We will focus on these 3 classic experiments highlighted in this chapter…… Griffith (Frederick) Hershey and Chase (Alfred and Martha) Meselsen and Stahl (Matt and Frank)

There are unknown heritable substances… Griffith There are unknown heritable substances… Turned to a bacterial pathogen…Streptococcus pneumoniae Is it gram positive or negative? http://en.wikipedia.org/wiki/File:Fred_Griffith_and_%22Bobby%22_1936.jpg

Work by Avery identified the transforming substance as DNA Figure 13.2 Experiment Mixture of heat-killed S cells and living R cells Living S cells (control) Living R cells (control) Heat-killed S cells (control) Figure 13.2 Inquiry: Can a genetic trait be transferred between different bacterial strains? Results Mouse dies Mouse healthy Mouse healthy Mouse dies Work by Avery identified the transforming substance as DNA Living S cells

Transformation-did not really understand mechanism Can we do this-pick up DNA from our environment?

Hershey and Chase (1952) Their work pointed to DNA rather than proteins… Bacteriophages what are they???? (worked with one called T2)

Some phages grown in media for a couple hrs with radioactive Sulphur…(which should be incorporated into some proteins Methionine, Cysteine) Other phages grown in media for a couple hrs with radioactive Phosphorus….(which should be incorporated into DNA)

Batch 1: Radioactive sulfur (35S) in phage protein Figure 13.4 Experiment Batch 1: Radioactive sulfur (35S) in phage protein 1 Labeled phages infect cells. 2 Agitation frees outside phage parts from cells. 3 Centrifuged cells form a pellet. Radioactive protein 4 Radioactivity (phage protein) found in liquid Centrifuge Pellet Batch 2: Radioactive phosphorus (32P) in phage DNA Radioactive DNA Figure 13.4 Inquiry: Is protein or DNA the genetic material of phage T2? Centrifuge 4 Radioactivity (phage DNA) found in pellet Pellet 7

Watson-Crick Model predicted…. Each of two daughter molecules would have one parental strand and one newly made! Meselson and Stahl-clever experiment…What did they do??

Experiment Bacteria cultured in medium with 15N (heavy isotope) Figure 13.11 Experiment 1 Bacteria cultured in medium with 15N (heavy isotope) 2 Bacteria transferred to medium with 14N (lighter isotope) Results 3 DNA sample centrifuged after first replication 4 DNA sample centrifuged after second replication Less dense More dense Conclusion Predictions: First replication Second replication Conservative model Figure 13.11 Inquiry: Does DNA replication follow the conservative, semiconservative, or dispersive model? Semiconservative model Dispersive model 9

Figure 13.1 Watson and Crick http://www.ted.com/talks/james_watson_on_how_he_discovered_dna.html Figure 13.1 How was the structure of DNA determined?

What do these terms refer to… How does this replication thing work?? origin of replication helicase topoisomerase replication fork primase and the RNA primer single stranded binding proteins DNA polymerase Search online for stronger and weaker video clips-which one is the very best and the very worst? Email me the links to your very best and worst (with your group members names) Jot down on the board enough of the web address that we can distinguish which ones are the same

1 2 5 end 3 end 3 end 5 end T A G C C G A T Figure 13.7b Figure 13.7b The double helix (part 2: chemical structure) A T 3 end 5 end 12

Single-strand binding proteins Figure 13.12 Primase Topoisomerase 3 RNA primer 5 3 5 Replication fork 3 Helicase 5 Single-strand binding proteins

Overall directions of replication Figure 13.15 Overview Leading strand Origin of replication Lagging strand Primer Leading strand Lagging strand Overall directions of replication Origin of replication 3 5 5 RNA primer 3 3 Sliding clamp DNA pol III Parental DNA Figure 13.15 Synthesis of the leading strand during DNA replication 5 3 5 5 Continuous elongation in the 5 to 3 direction 3 3 5 14

Overall directions of replication Figure 13.16a Overview Lagging strand Origin of replication Leading strand Lagging strand Leading strand Overall directions of replication Figure 13.16a Synthesis of the lagging strand (part 1) What is going to latch on at #1? 15

Primase makes RNA primer. Template strand 1 3 5 3 5 Figure 13.16b-1 1 Primase makes RNA primer. 3 5 3 Template strand 5 Figure 13.16b-1 Synthesis of the lagging strand (part 2, step 1) 16

Primase makes RNA primer. Template strand RNA primer DNA pol III Figure 13.16b-2 1 Primase makes RNA primer. 3 5 3 Template strand 5 RNA primer for fragment 1 2 DNA pol III makes Okazaki fragment 1. 3 5 3 5 Figure 13.16b-2 Synthesis of the lagging strand (part 2, step 2) 17

Where is DNA pol III going to go next?? Figure 13.16b-3 1 Primase makes RNA primer. 3 5 3 Template strand 5 RNA primer for fragment 1 2 DNA pol III makes Okazaki fragment 1. 3 5 3 5 Figure 13.16b-3 Synthesis of the lagging strand (part 2, step 3) 3 DNA pol III detaches. 3 Okazaki fragment 1 5 3 5 Where is DNA pol III going to go next?? 18

