2.  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.

4.  T.H. Morgan ◦ worked with Drosophila ◦ associated phenotype with specific chromosome  white-eyed male had specific X chromosome 1908 | 1933

5.  Morgan’s conclusions ◦ genes on chromosomes ◦ BIG QUESTION – The Gene Wars: Is protein or DNA that are the genes?  Why all the fuss???

6. 1928 Frederick Griffith Observe the following data – explain what is going on

7. 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

8. Oswald Avery Maclyn McCarty Colin MacLeod

9.  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!

10. Alfred HersheyMartha Chase 1952 | 1969

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

12. Watson and Crick 1952

14.  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% 1947 Rules A = T C = G

15. Review with Hank!

17. ◦ base pairing suggests that each side can serve as a template for a new strand “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 * DNA Replication Machinery – 1:45DNA Replication Machinery

18.  Prokaryotes: DNA usually circular (1 fork)  Eukaryotes: DNA linear (many forks)

19. BBacterial DNA is circular - Animation EEukaryotic DNA is linear ◦C◦C an you think of any problems this may pose in the successful completion of replication? Animation

21. 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.

22. 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

23. 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

24. proteinscellsbodies How does DNA code for cells & bodies? DNA

25.  Flow of genetic information in a cell ◦ DNA to proteins? transcription translation replication protein RNA DNAtrait

26.  suggest genes code for enzymes ◦ Disruptions in pathways result in  lack of an enzyme  disease  variation of phenotype ABCDE disease metabolic pathway

27. Make a model! o Steps o Structures

28.  ribose sugar  N-bases ◦ uracil instead of thymine ◦ U : A ◦ C : G  single stranded  many RNAs ◦ mRNA, tRNA, rRNA, siRNA… RNADNA transcription

29.  Making mRNA ◦ transcribed DNA strand = template strand ◦ untranscribed DNA strand = coding strand ◦ synthesize complementary RNA strand  transcription bubble ◦ Enzymes involved  RNA polymerase  Helicase template strand mRNA RNA polymerase coding strand DNA C C C C C C C CC C G G G GG GG G G G A A A AA A A A A A A A A T T T T T T T T T T T T UU 5 3 5 3 3 5 build RNA 5  3

30.  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**

31.  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

32.  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 eukaryotic DNA exon = coding (expressed) sequence intron = noncoding (inbetween) sequence primary mRNA transcript mature mRNA transcript pre-mRNA spliced mRNA ~10,000 bases ~1,000 bases

33. snRNPs exon intron snRNA 5'3' spliceosome exon excised intron 5' 3' lariat exon mature mRNA 5'  snRNPs “snurps” ◦ small nuclear RNA ◦ Proteins  Spliceosome ◦ several snRNPs ◦ recognize splice site sequence  cut & paste gene

34. A A A A A 3' poly-A tail mRNA 5' 5' cap 3' G P P P 50-250 A’s  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

35. Make a model! o Steps o Structures

36. TAC GCA CAT TTA CGT ACG CGG DNA AUG CGU GUA AAU GCA UGC GCC mRNA Met Arg Val Asn Ala Cys Ala protein ? How can you code for 20 amino acids with only 4 nucleotide bases (A,U,G,C)? 4 4 20 ATCG AUCG

37.  20 different amino acids  aa’s coded for by THREE nucleotides –codons  4 bases, 3 per codon: 4 3 = 64 total possible combinations

38. 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

40.  “Clover leaf” structure ◦ anticodon on “clover leaf” end ◦ amino acid attached to 3 end

41.  Aminoacyl tRNA synthetase - enzyme bonds aa’s to tRNA ◦ requires energy  ATP  AMP  bond is unstable  can easily release amino acid at ribosome activating enzyme anticodon tRNA Trp binds to UGG codon of mRNA Trp mRNA C=O OH H2OH2O O tRNA Trp tryptophan attached to tRNA Trp C=O O

42. RIBOSOMES!!!  Facilitate binding of tRNA anticodon to mRNA codon Organelle or enzyme??  Structure ◦ rRNA & proteins ◦ 2 subunits  large  small ◦ 3 sites

43.  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) ◦ empty tRNA leaves ribosome from exit site Met U U A C A G APE 3' U U A C A G APE 5'

44.  Initiation ◦ brings together mRNA, ribosomal subunits, initiator tRNA  Elongation ◦ adding amino acids based on codon sequence  Termination ◦ end codon 123

46.  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

47.  Transcription & translation simultaneous in bacteria ◦ DNA in cytoplasm ◦ no mRNA editing ◦ ribosomes read mRNA as transcribed ◦ Faster than in eukaryotes (DNA to protein ~1hr)

48.  Transcription Transcription  mRNA processing mRNA processing  mRNA splicing mRNA splicing

49.  Translation Animation Translation Animation  Protein Synthesis Protein Synthesis