3 Identifying DNA as a unique molecule. 1869- Friedrich Miescher was a Swiss chemist and was the first to identify DNA as a unique molecule.Friedrich Miescher ( ) was a Swiss chemist. He studied tissue chemistry under Felix Hoppe-Seyler at the University of Tubingen, Germany. His field of study included the molecules found in blood cells. He showed that when pepsin (an enzyme that digested) proteins was used on the nucleus of cells a strange phosphorous-containing material remained. He also found that there was general ratio of phosphorous to nitrogen in this molecule. He called this molecule nuclein. Mieischer did not know that DNA or nuclein (later called nucleic acid and finally deoxyribonucleid acid) was the molecule of heredity. He still thought that proteins were the molecules of heredity.Students are not responsible for information about Miescher
4 Staining of DNA reveals somatic cells have the same amount of DNA and half as much as gametes. 1914-Robert Feulgen, a German chemist, found a staining technique that stains more or less strongly based in the amount of DNA present (called Feulgen stain). He found that all cells in an organism had the same amount of DNA except gametes, which had half the normal amount.Robert Feulgen ( ) was a German chemist.It is possible to use an instrument known as a microspectrophometer to actually measure the intensity of the pink stain for the nucleus. Using this procedure, it was easily determined that interphase cells were composed of two populations, those with haploid DNA in gametes and those with diploid DNA in the somatic cells. The nuclei looked identical, but the somatic cells contained twice as much DNA as the gametes.Later on it was found that cells that just completed mitosis had half the amount of DNA as those cells that were just about to enter mitosis. This supports the cell cycle and the DNA is replicated in interphase (S) and not during mitosis itself.Students are not tested on this historical information.
5 Staining of DNA reveals somatic cells have the same amount of DNA and half as much as gametes. Cells stained with Feulgen stain. It is the DNA and not the proteins that are visible under the microscope.
6 History of DNAFred Griffith demonstrated that bacteria could be “transformed” from one strain to another by transferring genetic factor from one organism to another. He used two different strains of the same bacteria. One could cause pneumonia and the other could not.Fred Griffith ( ) 1928-Fred Griffith performed an experiment with 2 different strains of Pneumococcus. One was virulent and the other was not. The virulent strain had a smooth polysaccharide capsule which protected from the immune system. This allowed to caused pneumonia in mice and killed them. The other strain did not have the capsule and was "rough.” This Strain could not cause pneumonia in mice. When Griffith injected the rough strain of bacteria in mice they lived, and when the smooth strain of bacteria was injected into the mice they died. He killed some of the smooth bacteria by heating them and then injecting them into the mice. The mice lived. He then took some of the killed smooth bacteria and some of the rough bacteria and mixed them together. This bacteria then had the ability to kill mice. This is because the rough bacteria had been "transformed" by taking up some of the DNA from the smooth bacteria. Griffith did not identify DNA as the molecule that was taken up but that some genetic factor has transformed the bacteria.
7 Griffith’s Experiment Fred Griffith ( ) 1928-Fred Griffith performed an experiment with 2 different strains of Pneumococcus. One was virulent and the other was not. The virulent strain had a smooth polysaccharide capsule which protected from the immune system. This protection allowed the bacteria to caused pneumonia in mice and kill them. The other strain did not have the capsule and was "rough.” The R strain could not cause pneumonia in mice. When Griffith injected the rough strain of bacteria in mice they lived, and when the smooth strain of bacteria was injected into the mice they died. He killed some of the smooth bacteria by heating them and then injecting them into the mice. The mice lived. He then took some of the killed smooth bacteria and some of the rough bacteria and mixed them together. These bacteria then had the ability to kill mice. This is because the rough bacteria had been "transformed" by taking up some of the DNA from the smooth bacteria. Griffith did not identify DNA as the molecule that was taken up but that some genetic factor has transformed the bacteria.The conclusion was that the bacteria had incorporated heredity factor from a source and in doing so expressed a new smooth trait.
8 Extending Griffith’s Experiment and Identifying DNA as the Transforming Factor Avery, MacLeod and McCarty examined the various molecules found in the S-strain Pneumococcus cells to prove that DNA was responsible for the transformation of the bacterial cells.
