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The Molecular Basis of Inheritance Chapter 16 – AP Biology The Search for the Genetic Material • From Previous Chapters: – Sutton said the genes were on the chromosomes – Morgan proved this with his work with fruit flys • Still, it was unknown what PART of the chromosomes carried the genes… Chromosome Structure • Chromosomes are composed of two main chemicals – Nucleic Acid (DNA) – Protein Which part of the Chromosomes carries the Genetic Material? • While it was widely believed (thanks to Sutton and Morgan) that the genes were located on the chromosomes, it was unknown which chemical component (nucleic acid or protein) actually carried them. • Up until the middle part of the 20th century, it was thought that the protein parts of the chromosomes were most likely the genetic material. – This was because proteins were being found at Experiments Leading to DNA as the Genetic Material • (Bacteria were found to be the organisms best suited for determining the nature of the genetic material) Griffith and Avery • Griffith killed virulent (disease causing bacteria • He mixed the remains of the killed bacteria with living, harmless bacteria • Upon exposure to the dead virulent bacteria, the harmless bacteria were transformed into virulent bacteria • Griffith concluded that some component of the dead cells was causing an inheritable change in the living, harmless cells. • This was called transformation. • The question remained: – What was causing the transformation? Griffith and Avery • Avery took Griffith’s work further. • He isolated and purified the different chemicals in the killed bacteria • He exposed the harmless bacteria to each different chemical that he obtained from the killed virulent bacteria • The only chemical that caused the harmless bacteria to transform was the DNA. • Still, the results were not widely Diagram: Griffith and Avery’s Experiment Fred Griffith (with “Bobby”) and Oswald Avery Griffith and Avery • They never met, though they did respect each other’s work • Griffith was English and was killed in the Blitz in London in 1941. • Avery was American and lived from 1877 to 1955. • For more click HERE. Hershey and Chase • Worked with Bacteriophages – Viruses that infect bacteria – Viruses are composed of • Protein • Nucleic Acid Alfred Hershey and Martha Chase Hershey and Chase • Designed and experiment to determine, without question, which component of a bacteriophage infected a bacterium – the protein or the nucleic acid Hershey and Chase • Tagged the protein coat of the virus with one type of radioactive marker that they could see and follow • Tagged the DNA of the virus with a different radioactive marker that they could see and follow • Allowed bacteria to be infected by the tagged viruses • Observed which markers actually ended up inside the bacteria Diagram: Hershey and Chase’s Experiment Discovering the Structure of DNA • Once it was determined that DNA was the genetic material, the next step was to determine the structure of the molecule • It was hoped once the structure was determined, clues would be evident about how the molecule worked. Scientists Working on the DNA Molecule in the 1950s • Linus Pauling – Very well known scientist – Made many important discoveries in molecular biology • Especially important discoveries about proteins • Discovered the alpha helix shape of some protein molecules – Still, he ultimately “lost” the race in determining the structure of DNA Scientists Working on the DNA Molecule in the 1950s • Rosalind Franklin – Working the the lab of Maurice Wilkins at King’s College (in England) – Made excellent X-ray crystallography images of the DNA molecule that were ultimately used to discover DNA’s structure Photo 51 • The x-ray diffraction image made by Franklin and shown to Watson by Maurice Wilkins Scientists Working on the DNA Molecule in the 1950s • Maurice Wilkins – It was Wilkins’ lab in which Rosalind Franklin came to work at King’s College – Was, along with Watson and Crick, awarded the Nobel Prize. Scientists Working on the DNA Molecule in the 1950s • James Watson – Very young American – Came to study at the Cavendish in England – Interested in DNA – Using Rosalind Franklin’s DNA images, he and Francis Crick determined DNA’s structure and were awarded the Nobel Prize. Scientists Working on the DNA Molecule in the 1950s • Francis Crick – British – Ph.D student at at the Cavendish Laboratory in England. – Shared a common interest with James Watson in DNA – Using Rosalind Franklin’s DNA images, he and Watson determined DNA’s structure and were awarded the Nobel Prize Puzzle of DNA Structure • Initially, Watson tried to make the bases fit together “like with like” • Purine + purine was too wide and pyrimidine + pyrimidine was too narrow – the structure simply could not fit together. • Eventually he realized that purine and pyrimidine would fit together were like with like would not. Puzzle of DNA Structure • Figuring out the A-T and C-G arrangement of DNA also explained “Chargaff’s Rules”. – That A’s and T’s occurred in a 1:1 ratio as did C’s and G’s. DNA Structure • Though the rungs of the DNA ladder must always be A-T and C-G, the SEQUENCE of these bases along the length of the strand is NOT restricted. • Thus the sequence can be varied in many ways, allowing for nearly unlimited variety among organisms. Announcement of the Structure of DNA • Watson and Crick announced their findings in the April 1953 issue of the journal “Nature”. • Their article was only one page long. • The beauty of the model that Watson and Crick described was that it immediately suggested a mechanism by which DNA could replicate itself DNA Structure • DNA is a NUCLEIC ACID • Nucleic Acids are POLYMERS • The MONOMERS that make up Nucleic Acid polymers are NUCLEOTIDES. Nucleotide Structure • Each nucleotide is composed of 3 parts – Sugar – Nitrogenous base – Phosphate group • Variation only occurs only in nitrogenous base The Bases - Two Families – Purines - LARGER (TWO Rings) • Adenine • Guanine – Pyrimidines -(smaller and one ring) • Cytosine • Thymine - (DNA) • Uracil - (RNA) The Sugar • 5 carbon sugar (also known as a pentose sugar) – DNA - deoxyribose – RNA - ribose • carbons are numbered to indicate position The