Molecular Genetics


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Molecular Genetics

  1. 1. Molecular Genetics The Central Dogma of Biology
  2. 2. What is a Genetic “Factor”? <ul><li>From Mendel: </li></ul><ul><ul><li>we now accepted that there was genetic transmission of traits. </li></ul></ul><ul><li>Traits are transmitted by “factors” </li></ul><ul><ul><li>Organisms carry 2 copies of each “factor” </li></ul></ul><ul><li>The question now was: what is the factor that carries the genetic information? </li></ul>
  3. 3. Requirements of Genetic Material <ul><li>Must be able to replicate , so it is reproduced in each cell of a growing organism. </li></ul><ul><li>Must be able to control expression of traits </li></ul><ul><ul><li>Traits are determined by the proteins that act within us </li></ul></ul><ul><ul><li>Proteins are determined by their sequences </li></ul></ul><ul><li>Therefore, the genetic material must be able to encode the sequence of proteins </li></ul><ul><li>It must be able to change in a controlled way, to allow variation, adaptation, thus survival in a changing environment. </li></ul>
  4. 4. Chromosomes – The First Clue <ul><li>First ability to visualize chromosomes in the nucleus came at the turn of the century </li></ul><ul><ul><li>construction of increasingly powerful microscopes </li></ul></ul><ul><ul><li>the discovery of dyes that selectively colored various components of the cell </li></ul></ul><ul><li>Scientists examined cellular nuclei and observed nuclear structures, which they called chromosomes </li></ul><ul><li>Observation of these structures suggested their role in genetic transmission </li></ul>
  5. 5. Chromosome Observations <ul><li>Variety of chromosome types found in the nucleus </li></ul><ul><li>2 copies of each type of chromosome in most cells. </li></ul><ul><li>All of the cells of an organism, except gametes and rbc’s, have the same number of chromosomes. </li></ul><ul><li>All organisms of the same species have the same number of chromosomes. </li></ul><ul><li>The number of chromosomes in a cell doubles immediately prior to cell division </li></ul><ul><li>Gametes have exactly half of the number of chromosomes as the somatic cells of any organism. </li></ul><ul><ul><li>Gametes have just one copy of each chromosome type. </li></ul></ul><ul><ul><li>Fertilization produces a diploid cell (a zygote), with the same number of chromosomes as somatic cells of that organism. </li></ul></ul>
  6. 6. Implications <ul><li>Chromosomes behaved like Mendel’s “factors” </li></ul><ul><ul><li>Mendel's hereditary factors were either located on the chromosomes or were the chromosomes themselves. </li></ul></ul><ul><li>Proof chromosomes were hereditary factors – 1905: </li></ul><ul><li>The first physical trait was linked to the presence of specific chromosomal material </li></ul><ul><ul><li>conversely, the absence of that chromosome meant the absence of the particular physical trait. </li></ul></ul><ul><li>Discovery of the sex chromosomes </li></ul><ul><ul><li>&quot;X&quot; and &quot;Y.&quot; </li></ul></ul><ul><ul><li>distinguished from other chromosomes and from each other </li></ul></ul>
  7. 7. Importance of Sex Chromosomes <ul><li>Somatic cells taken from female donors always contained 2 copies of the X chromosome </li></ul><ul><li>Somatic cells taken from male donors always contained one copy of the X chromosome and one copy of the Y chromosome </li></ul><ul><li>All of the other chromosomes in the nucleated cells of both male and female donors appeared identical </li></ul><ul><li>Thus, gender was shown to be the direct result of a specific combination of chromosomal material </li></ul><ul><ul><li>The first phenotype (physical characteristic) to be assigned a chromosomal location </li></ul></ul><ul><ul><li>Specifically the X and Y chromosomes. </li></ul></ul>
