12 E. coli grown in 15 N medium E. coli shifted to 14 N medium Samples taken at 0, 1 generation, and 2 generations (replication rounds) samples centrifuged at high speeds The positions of radioactive 15 N in the centrifuge tubes are visualized by placing tubes against photographic film
25 The Genetic Material Frederick Griffith, 1928 studied Streptococcus pneumoniae, a pathogenic bacterium causing pneumonia there are 2 strains of Streptococcus: - S strain is virulent - R strain is nonvirulent Griffith infected mice with these strains hoping to understand the difference between the strains
26 The Genetic Material Griffith’s results: - live S strain cells killed the mice - live R strain cells did not kill the mice - heat-killed S strain cells did not kill the mice - heat-killed S strain + live R strain cells killed the mice
27 The Genetic Material Griffith’s conclusion: - information specifying virulence passed from the dead S strain cells into the live R strain cells - Griffith called the transfer of this information transformation
28 The Genetic Material Avery, MacLeod, & McCarty, 1944 repeated Griffith’s experiment using purified cell extracts and discovered: - removal of all protein from the transforming material did not destroy its ability to transform R strain cells - DNA-digesting enzymes destroyed all transforming ability - the transforming material is DNA
29 The Genetic Material Hershey & Chase, 1952 - investigated bacteriophages: viruses that infect bacteria - the bacteriophage was composed of only DNA and protein - they wanted to determine which of these molecules is the genetic material that is injected into the bacteria
30 The Genetic Material - Bacteriophage DNA was labeled with radioactive phosphorus ( 32 P) - Bacteriophage protein was labeled with radioactive sulfur ( 35 S) - radioactive molecules were tracked - only the bacteriophage DNA (as indicated by the 32 P) entered the bacteria and was used to produce more bacteriophage - conclusion: DNA is the genetic material
31 DNA Structure DNA is a nucleic acid. The building blocks of DNA are nucleotides, each composed of: –a 5-carbon sugar called deoxyribose –a phosphate group (PO 4 ) –a nitrogenous base adenine, thymine, cytosine, guanine
32 DNA Structure The nucleotide structure consists of –the nitrogenous base attached to the 1’ carbon of deoxyribose –the phosphate group attached to the 5’ carbon of deoxyribose –a free hydroxyl group (-OH) at the 3’ carbon of deoxyribose
33 DNA Structure Nucleotides are connected to each other to form a long chain phosphodiester bond: bond between adjacent nucleotides –formed between the phosphate group of one nucleotide and the 3’ –OH of the next nucleotide The chain of nucleotides has a 5’ to 3’ orientation.
34 DNA Structure Determining the 3-dimmensional structure of DNA involved the work of a few scientists: –Erwin Chargaff determined that amount of adenine = amount of thymine amount of cytosine = amount of guanine This is known as Chargaff’s Rules
35 DNA Structure Rosalind Franklin and Maurice Wilkins –Franklin performed X-ray diffraction studies to identify the 3-D structure –discovered that DNA is helical –discovered that the molecule has a diameter of 2nm and makes a complete turn of the helix every 3.4 nm
36 DNA Structure James Watson and Francis Crick, 1953 –deduced the structure of DNA using evidence from Chargaff, Franklin, and others –proposed a double helix structure
37 DNA Structure The double helix consists of: –2 sugar-phosphate backbones –nitrogenous bases toward the interior of the molecule –bases form hydrogen bonds with complementary bases on the opposite sugar-phosphate backbone
38 DNA Structure The two strands of nucleotides are antiparallel to each other –one is oriented 5’ to 3’, the other 3’ to 5’ The two strands wrap around each other to create the helical shape of the molecule.
39 DNA Replication Matthew Meselson & Franklin Stahl, 1958 investigated the process of DNA replication considered 3 possible mechanisms: –conservative model –semiconservative model –dispersive model
40 DNA Replication Bacterial cells were grown in a heavy isotope of nitrogen, 15 N all the DNA incorporated 15 N cells were switched to media containing lighter 14 N DNA was extracted from the cells at various time intervals
41 DNA Replication The DNA from different time points was analyzed for ratio of 15 N to 14 N it contained After 1 round of DNA replication, the DNA consisted of a 14 N- 15 N hybrid molecule After 2 rounds of replication, the DNA contained 2 types of molecules: –half the DNA was 14 N- 15 N hybrid –half the DNA was composed of 14 N
42 DNA Replication Meselson and Stahl concluded that the mechanism of DNA replication is the semiconservative model. Each strand of DNA acts as a template for the synthesis of a new strand.
43 DNA Replication DNA replication includes: –initiation – replication begins at an origin of replication –elongation – new strands of DNA are synthesized by DNA polymerase –termination – replication is terminated differently in prokaryotes and eukaryotes
44 Prokaryotic DNA Replication The chromosome of a prokaryote is a circular molecule of DNA. Replication begins at one origin of replication and proceeds in both directions around the chromosome.
45 Prokaryotic DNA Replication The double helix is unwound by the enzyme helicase DNA polymerase III (pol III) is the main polymerase responsible for the majority of DNA synthesis DNA polymerase III adds nucleotides to the 3’ end of the daughter strand of DNA
46 Prokaryotic DNA Replication DNA replication is semidiscontinuous. –pol III can only add nucleotides to the 3’ end of the newly synthesized strand –DNA strands are antiparallel to each other leading strand is synthesized continuously (in the same direction as the replication fork) lagging strand is synthesized discontinuously creating Okazaki fragments
47 Prokaryotic DNA Replication The enzymes for DNA replication are contained within the replisome. The replisome consists of –the primosome - composed of primase and helicase –2 DNA polymerase III molecules The replication fork moves in 1 direction, synthesizing both strands simultaneously.
48 Eukaryotic DNA Replication The larger size and complex packaging of eukaryotic chromosomes means they must be replicated from multiple origins of replication. The enzymes of eukaryotic DNA replication are more complex than those of prokaryotic cells.
49 Eukaryotic DNA Replication Synthesizing the ends of the chromosomes is difficult because of the lack of a primer. With each round of DNA replication, the linear eukaryotic chromosome becomes shorter.
50 Eukaryotic DNA Replication telomeres – repeated DNA sequence on the ends of eukaryotic chromosomes –produced by telomerase telomerase contains an RNA region that is used as a template so a DNA primer can be produced
51 DNA Repair - DNA-damaging agents - repair mechanisms - specific vs. nonspecific mechanisms
52 DNA Repair Mistakes during DNA replication can lead to changes in the DNA sequence and DNA damage. DNA can also be damaged by chemical or physical agents called mutagens. Repair mechanisms may be used to correct these problems.
53 DNA Repair DNA repair mechanisms can be: –specific – targeting a particular type of DNA damage photorepair of thymine dimers –non-specific – able to repair many different kinds of DNA damage excision repair to correct damaged or mismatched nitrogenous bases