DNA. Griffith s Transforming Principle Experiment 11/30/2006 DNA 2

Transcription

1 DNA Griffith s Transforming Principle Experiment 11/30/2006 DNA 2 1

2 Avery, McCarty, & MacLeod 1944 Extended Griffith s work 16 years later Search for the transforming factor Live rough cells + Protein from heat killed smooth cells Carbohydrates from heat killed smooth cells Lipids from heat killed smooth cells DNA from heat killed smooth cells This one Which was virulent? 11/30/2006 DNA 3 Hershey Chase Experiment 11/30/2006 DNA 4 2

3 Meselson~Stahl Experiment culture bacteria in N 15 for several generations all DNA with N 15 culture in N 14 media after 20 min (1 replication cycle): all DNA hybrid N 14/N 15 discount conservative hypothesis support semiconservative and dispersive hypotheses after 40 min (2 replication cycles): DNA either all 14 or all N 15 discount dispersive hypothesis and support SEMICONSERVATIVE HYPOTHESIS 11/30/2006 DNA 5 Origins of Replication Specific DNA sequence recognized by initiation proteins 1 origin in bacterial chromosome (single circular DNA strand) Multiple origins in eukaryotic chromosomes. bubbles Replication proceeds in both directions at replication forks Elongation catalyzed by DNA polymerase Nucleoside triphosphates lose a pyrophospate (2 phosphates) in a hydrolysis reaction when incorporated into DNA 11/30/2006 DNA 6 3

4 DNA Strands are Anti Parallel 11/30/2006 DNA 7 Replication in both directions Leading strand elongates continuously in 5 3 direction Lagging strand grows in 3 5 overall direction via addition of Okazaki fragments short segments that grow individually in 5 3 direction DNA polymerase extends strand by adding nucleotides at 3 end of already started strand Ligase connects the fragments 11/30/2006 DNA 8 4

5 Priming Since DNA cannot initiate but only add to 3 end of already started strand Primer = short RNA strand synthesized by primase Primer eventually replaced by DNA 11/30/2006 DNA 9 DNA Replication: Proteins Involved 11/30/2006 DNA 10 5

6 DNA Replication: Summary 11/30/2006 DNA 11 Proofreading and DNA Repair Mismatch Repair Mistakes fixed by DNA polymerase during replication Excision Repair Enzyme team detects damage Example: Covalent linking of adjacent thymine bases Thymine dimers induced by UV Cause DNA to buckle and interferes with replication Repair enzymes excise damaged region and replace with normal DNA segment Especially important in skin cells 11/30/2006 DNA 12 6

7 Beadle and Tatum nutritional mutants in Neurospora crassa developed 1 gene~1 enzyme hypothesis 11/30/2006 DNA 13 Variation on the Transcription~Translation Theme 11/30/2006 DNA 14 7

8 Triplet Code 11/30/2006 DNA 15 Stages of Transcription Initiation RNA polymerase binds to promoter AFTER binding of transcription factors (proteins) (transcription initiation complex) Unwinds 2 DNA strands Initiates RNA synthesis Elongation Grows RNA in 5 3 direction DNA strands reform (close) in wake of RNA elongation Termination Terminator section transcribed nucleotide sequence that signals end of transcription unit RNA released RNA polymerase dissociates Prokaryotic mrna immediately usable Eukaryotic mrna is processed 11/30/2006 DNA 16 8

9 Eukaryotic mrna processing snrnps small nuclear ribonucleoproteins combine with RNA transcript and other proteins to form spliceosome RNA transcript cut to release INTRON EXONS spliced together Spliceosome comes apart and releases mrna RNA transcript may contain more than one intron Processing occurs in the nucleus 11/30/2006 DNA 17 Translation 11/30/2006 DNA 18 9

10 Anatomy of a Ribosome Initiation: Small ribosomal subunit binds to mrna at AUG Large subunit arrives and completes initiation complex Initiator trna is in P site A site available to next trna Initiation factors (proteins) and GTP required 11/30/2006 DNA 19 Stages of Translation 11/30/2006 DNA 20 10

11 Final Stage of Translation: Termination 11/30/2006 DNA 21 Polyribosome: Ribosome Clusters that simultaneously transcribe a mrna strand Polyribosomes are found in prokaryotic and eukaryotic cells 11/30/2006 DNA 22 11

12 Synthesis of secretory protein and simultaneous import into endoplasmic reticulum 1. Begins on ribosome in cytosol 2. Signal recognition protein (SRP) binds to signal peptide 3. This binds to receptor protein (part of translocation complex) in ER membrane 4. SRP released and growing polypeptide translocates across 5. Signal cleaving enzyme cuts off peptide 6. Completed polypeptide leaves ribosome and folds into final conformation 7. Secretion via golgi apparatus etc.. 11/30/2006 DNA 23 Transcription and Translation in Prokaryotes Translation begins as soon as leading 5 end of mrna peels away from DNA template Remember: no processing required for prokaryotic mrna 11/30/2006 DNA 24 12

13 11/30/2006 DNA 25 Mutations 11/30/2006 DNA 26 13

14 11/30/2006 DNA 27 11/30/2006 DNA 28 14

15 11/30/2006 DNA 29 11/30/2006 DNA 30 15

16 11/30/2006 DNA 31 11/30/2006 DNA 32 16

17 11/30/2006 DNA 33 11/30/2006 DNA 34 17

18 Detecting Genetic Recombination in Bacteria Mutant arg and trp cannot grow on minimal medium arg+ and trp cannot make trp and thus cannot grow arg and trp+ cannot make arg and thus cannot grow Mixture of arg+ and trp with arg and trp+ results in some arg+ and trp+ colonies, indicating transformation Shades of Griffith and streptococcus 11/30/2006 DNA 35 Transduction: phages carry bacterial genes from one cell to another Generalized: random pieces of host chromosome carried Specialized: prophage carries adjacent bacterial genes 11/30/2006 DNA 36 18

19 Bacterial Conjugation 11/30/2006 DNA 37 Bacterial Conjugation continued 11/30/2006 DNA 38 19

20 11/30/2006 DNA 39 11/30/2006 DNA 40 20

21 11/30/2006 DNA 41 11/30/2006 DNA 42 21

22 11/30/2006 DNA 43 Making Antibodies How does the body make so many different kinds? 11/30/2006 DNA 44 22

23 11/30/2006 DNA 45 11/30/2006 DNA 46 23

24 Genetic Changes turning Proto Oncogenes into Oncogenes 11/30/2006 DNA 47 Regulation of Cell Growth 11/30/2006 DNA 48 24

25 Multi step model for the development of colo rectal cancer 11/30/2006 DNA 49 Cloning Overview 11/30/2006 DNA 50 25

26 Restriction Enzymes 11/30/2006 DNA 51 11/30/2006 DNA 52 26

27 11/30/2006 DNA 53 11/30/2006 DNA 54 27

28 11/30/2006 DNA 55 28