Phosphate Group • The phosphate group is always attached to the 5’ carbon Linking Nucleotides together to make DNA • Covalent bonds link one nucleotide to the next – a phosphodiester bond • Formed between sugar of one nucleotide and phosphate group of the next nucleotide Double Helix • The DNA molecule is composed of two “polynucleotide” molecules that spiral around an imaginary axis. • This is known as a “double helix”. • Click here for another DNA model site. Double Helix Structure • Sugar/phosphate (backbones) are on the outside of the helix • Nitrogenous bases are paired in the inside of the helix • Hydrogen bonds between bases hold the two strands of DNA together Double Helix Structure • Only certain bases are compatible with each other – Purine + Pyrimidine • Cytosine + Guanine • Adenine + Thymine • (RNA = Adenine matches with Uracil) Double Helix Structure • Complementarity • – The two strands of the double helix are said to be complementary. – This means that each is a predictable counterpart of the other. Antiparallel means that sugar/phosphate backbones run in opposite directions. DNA Replication • During replication, the pairing of the bases enables existing DNA strands to serve as templates for new, complementary strands. DNA Replication • The first step in replication is the separation of the strands DNA Replication • The second step in replication – Each “old” strand serves as a template that the determines the order of nucleotides along the new complementary strands. DNA Replication • The third step in replication – The nucleotides are connected to for the sugar/phosphate backbones of the new strands. Each DNA molecule now consists of one old strand and one new strand DNA Replication • Each DNA molecule completed in replication is identical to the parent molecule • Term for the two daughter DNA molecules that are composed of one OLD strand and one NEW strand = SEMICONSERVATIVE Semiconservative Model of Replication (diagram b) How replication of DNA is carried out • A large team of Enzymes and other proteins carries out the process of DNA replication Keep in mind… • There are 46 DNA molecules (that is, chromosomes) in each of your cells • That’s 6 billion base pairs • It would take about 900 AP Biology books to print it all out (A’s, T’s, C’s and G’s) • It takes a cell just a few hours to copy all of that information • And the cells are VERY good at it – only 1 error per BILLION nucleotides, on Replication – the Process – Page 301 • Origins of Replication – Sites on the chromosome where replication begins – A bacterial chromosome has only one origin of replication – Eukaryotic chromosomes have multiple origin sites. • This makes replication faster Origin of Replication • stretch of DNA that has a specific sequence of nucleotides • Proteins that initiate DNA replication recognize this sequence and attach to the DNA at these points. • This separates the DNA into two strands and opens up a replication bubble. Origins of Replication - Diagram What happens at a replication bubble? • Replication occurs in both directions from the origin • Eukaryotic cells - multiple (hundreds of thousands) replication bubbles fuse. – Multiple bubbles speeds up replication What is a replication fork? • Y-shaped region at either end of a replication bubble • New strands of DNA are being elongated at these points. Elongating the DNA Strand • DNA Polymerase – the enzyme that catalyzes the elongation of new DNA at a replication fork. • elongates a DNA strand at rates of – 500 nucleotides/sec in bacteria – 50 nucleotides/sec in human cells Elongating the DNA Strand - Energy • a nucleotide is a triphosphate. • Cleavage of phosphate groups from the nucleotides themselves provide energy for their attachment to the strand. • Remember ATP! Explanation of Antiparallel Strands • two sugar/phosphat e backbones that make up a DNA double helix are arranged “upside down” or antiparallel to each other. Why Do We Care?? • Important in the mechanism of replication • DNA Polymerase can ONLY ADD NUCLEOTIDES TO THE 3’ END OF AN ELONGATING DNA MOLECULE – new DNA strand can elongate ONLY IN THE 3’ DIRECTION – NEVER elongate in the 5’ DIRECTION First Look at Replication • VIEW ANIMATION 54 How DNA Polymerase works… • Along ONE of the template (old/parent) strands of DNA, DNA Polymerase can create a CONTINUOUS complementary strand – elongating the new DNA in the 5’ to 3’ direction – CALLED the LEADING strand. How DNA Polymerase works… • On the OTHER side of the replication fork, the process is DIFFERENT. – This is because DNA Polymerase can ONLY add to the 3’ end of the elongating strand. • DNA Polymerase must work in the Tet direction leading AWAY from the • The strand fork. replication made in this direction is called the LAGGING strand. How the Lagging Strand is Made… • Replication bubble opens • DNA polymerase molecule can work away from the fork and make a short segment of DNA. • As the bubble opens up a bit more, polymerase can leap frog back up the fork and slide back out of the fork again until it bumps into the strand it just made • Thus, the lagging strand is made in a series of short segments – Okazaki fragments – 100-200 nucleotides long in eukaryotes More Problems for DNA Polymerase… • Additionally, DNA polymerase CANNOT initiate the synthesis of a strand of DNA • DNA Polymerase can ONLY add nucleotides to a pre-existing polynucleotide Solution… • Another enzyme, this one called primase, places a short stretch of RNA that is complementary to the template strand. • Provides a “hook” upon which DNA polymerase can “hang” the nucleotides needed to elongate the new DNA strand • RNA “hook” called the “Primer” More on Primers and Primase • Only 1 primer is required for the synthesis of the LEADING DNA strand. • However, for the LAGGING strand, each new segment created must have its own primer Other Enzymes and Proteins in Replication – Page 304 • Helicase – enzyme that untwists the DNA molecule at the site of the replication fork and separates the two “old” strands. • Single strand binding proteins – enzymes that hold the open strands of the template DNA apart while new strands are being made. • DNA Ligase – joins the 3’ end of DNA to rest of the strand Repairing Enzymes – page 305 • Nuclease – cuts out damaged strands of DNA