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-Chapter 11 DNA: The Carrier of Genetic Information Lecture Outline I. Most genes carry information for making proteins A. 1908, Archibald Garrod proposed that genes are responsible for enzymes “Inborn Errors of Metabolism” B. George Beadle and Edward Tatum in the 1940s worked on Neurospora 1. One mutant gene affected 1 single step in a metabolic pathway 2. The “one gene, one enzyme hypothesis” a. Now known that some genes encode proteins that are not enzymes; work of Pauling and others b. Also known that one gene is responsible for one polypeptide chain 3. The “one gene, one polypeptide hypothesis” II. Evidence that DNA was the hereditary material was first found in microorganisms A. Early biologist believed that the genetic material was a protein B. Frederick Griffith in 1928 studied pneumococcus in mice 1. Smooth (S) strain was virulent 2. Rough (R) strain was not virulent 3. Heat-killed S strain was not virulent 4. A combination of heat-killed S and R strain was virulent 5. Something caused the R strain to become virulent (i.e. transformation) C. This “transforming principle” was later identified by Avery and colleagues in 1944 as DNA D. Alfred Hershey and Martha Chase’s experiments on bacteriophages 1. Labeled the protein coat with radioactive sulfur, the DNA with radioactive phosphorus 2. Found that only the phosphorus was found in the bacterium after infection with the bacteriophages; providing further evidence that DNA is the genetic material III. The structure of DNA allows it to carry information and to be faithfully duplicated A. Nucleotides can be covalently linked in any order to form long polymers 1. DNA is a nucleotide-based molecule containing deoxyribose, phosphate, and a nitrogen-containing base 2. The bases of DNA are purines (adenine and guanine) and pyrimidines (thymine and cytosine) 3. Nucleotides are covalently bonded with a sugar-phosphate backbone a. The linkage forms a 3’, 5’ phosphodiester linkage b. One end of the molecule has a free 5’ carbon; the other end has a free 3’ carbon B. DNA is made of two polynucleotide chains intertwined to form a double helix 1. Rosalind Franklin and Maurice Wilkins did xray diffraction analysis on DNA a. Results showed that the molecule was helical, and had several repetitive elements at 0.34 nm, 3.4 nm, and 2.0 nm apart b. They deduced that the relatively flat base molecules were stacked like the rungs of a ladder For 3d image of DNA click the following link: http://www.google.com/imgres?imgurl=http://www.worldofmolecules.com/life/dna.gif&imgrefurl=h ttp://www.worldofmolecules.com/life/dna.htm&h=341&w=457&sz=32&tbnid=9Ub1J9karkMJ::&tb nh=96&tbnw=128&prev=/images%3Fq%3Ddna%2Bmolecule%2Bdimensions%2Bimage&usg=__ EyQV_Lkvc5BFEAAJUCISad0C7-s=&sa=X&oi=image_result&resnum=4&ct=image&cd=1 2. James Watson and Francis Crick deduced the correct structure of DNA a. The molecule is a double helix of antiparallel strands made up of a backbone of sugars and phosphates b. The 0.34 nm repeat is due to the distance between the bases c. The 3.4 nm repeat is due to the distance between a complete “turn” of the helix d. The 2.0 nm represents the width of the molecule C. In double-stranded DNA, hydrogen bonds form between adenine and thymine and between guanine and cytosine 1. Edwin Chargaff determined that the ratio of adenine to thymine and the ratio of guanine to cytosine was always the same in all organisms tested 2. Pyrimidines are single-ring molecules; purines have double-rings a. Two hydrogen bonds form between thymine and adenine b. Three hydrogen bonds form between cytosine and guanine 3. The bonding of bases is complimentary; the sequence in one chain dictates the sequence in the opposite chain IV. DNA replication is semi conservative A. The structure of DNA clearly indicated the copying mechanism 1. “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material” (quote from their Nature article) 2. Matthew Meselson and Franklin Stahl (1957), using e-coli disproved the model of dispersive replication and showed semi conservative replication a. Grew the bacteria in medium with nitrogen-15 b. Transferred to medium with nitrogen-14 c. Isolated the DNA and found that some of the DNA had only nitrogen-15, some had only nitrogen-14, and some had both B. DNA replication is complex and has a number of unique features 1. Proteins and enzymes work together in replication 2. DNA strands must be unwound during replication a. DNA helicase enzymes unwind the strands b. Helix destabilizing proteins prevent immediate reformation of the double helix c. Topoisomerases break and rejoin the strands, “untying” the knots that form 3. DNA synthesis always proceeds in a 5’3’ direction a. DNA polymerases can add only at the 3’ end b. Nucleotides become polymerized and two phosphates are removed in the process 4. DNA synthesis requires an RNA primer The RNA primer is first synthesized by the primosome 5. DNA replication is discontinuous in one strand and continuous in the other a. Replication begins at replication forks b. Synthesis of leading strand is continuous, the lagging strand is synthesized in pieces called Okazaki fragments c. Okazaki fragments are between 100 and 2000 nucleotides in length, each is initiated by a separate RNA primer d. Okazaki fragments are joined together by DNA ligase 6. Most DNA synthesis is bidirectional a. In prokaryotes, the circular DNA is opened up, and synthesis occurs in both directions b. In eukaryotes, the linear DNA has many replication forks c. The ends of eukaryotic chromosomes, the telomeres, present problems in replication V. DNA in chromosomes is packaged in a highly organized way A. The genome of E. Coli consists of about 4 x 106 base pairs – about 1.35 mm in length B. The haploid DNA of a human cell is made of 3 x 109 base pairs – about 1 m ( over 3 ft.) in length C. In eukaryotes, DNA is associated with positively charged histone proteins to form bead-like nucleosomes 1. Each nucleosome is composed of about 140 base pairs wrapped around 8 histones, plus a linker segment of DNA and another histone 2. Other scaffolding proteins aid in the formation of chromosomes