9 DNA is the Molecule of Heredity. When various isolated chemical components of the S-strain Pneumococcus cells was mixed the R-strain Pneumococcus cells, it was shown that the DNA from the S-strain cells, that caused trans-formation.1944-Avery, MacLeod and McCarty tried mixing the rough strain with different isolated molecules from the S strain and it was found the DNA extracted from the smooth-strain and transformed the rough strain.***Students need to know this experiment and how it was conducted and its conclusion. It needs to be pointed out that it was extending the work of Fred Griffith.Many scientists did not support the conclusion of Avery, MacLeod and McCarty and their work was ignored by many scientists.
10 Experiment of Hershey and Chase Alfred Hershey and Martha Chase demonstrated the genetic material is DNA by using viruses that infect bacteria. These viruses only stay on the outside of the cell when infecting the cells. Also viruses are composed of protein and DNA. It is known that the virus injects its genetic material into the bacterium which had to DNA or proteins.*** Students need to be familiar with this experiment.
11 Experiment of Hershey and Chase Trial 1Replicate phages with radioactive sulfur (labels nucleotides) which is found in proteins and not DNA. These are now labeled phages for their radioactive proteins.Mix radioactively phages with bacteria. Allow them to infect cells.Put mixture in blender to separate infected cells from the viruses on the outside of the bacteria.Centrifuge the mixture to separate and layer viruses and infected bacteria. Determine if the shell of the virus is radioactive OR if the bacteria are radioactive.ConclusionIf the bacteria are radioactive, then proteins are the molecules of heredity. The bacteria were not radioactive the empty viral coats were radioactive.Trial 2Repeat the experiment above but this time replicate the viruses and with radioactive phosphorus. This labels the DNA and not the proteins. These viruses are now labeled phages for their radioactive DNA.When the experiment is complete and if the bacteria are radioactive, then DNA are the molecules of heredity. The bacteria were radioactive and the empty viral coats were not radioactive.ResultsIn the second trial, the bacteria were radioactive. So it can be concluded that DNA is the molecule of heredity.This is a great experiment because the experiment took advantage of differences the structure of DNA and proteins.It demonstrated that DNA is the material that genes are made of and not protein.
12 Chargaff's Rule -> A+G=C+T=50% Experiment ChargaffChargaff's Rule -> A+G=C+T=50%Percentage of Various Nucleotides in GenomeOrganismsATGCHumans30.929.419.919.5Wheat27.327.122.722.8Sarcina lutea13.412.437.1T726.32623.823.9Erwin Chargaff ( ) was a Austrian biochemist that immigrated to the U.S. during W.W. II. He was a professor at Columbia University. Using paper chromatography and using a ultraviolet spectrophotometer, Chargaff was able to demonstrate that in a given organism the number of adenine nucleotides was approximately the same as the number of guanine nucleotides and that the number of cytosine nucleotides was approximately the same as the number of thiamine nucleotides. This is called Chargaff’s first rule. His second rule was based on the observation that these percentages were unique for various species.Students are responsible for this information.Based on the observations above, two rules can be deducedA+G=C+T=50%.The percentages of the nucleotide vary for different species
13 Work of Rosalind Franklin Rosalind Franklin worked in a lab with Maurice Wilkins. She was investigating the structure of DNA using x-ray crystallography. She determined that the phosphate groups were on the outside of the double helix. She did not determine the base of DNA pairing of DNA or their role in the structure of DNA.Rosalind Franklin used x-ray crystallography to determine that DNA was double stranded, a helix, phosphates were on the outside and three distances, 2.0 nm, .34 nm, and 3.4 nm showed up in a pattern over and over again in the diffraction pattern.
14 Work of James Watson and Francis Crick Based on the rules of Chargaff and the information from the work of Franklin, James Watson and Francis Crick, determined the structure of DNA by making models.Determined that the sugar and phosphates were on the outside.2. Determined that the nitrogenous bases were forming the rungs of the ladder.Watson and Crick determined that 2.0 nm was the distance from one strand to the other nm was the distance from one base pair to another and finally 3.4 nm determined that there were 10 bases to a complete twist in the helix. So with 2.0 nm from one strand to the other, it was determined that the a purine had had to be base paired with a pyrimidine. Next problem to investigate is why did adenine base pair with guanine and why did cytosine base pair with thymine? The answer had to do with the hydrogen bonding, as adenine base paired with thymine because they could form two hydrogen bonds and guanine base paired with cytosine because they could form three hydrogen bonds.Watson, Crick, and Wilken received the Nobel Prize in Unfortunately, Franklin died of cancer at age 38 in Watson, Crick, and Wilkens received the Nobel Prize in 1962.