Figure 13.16c-1 RNA primer for fragment 2 Okazaki fragment 2 5 4 DNA pol III makes Okazaki fragment 2. 3 3 5 Now you have all these bits what has to happen next? And who does that? Figure 13.16c-1 Synthesis of the lagging strand (part 3, step 1) 19

RNA primer for fragment 2 Figure 13.16c-2 RNA primer for fragment 2 Okazaki fragment 2 5 4 DNA pol III makes Okazaki fragment 2. 3 3 5 5 5 DNA pol I replaces RNA with DNA. 3 3 5 Figure 13.16c-2 Synthesis of the lagging strand (part 3, step 2) 20

RNA primer for fragment 2 Figure 13.16c-3 RNA primer for fragment 2 Okazaki fragment 2 5 4 DNA pol III makes Okazaki fragment 2. 3 3 5 5 5 DNA pol I replaces RNA with DNA. 3 3 5 6 DNA ligase forms bonds between DNA fragments. Figure 13.16c-3 Synthesis of the lagging strand (part 3, step 3) 5 3 3 5 Overall direction of replication 21

Overall directions of replication Figure 13.16 Overview Lagging strand Origin of replication Leading strand Lagging strand Leading strand RNA primer for fragment 2 Overall directions of replication Okazaki fragment 2 5 4 DNA pol III makes Okazaki fragment 2. 3 1 Primase makes RNA primer. 3 5 3 3 Template strand 5 5 5 5 DNA pol I replaces RNA with DNA. 3 RNA primer for fragment 1 2 DNA pol III makes Okazaki fragment 1. 3 Figure 13.16 Synthesis of the lagging strand 5 3 3 5 6 DNA ligase forms bonds between DNA fragments. 5 5 3 3 DNA pol III detaches. 3 Okazaki fragment 1 5 3 5 3 5 Overall direction of replication 22

Leading strand template Single-strand binding proteins Leading strand Figure 13.17 Overview Origin of replication Leading strand template Leading strand Lagging strand Single-strand binding proteins Lagging strand Leading strand Overall directions of replication Leading strand Helicase DNA pol III 5 3 Primer 5 3 Primase 3 Parental DNA Lagging strand DNA pol III Figure 13.17 A summary of bacterial DNA replication 5 Lagging strand template 3 DNA pol I DNA ligase 5 3 5 23

Overall directions of replication Figure 13.17a Overview Origin of replication Leading strand Lagging strand Lagging strand Leading strand Figure 13.17a A summary of bacterial DNA replication (part 1) Overall directions of replication 24

Leading strand template Single-strand binding proteins Leading strand Figure 13.17b Leading strand template Single-strand binding proteins Leading strand Helicase DNA pol III 5 Primer 3 5 Primase 3 3 Figure 13.17b A summary of bacterial DNA replication (part 2) Parental DNA Lagging strand template 25

Lagging strand DNA pol III 5 3 DNA pol I DNA ligase 5 3 5 Figure 13.17c Lagging strand DNA pol III 5 3 DNA pol I DNA ligase 5 3 5 Figure 13.17c A summary of bacterial DNA replication (part 3) 26

DNA poly- merase Pyro- phosphate Figure 13.14 New strand Template strand 5 3 5 3 Sugar A T A T Base Phosphate C G C G DNA poly- merase G C G C 3 A T A T Figure 13.14 Addition of a nucleotide to a DNA strand P P P P i P C 3 C Pyro- phosphate Nucleotide 5 5 2 P i 27

Question 1. True of Leading strand, Lagging strand, or Both???? Daughter strand elongates away from replication fork Synthesizes 5’ to 3’ Multiple primers needed Made in segments Made continuously Daughter strand elongates toward replication fork

True of Leading strand, Lagging strand, or Both???? Daughter strand elongates away from replication fork Lag Synthesizes 5’ to 3’ Both Multiple primers needed Lagg Made in segments Lag Made continuously Lead Daughter strand elongates toward replication fork from Lead

Question 2. The diagram below shows a replication bubble with synthesis of the leading and lagging strands on both sides of the bubble. The parental DNA is shown in dark blue, the newly synthesized DNA is light blue, and the RNA primers associated with each strand are red. The origin of replication is indicated by the black dots on the parental strands. Rank the primers in the order they were produced. If two primers were produced at the same time, overlap them.

Question 3. The lagging strand is synthesized as a series of segments called Okazaki fragments Fragment A is the most recently synthesized and Fragment B will be synthesized next in the space between primers A and B. -----Start DNA polymerase III binds to 3’ end of primer B A. DNA polymerase I replaces primer with DNA B. DNA polymerase I binds to 5’ end of primer A C. DNA polymerase III moves 5’ to 3’ adding DNA nucleotides to primer B D. DNA ligase links fragments A and B

In an analysis of the nucleotide composition of DNA, which of the following will be found? A = G and C = T G + C = T + A A = C A + C = G + T

Cytosine makes up 42% of the nucleotides in a sample of DNA from an organism. Approximately what percentage of the nucleotides in this sample will be thymine? 31% 42% 8% 16% It cannot be determined from the information provided.