribe genes into mRNA • 3. RNA polymerase 3 • transcribe tRNA genes ONLY • **each has a specific promoter sequence it recognizes**

  29. “What is a promoter?” you may ask • Promoter region - site marking the start of gene • TATA box binding site • transcription factors(ie. proteins, hormones?) - on/off switch; trigger binding of RNA pol • RNA polymerase • Enhancer region • binding site far upstream • turns transcription on HIGH

  30. intron = noncoding (inbetween) sequence exon = coding (expressed) sequence mRNA Processing • Eukaryotic genes contain “fluff” – spliced • exons = expressed / coding DNA • introns = the junk; inbetween sequence; now thought to be involved in switches • 5’ Cap & PolyA tail added ~10,000 bases eukaryotic DNA pre-mRNA primary mRNA transcript ~1,000 bases mature mRNA transcript spliced mRNA

  31. snRNPs snRNA intron exon exon 5' 3' spliceosome 5' 3' lariat 5' 3' exon exon mature mRNA excised intron 5' 3' The splicing process… • snRNPs “snurps” • small nuclear RNA • Proteins • Spliceosome • several snRNPs • recognize splice site sequence • cut & paste gene

  32. 3' poly-A tail 3' A A A A A mRNA 50-250 A’s 5' cap P P P 5' G The fancy cap & tail… • Enzymes in cytoplasm attack mRNA – protection is needed • add 5 GTP cap • add poly-A tail • longer the tail, longer mRNA lasts: produces more protein

  33. Translation • Make a model! • Steps • Structures

  34. DNA TAC GCA CAT TTA CGT ACG CGG mRNA AUG CGU GUA AAU GCA UGC GCC Met ArgVal AsnAlaCysAla protein ? How does mRNA code for proteins? 4 ATCG 4 AUCG 20 How can you code for 20 amino acids with only 4 nucleotide bases (A,U,G,C)?

  35. 20 different amino acids • aa’s coded for by THREE nucleotides –codons • 4 bases, 3 per codon: 43 = 64 total possible combinations

  36. Why don’t these numbers match? 20 amino acids, 64 options?? WOBBLE • Code is redundant • several codons for each amino acid • 3rd base “wobble” • Most codons = aa’s • Start codon • AUG • methionine • Stop codons • UGA, UAA, UAG

  37. How are codons matched to amino acids?

  38. A little more on tRNA • “Clover leaf” structure • anticodon on “clover leaf” end • amino acid attached to 3 end

  39. Loading “naked” tRNA’s • AminoacyltRNAsynthetase -enzyme bonds aa’s to tRNA • requires energy • ATP  AMP • bond is unstable • can easily release amino acid at ribosome Trp C=O Trp Trp C=O H2O OH O OH C=O O activating enzyme tRNATrp mRNA anticodon tryptophan attached to tRNATrp tRNATrpbinds to UGG codonof mRNA

  40. So where’s the protein making factory? • RIBOSOMES!!! • Facilitate binding of tRNAanticodon to mRNA codon • Organelle or enzyme?? • Structure • rRNA & proteins • 2 subunits • large • small • 3 sites

  41. 3 ribosomal sites… • A site(aminoacyl-tRNA site) • tRNA carrying next aa to be added to chain binds here • P site (peptidyl-tRNA site) • holds tRNA carrying growing polypeptide chain • E site (exit site) • emptytRNAleaves ribosome from exit site Met Met 5' C A U C A U G U A G U A 3' E P A E P A

  42. 3 2 1 Let’s translate… • Initiation • brings together mRNA, ribosomal subunits, initiator tRNA • Elongation • adding amino acids based on codon sequence • Termination • end codon

  43. Full circle: metabolic pathways

  44. Do you think this process is the same for prokaryotes & eukaryotes? Explain your ideas. • Prokaryotes • DNA in cytoplasm • circular chromosome • naked DNA • no introns • continuous process • Eukaryotes • DNA in nucleus • linear chromosomes • DNA wound on histone proteins • introns vs. exons • mRNA processing

  45. Translation in Prokaryotes • Transcription & translation simultaneous in bacteria • DNA in cytoplasm • no mRNA editing • ribosomes read mRNA as transcribed • Faster than in eukaryotes (DNA to protein ~1hr)

  46. Review Videos • Transcription • mRNA processing • mRNA splicing

  47. Let’s review • Translation Animation • Protein Synthesis

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