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1 DNA: The Genetic Material A Short history of DNA.

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1 1 DNA: The Genetic Material A Short history of DNA

2 2 Classic Genetics When Mendel came out with his laws in 1865, he used the term factors to describe what we today call genes. Genes were not known at that time.Actually not much about cell was known even when his work was rediscovered in Then when scientists started to explore more about genetic factors they thought the genes were located in the cytoplasm. ( cytoplasmic theory of inheritance) Chromosomes were known but most scientists belived genes were located somewhere in cytoplasm. Not much about chromosomes cell division was known which added to the strength of that theory. Then Sutton (1902) came out with his chromosomal theory of inheritance which stated that genes were located on the chromosomes. Many scientists followed with many experiments which established the chromosomal theory of inheritance as a irrefutable fact.

3 3 Suttons Reasoning Sutton made two very important points in favour of his theory. It was known from breeding experiments that both parents contributed equally to the offspring. If that was the case cytoplasm couldnot have the genes as the egg and the sperms have very different amount of cytoplasm. Eggs are very large and mostly cytoplasm. Sperms are very small with almost no cytoplasm. ( very minimal cytoplasm). However the nucleus of both had the same amount of materials. He reasoned that if both parents contributed the same it the genes had to come from the nucleus. Parallelism between meiosis and the behavior of the genes. Genes are in pairs as do the chromosomes. Half genes come from one parent the other half from the other parent as in fertilization- half chromosomes come from egg the other half from the sperm. There are many other parallelisms between the behavior of genes and chromosomes during meiosis.

4 4 Road to DNA Discovery Hammerling's experiment with the single celled green algae, Acetabularia, showed that the nucleus of a cell contains the genetic information that directs cellular development. A. mediterranea has a smooth, disc shaped cap, while A. crenulata has a branched, flower-like cap. Each Acetabularia cell is composed of three segments: the "foot" or base which contains the nucleus, the "stalk," and the "cap." In his experiments, Hammerling grafted the stalk of one species of Acetabularia onto the foot of another species. In all cases, the cap that eventually developed on the grafted cell matched the species of the foot rather than that of the stalk. In this example, the cap that is allowed to grow on the grafted stalk looks like the base species one... A. mediterranea. This experiment shows that the base is responsible for the type of cap that grows. The nucleus that contains genetic information is in the base, so the nucleus directs cellular development.genetic information

5 5 Hammerling's Acetabularia

6 6 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

7 7 The Genetic Material Griffiths 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

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9 9 The Genetic Material Griffiths 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

10 10 The Genetic Material Avery, MacLeod, & McCarty, 1944 repeated Griffiths 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

11 11 The Genetic Material Hershey & Chase, 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

12 12 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

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14 14 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

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16 16 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

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18 18 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.

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20 20 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 Chargaffs Rules

21 21 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

22 22 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

23 23 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

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25 25 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.

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27 27 DNA Replication Matthew Meselson & Franklin Stahl, 1958 investigated the process of DNA replication considered 3 possible mechanisms: –conservative model –semiconservative model –dispersive model

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29 29 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

30 30 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

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32 32 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.

33 33 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

34 34 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.

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36 36 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

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38 38 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

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40 40 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.

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42 42 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.

43 43 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.

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45 45 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

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47 47 DNA Repair - DNA-damaging agents - repair mechanisms - specific vs. nonspecific mechanisms

48 48 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.

49 49 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

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