  8. 9. What Carries the Genetic Information? <ul><li>Chromosomes are about 40% DNA & 60% protein. </li></ul><ul><ul><li>Protein is the larger component </li></ul></ul><ul><li>Protein molecules are composed of 20 different subunits </li></ul><ul><li>DNA molecules are composed of only four </li></ul><ul><li>Therefore protein molecules could encode more information, and a greater variety of information </li></ul><ul><ul><li>protein had the possibility for much more diversity than in DNA </li></ul></ul><ul><li>Therefore, scientists believed that the protein in chromosomes must carry the genetic information </li></ul>
  9. 10. The Discovery of DNA <ul><li>First identified in 1868 by Friedrich Miescher, a Swiss biologist, in the nuclei of pus cells obtained from discarded surgical bandages. </li></ul><ul><li>He called the substance nuclein </li></ul><ul><li>Noted the presence of phosphorous </li></ul>
  10. 11. The Transforming Principle <ul><li>Fredrick Griffith - 1928 </li></ul><ul><li>Discovered that different strains of the bacterium Strepotococcus pneumonae had different effects on mice </li></ul><ul><ul><li>One strain could kill an injected mouse (virulent) </li></ul></ul><ul><ul><li>Another strain had no effect (avirulent) </li></ul></ul><ul><ul><li>When the virulent strain was heat-killed and injected into mice, there was no effect. </li></ul></ul><ul><ul><li>But when a heat-killed virulent strain was co-injected with the avirulent strain, the mice died. </li></ul></ul><ul><li>Concluded that some factor in the heat killed bacteria was transforming the living avirulent to virulent? </li></ul><ul><li>What was the transforming principle and was this the genetic material? </li></ul>
  11. 13. The Transforming Principle is DNA <ul><li>Avery, Macleod, & McCarty – 1943 </li></ul><ul><li>Attempted to identify Griffith’s “transforming principle” </li></ul><ul><li>Separated the dead virulent cells into fractions </li></ul><ul><ul><li>The protein fraction </li></ul></ul><ul><ul><li>DNA fraction </li></ul></ul><ul><li>Co-injected them with the avirulent strain. </li></ul><ul><ul><li>When co-injected with protein fraction, the mice lived </li></ul></ul><ul><ul><li>with the DNA fraction, the mice died </li></ul></ul><ul><li>Result was IGNORED </li></ul><ul><ul><li>Most scientists believed protein was the genetic material. </li></ul></ul><ul><ul><li>They discounted this result and said that there must have been some protein in the fraction that conferred virulence. </li></ul></ul>
  12. 14. The Hershey-Chase Experiment <ul><li>Hershey & Chase – 1952 </li></ul><ul><li>Performed the definitive experiment that showed that DNA was the genetic material. </li></ul><ul><li>Bacteriphages = viruses that infect bacteria </li></ul><ul><li>Bacteriphage is composed only of protein & DNA </li></ul><ul><li>Inject their genetic material into the host </li></ul>
  13. 16. The Experiment <ul><li>Prepared 2 cultures of bacteriophages </li></ul><ul><li>Radiolabeled sulphur in one culture </li></ul><ul><ul><li>there is sulphur in proteins, in the amino acids methionine and cysteine </li></ul></ul><ul><ul><li>there is no sulphur in DNA </li></ul></ul><ul><li>Radiolabeled phosphorous in the second culture </li></ul><ul><ul><li>there is phosphorous in the phosphate backbone of DNA </li></ul></ul><ul><ul><li>none in any of the amino acids. </li></ul></ul><ul><li>So this one culture in which only the phage protein was labeled, and one culture in which only the phage DNA was labeled. </li></ul>
  14. 17. Experiment Summary <ul><li>Performed side by side experiments with separate phage cultures in which either the protein capsule was labeled with radioactive sulfur or the DNA core was labeled with radioactive phosphorus. </li></ul><ul><li>The radioactively labeled phages were allowed to infect bacteria. </li></ul><ul><li>Agitation in a blender dislodged phage particles from bacterial cells. </li></ul><ul><li>Centrifugation pelleted cells, separating them from the phage particles left in the supernatant. </li></ul>