15 Determining the Nitrogen Base Pairing Based on the work Franklin’s x-ray crystallography, Watson and Crick found the bonding;two purines are too wide and would overlap.two pyrimidines are too far apart to form the hydrogen bonds.a purine and a pyrimidine however, are just right!Also due to the hydrogen bonding, A and T forms two hydrogen bonds and C and G forms three hydrogen bonds. That is the only way they hydrogen bonds will fit.
16 Chargaff’s SnubChargaff felt there had been an injustice done when he did not receive the Nobel Prize in 1962 along with Watson, Crick and Wilkins. Wilkins’ contribution to the structure of DNA was to show James Watson the work of Rosalind Franklin without her permission. Franklin did not share the Nobel Prize as she passed away from ovarian cancer in 1958 and posthumous nominations are forbidden.Chargaff is quoted as saying, “I told them all I knew. If they had heard before about the pairing rules, they concealed it. But as they did not seem to know much about anything, I was not unduly surprised. I mentioned our early attempts to explain the complementarity relationships by the assumption that, in the nucleic acid chain, adenylic was always next to thymidylic acid and cytidylic next to guanylic acid...I believe that the double-stranded model of DNA came about as a consequence of our conversation.*”
17 Structure of a Nucleotide DNA is the longest molecule found in the cell, yet its structure is quite simple. The human cell contains 5-6 feet of DNA in every cell autosomal cell. The basic building blocks of nucleic acids are the nucleotides. There are 3 billion base pairs or 6 billion nucleotides in a human cell.Have the students compare and contrast the differences between purines and pyrimidines and point out the differences. Purines have two rings whereas pyrimidines only have one ring. Adenine and guanine are purines and cytosine, thymine, and uracil are pyrimidinesHave the students compare and contrast the differences between ribose and deoxyribose. Deoxyribose is missing an atom of oxygen.Be sure to go over how the carbons are numbered and emphasize that # 3 and # 5 are important and termed 3’ or three prime and 5’ or five prime.The significance of the various carbons are listed below:Carbon #1 is where the nitrogenous base is attached.Carbon #2 is what differentiates between ribose (C5H10O5) which has a hydroxyl group attached (OH) and deoxyribose (C5H10O4) has only hydrogen (H). Hence the name deoxyribose makes sense.Carbon #3 is where the next nucleotide will attach.Carbon #5 is the phosphate group is attached.
18 Sides of the LadderDNA is a double stranded and analogous to a ladder. The sides of the ladder are composed of alternating sugars (deoxyribose) and phosphate groups that run antiparallel to one another. On the left side (next card) the first carbon found on the strand is #5 and moving on down the last carbon is carbon # 3. This side is said to be 5'-3'. The opposite side is upside down compared to the other side. The right hand side, the first carbon found on the strand is #3 and moving on down the last carbon is carbon # 5. This side is said to be 3'-5'.
19 Hydrogen Bonding and Nitrogenous Bases The nitrogenous bases form the rungs of the ladder. Thymine will base pair with adenine on the opposite side, which is a pyrimidine base paired with a purine. This will form 2 hydrogen bonds. Hydrogen bonds are weak but millions of them together will keep the two strands together.
20 Hydrogen Bonding and Nitrogenous Bases Guanine will base pair with cytosine on the opposite side. This is a pyrimidine base paired with a purine. This will form 3 hydrogen bonds instead of 2 hydrogen bonds.
21 Hydrogen Bonding and Nitrogenous Bases This will continue for billions of base pairings forming a molecule of DNA.
22 Hydrogen Bonding and Nitrogenous Bases The DNA molecule once formed will make a double helix. There are 10 base pairs in one complete turn of the DNA molecule.
23 Forming the Double Helix There are 10 base pairs in one turn of the DNA molecule. The length of one complete turn is 3.4 nm. The radius of the spiral is 1 nm. The distance between nitrogenous bases is .34 nm.The spiral shows a large groove called the major groove and a small groove call the minor groove. Students do not have to know the measurements or the information about the grooves.
24 DNA Forming Chromosomes Structure in eukaryotes.the DNA is wrapped around proteins called histones forming nucleosomes.This forms a fiber known as chromatin.This forms a coil within a coil.The final structure is a chromosome which can only be seen during mitosis. During the rest of the cell cycle, the DNA is in chromatin state.
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