  15. 18. Results Summary <ul><li>Radioactive sulfur was found predominantly in the supernatant. </li></ul><ul><li>Radioactive phosphorus was found predominantly in the cell fraction, from which a new generation of infective phage was generated. </li></ul><ul><li>Thus, it was shown that the genetic material that encoded the growth of a new generation of phage was in the phosphorous-containing DNA. </li></ul>
  16. 20. Chargaff’s Rule <ul><li>Once DNA was recognized as the genetic material, scientists began investigating its mechanism and structure. </li></ul><ul><li>Erwin Chargaff – 1950 </li></ul><ul><ul><li>discovered the % content of the 4 nucleotides was the same in all tissues of the same species </li></ul></ul><ul><ul><li>percentages could vary from species to species. </li></ul></ul><ul><li>He also found that in all animals (Chargaff’s rule): </li></ul><ul><li>%G = %C %A = %T </li></ul><ul><li>This suggested that the structure of the DNA was specific and conserved in each organism. </li></ul><ul><li>The significance of these results was initially overlooked </li></ul>
  17. 21. Base Pairing in DNA <ul><li>Not understood ‘til Watson & Crick described double helix </li></ul><ul><li>Adenine & guanine are purines </li></ul><ul><ul><li>2 organic rings </li></ul></ul><ul><li>Cytosine & guanine are pyrimidines </li></ul><ul><ul><li>1 organic ring </li></ul></ul><ul><li>Pairing a purine & a pyrimidine creates the correct 2 nm distance in the double helix </li></ul><ul><li>A – T joined by 2 hydrogen bonds </li></ul><ul><li>G – C joined by 3 hydrogen bonds </li></ul>
  18. 22. The Double Helix <ul><li>James Watson and Francis Crick – 1953 </li></ul><ul><li>Presented a model of the structure of DNA. </li></ul><ul><li>It was already known from chemical studies that DNA was a polymer of nucleotide (sugar, base and phosphate) units. </li></ul><ul><li>X-ray crystallographic data obtained by Rosalind Franklin, combined with the previous results from Chargaff and others, were fitted together by Watson and Crick into the double helix model. </li></ul>
  19. 23. Watson and Crick shared the 1962 Nobel Prize for Physiology and Medicine with Maurice Wilkins. Rosalind Franklin died before this date.
  20. 24. DNA Structure <ul><li>The double helix is formed from two strands of DNA </li></ul><ul><li>DNA strands run in opposite directions </li></ul><ul><li>They are complementary </li></ul><ul><ul><li>attached by hydrogen bonds between complimentary base pairs: </li></ul></ul><ul><ul><li>A - T and G - C </li></ul></ul><ul><li>This complementary pairing of the bases suggests that, when DNA replicates, an exact duplicate of the parental genetic information is made. </li></ul><ul><ul><li>The polymerization of a new complementary strand takes place using each of the old strands as a template. </li></ul></ul>
  21. 26. Messelson and Stahl <ul><li>How does DNA replicate? </li></ul><ul><li>Matthew Meselson and Franklin Stahl - 1957 </li></ul><ul><li>Did an experiment to determine which model best represented DNA replication: </li></ul><ul><li>semiconservative replication </li></ul><ul><ul><li>the two strands unwind and each acts as a template for new strands </li></ul></ul><ul><ul><li>each new strand is half comprised of molecules from the old strand </li></ul></ul><ul><li>conservative replication </li></ul><ul><ul><li>the strands do not unwind, but somehow generate a new double stranded DNA copy of entirely new molecules </li></ul></ul>
  22. 29. The Experiment <ul><li>The original DNA strand was labeled with the heavy isotope of nitrogen, N-15. </li></ul><ul><li>This DNA was allowed to go through one round of replication with N-14 (non-labeled) </li></ul><ul><li>the mixture was centrifuged so that the heavier DNA would form a band lower in the tube </li></ul><ul><li>the intermediate (one N-15 strand and one N-14 strand) and light DNA (all N-14) would appear as a band higher in the tube </li></ul>
  23. 30. The expected results for each model were:
  24. 31. The actual results were as expected for the semiconservative model and thus Watson and Crick's suspicion was borne out.
  25. 32. Enzymes & Replication <ul><li>DNA replication is not a passive, spontaneous process. </li></ul><ul><li>More than a dozen enzymes & other proteins are required to unwind the double helix & to synthesize & finalize a new strand of DNA. </li></ul><ul><li>The molecular mechanism of DNA replication can best be understood from the point of view of the machinery required to accomplish it. </li></ul><ul><li>The unwound helix, with each strand being synthesized into a new double helix, is called the replication fork . </li></ul>
  26. 33. The Enzymes of DNA Replication <ul><li>Topoisomerase </li></ul><ul><ul><li>Responsible for initiation of unwinding of DNA. </li></ul></ul><ul><ul><li>The tension holding the helix in its coiled and supercoiled structure is broken by nicking a single strand of DNA. </li></ul></ul><ul><li>Helicase </li></ul><ul><ul><li>Accomplishes unwinding of the original double strand, once supercoiling is eliminated by topoisomerase. </li></ul></ul><ul><ul><li>The two strands want to bind together because of hydrogen bonding affinity for each other, so helicase requires energy (ATP) to break the strands apart. </li></ul></ul>
  27. 34. Single-stranded Binding Proteins <ul><li>Important to maintain the stability of the replication fork. </li></ul><ul><li>Line up along unpaired DNA strans, holding them apart </li></ul><ul><li>Single-stranded DNA is very unstable, so these proteins bind bind to it while it remains single stranded and keep it from being degraded. </li></ul>
  28. 35. Beginning: Origins of Replication <ul><li>Replication begins at specific sites called origins of replication </li></ul><ul><li>In prokaryotes, the bacterial chromosome has a specific origin </li></ul><ul><li>In eukaryotes, replication begins at many sites on the large molecule </li></ul><ul><ul><li>100’s of origins </li></ul></ul><ul><li>Proteins that begin replication recognize the origin sequence </li></ul><ul><li>These enzymes attach to DNA, separating the strands, and opening a replication bubble </li></ul><ul><li>The end of the replication bubble is the “Y” shaped replication fork , where new strands of DNA elongate </li></ul>
  29. 37. Elongation <ul><li>Elongation of the new DNA strands is catalyzed by DNA polymerase </li></ul><ul><li>Nucleotides align with complimentary bases on the template strand, and are added by the polymerase, one by one, to the growing chain </li></ul><ul><ul><li>DNA polymerase proceeds along a single-stranded DNA molecule, recruiting free nucleotides to H-bond with the complementary nucleotides on the single strand </li></ul></ul><ul><li>Forms a covalent phosphodiester bond with the previous nucleotide of the same strand </li></ul><ul><ul><li>The energy stored in the triphosphate is used to covalently bind each new nucleotide to the growing second strand </li></ul></ul><ul><li>Replication proceeds in both directions </li></ul>
  30. 39. The Replication Fork
  31. 40. DNA Polymerase <ul><li>There are different forms of DNA polymerase </li></ul><ul><ul><li>DNA polymerase III is responsible for the synthesis of new DNA strands </li></ul></ul><ul><li>DNA polymerase is actually an aggregate of several different protein subunits, so it is often called a holoenzyme . </li></ul><ul><li>Primary job is adding nucleotides to the growing chain </li></ul><ul><li>Also has proofreading activities </li></ul>
  32. 41. Proofreading & Repair <ul><li>DNA polymerase proofreads each nucleotide added against its’ template as it is added </li></ul><ul><li>Removes incorrectly paired nucleotides & corrects </li></ul>
  33. 42. DNA Strands are Anti-parallel <ul><li>The sugar phosphate backbones of the 2 parent strands run in opposite directions </li></ul><ul><ul><li>“ upside down” to each other </li></ul></ul><ul><li>DNA is polar </li></ul><ul><ul><li>There is a 3’ end and a 5’ end </li></ul></ul><ul><ul><li>At the 3’ end, a OH is attached to the 3’ C of the last deoxyribose </li></ul></ul><ul><ul><li>At the other end a phosphate group is attached to the 5’ C of the last nucleotide </li></ul></ul><ul><li>DNA polymerase adds nucleotides only to the 3’ end of the growing chain </li></ul><ul><ul><li>So new DNA strand elongates only in the 5’  3” direction </li></ul></ul>
  34. 44. Why 5’  3’? <ul><li>Why can DNA polymerase only add nucleotides to the 3’ end ? </li></ul><ul><li>Needs a triphosphate to provide energy for the bond between an added nucleotide & the growing DNA strand. </li></ul><ul><li>This triphosphate is very unstable </li></ul><ul><ul><li>can easily break into a monophosphate and an inorganic pyrophosphate </li></ul></ul><ul><li>At the 5' end, this triphosphate can easily break </li></ul><ul><ul><li>It is not be able to attach new nucleotides to the 5' end once the phosphate has broken off </li></ul></ul><ul><li>The 3' end only has a hydroxyl group </li></ul><ul><ul><li>As long as new nucleotide triphosphates are brought by DNA polymerase, synthesis of a new strand continues, no matter how long the 3' end has remained free. </li></ul></ul>
  35. 45. Leading & Lagging Strands <ul><li>The new strand made by adding to the 3’ end = leading strand </li></ul><ul><ul><li>Parent strand is 3’  5’ </li></ul></ul><ul><ul><li>New strand is 5’  3’ </li></ul></ul><ul><li>How can DNA polymerase synthesize new copies of the 5'  3' strand, if it can only travel in one direction? </li></ul><ul><li>To elongate in the other direction, the process must work away from the replication fork </li></ul><ul><li>The new strand formed on the 5’  3’ parent strand is called the lagging strand </li></ul>
  36. 46. Building the Lagging Strand <ul><li>DNA polymerase makes a second copy in an overall 3’  5’ direction </li></ul><ul><li>First, it produces short segments, called Okazaki fragments </li></ul><ul><ul><li>These are built in a 5’ –3’ direction </li></ul></ul><ul><li>Okazaki fragments are joined together by ligase to produce the new 3’  5’ lagging strand </li></ul>
  37. 47. Ligase <ul><li>Catalyzes the formation of a phosphodiester bond given an unattached adjacent 3‘ OH and 5‘ phosphate. </li></ul><ul><li>This can join Okazaki fragments </li></ul><ul><li>This can also fill in the unattached gap left when an RNA primer is removed </li></ul><ul><li>DNA polymerase can organize the bond on the 5' end of the primer, but ligase is needed to make the bond on the 3' end. </li></ul>
  38. 49. Primers <ul><li>DNA polymerase cannot start synthesizing on a bare single strand. </li></ul><ul><ul><li>It only adds to an existing chain </li></ul></ul><ul><li>It needs a primer with a 3'OH group on which to attach a nucleotide. </li></ul><ul><li>The start of a new chain is not DNA, but a short RNA primer </li></ul><ul><ul><li>Only one primer is needed for the leading strand </li></ul></ul><ul><ul><li>One primer for each Okazaki fragment on the lagging strand </li></ul></ul>
  39. 50. Primase <ul><li>Part of an aggregate of proteins called the primeosome. </li></ul><ul><li>Attaches the small RNA primer to the single-stranded DNA which acts as a substitute 3'OH so DNA polymerase can begin synthesis </li></ul><ul><li>This RNA primer is eventually removed by RNase H </li></ul><ul><ul><li>the gap is filled in by DNA polymerase I. </li></ul></ul>
  40. 52. Ending the Strand <ul><li>DNA polymerase only adds to the 3’ end </li></ul><ul><li>There is no way to complete the 5’ end of the new strand </li></ul><ul><li>A small gap would be left at the 5’ end of each new strand </li></ul><ul><li>Repeated replication would then make the strand shorter and shorter, eventually losing genes </li></ul><ul><li>Not a problem in prokaryotes, because the DNA is circular </li></ul><ul><ul><li>There are no “ends” </li></ul></ul>
  41. 53. Telomeres <ul><li>Eukaryotes have a special sequence of repeated nucleotides at the end, called telomeres </li></ul><ul><li>Multiple repititions of a short nucleitide sequence </li></ul><ul><ul><li>Can be repeated hundreds, or thousands of times </li></ul></ul><ul><ul><li>In humans, TTAGGG </li></ul></ul><ul><li>Do not contain genes </li></ul><ul><li>Protects genes from erosion thru repeated replication </li></ul><ul><li>Precvents unfinished ends from activating DNA monitoring & repair mechanisms </li></ul>
  42. 54. Telomerase <ul><li>Catalyzes lengthening of telomeres </li></ul><ul><li>Includes a short RNA template with the enzyme </li></ul><ul><li>Present in immortal cell lines and in the cells that give rise to gametes </li></ul><ul><li>Not found in most somatic cells </li></ul><ul><li>May account for finite life span of tissues </li></ul>
  43. 56. Further Proofreading & Repair <ul><li>Some errors evade initial proofreading or occur after synthesis </li></ul><ul><li>DNA can be damaged by reactive chemicals, x-rays, UV, etc </li></ul><ul><li>Cells continually monitor DNA for mutations & repair </li></ul><ul><li>Contain 100’s of repair enzymes </li></ul>
  44. 57. Nucleotide Excision & Repair <ul><li>A segment of DNA containing damage is cut out by a nuclease (a DNA cutting enzyme) </li></ul><ul><li>The gap is filled & closed by DNA polymerase and ligase </li></ul><ul><li>Thymine dimers </li></ul><ul><li>Covalent linking of thymine bases </li></ul><ul><li>Causes DNA to “buckle” </li></ul><ul><li>One common problem corrected this way </li></ul>
  45. 59. Proteins of DNA Replication
  46. 60. Summary View of Replication