3_5_08_DNA - Clicker Question Clicker In the developing...

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Unformatted text preview: Clicker Question Clicker In the developing embryo, the fallopian tubes, In uterus and upper vagina develop from the uterus A) ovaries. B) Wolffian ducts. C) Mullerian ducts. D) Freudian ducts. E) labioscrotal swelling. Where are we? Where I have been discussing reproductive anatomy and have physiology in terms of “I am my genes”. physiology Today we will talk about DNA, the chemical that Today DNA the makes up the genes. DNA is the hereditary genes DNA material traditionally transferred from the male to material the female during sexual intercourse and transferred at conception, from the parents to the offspring. Does our DNA determine who we really really are? are? In the next series of lectures, I hope to help you In “prepare your mind” so that you can critically and deeply think about the influence of DNA on your future as a person. your Maupertuis (1753) Postulated that the Hereditary Material is Particulate Hereditary “If there are, in each of the If seminal seeds, particles particles predetermined to form the heart, the head, the entrails, the arms and the legs, if these particular particles had a special attraction for those which are to be their immediate neighbors in the animal body, this would lead to the formation of the fetus.” Robert Brown Serendipitously Discovered the Nucleus in 1831 while Studying Pollination Studying Nuclei in Orchid Epidermis Taken with Brown’s Microscope with Mathias Schleiden and Theodor Schwann: Promoters of the Cell Theory Schwann: Mathias Schleiden (1838) Mathias speculated that the nucleus was important for the formation of cells in plants. Schleiden’s idea attracted Schleiden’s the attention of a zoologist named Theodor Schwann who promoted the idea that the nucleus was as important in determining the development and morphology of animals as it was in plants. it The Nucleus Is Present in All Cells The The fact that a nucleus is The present in all plant and animal cells was not obvious until Theodor Hartig (1854) and Lord Osborne (1857) developed a stain made of carmine, which comes from the crushed bodies of Mexican cochineal insects. The nucleus then took on a new importance. new The Discovery of Nuclein The Friedrich Miescher Friedrich (1868) was a physician who became interested in investigating the chemical composition of nuclei. nuclei He isolated a He compound from the nuclei of pus cells pus that came from used bandages obtained from a hospital. The Chemistry of Nuclein The When Miescher removed When the fat from pus cells (white blood cells) with alcohol and removed the protein with pepsin-containing pig gastric mucosa, gastric something still remained in the nuclei. He called the new He compound, which contained carbon, nitrogen and phosphorus, nuclein. He phosphorus nuclein He determined that nuclein had the following chemical formula, C29H49N9P3O22. formula, The Chemical Basis of Identity? The • In a speculative note submitted In along with his other work Miescher (1869) suggested that the study of individual nucleins may reveal something about the differences between members of a group. members • However, the idea that one’s However, identity could have a identity chemical basis was beyond chemical belief and the note was rejected by the journal. rejected Visualizing Cell Reproduction Visualizing • About the same time, Eduard Flemming and About others stained cells and watched how the colored material (chromatin) iin the nucleus split n colored into many bodies. • He saw that the nucleus went through a He complicated process (mitosis) to divide the complicated to chromosomes in such a way that each somatic cell had not only the same number of chromosomes, but identical copies of the chromosomes. chromosomes. • During the formation of gametes, a special During division (meiosis) occurred, which resulted in a division occurred, halving (haploid) of the number of halving of chromosomes. During fertilization, the diploid fertilization the number of chromosomes was restored. chromosomes This led to the idea that the chromosomes This contained the self-perpetuating particulate information necessary to build an organism. information E. B. Wilson E. B. Wilson (1895) wrote about the possible function of chromatin and nuclein: chromatin “The precise equivalence of the chromosomes contributed The by the two sexes is a physical correlative of the fact that the two sexes play… equal parts in hereditary transmission, and it seems to show that the chromosomal substance, the chromatin, is to be regarded as the physical basis of inheritance. Now, chromatin is known physical Now, to be closely similar to… a substance known as nuclein nuclein (C29H49N9P3O22, according to Miescher), which analysis (C according shows to be a tolerably definite chemical composed of nucleic acid (a complex organic acid rich in phosphorous) and albumin. And thus we reach the remarkable conclusion that inheritance may, perhaps, be effected by the physical transmission of a particular chemical compound from parent to offspring.” compound Emil Fischer: Synthetic Chemist Emil Reading Emil Fischer’s Reading Nobel Prize acceptance speech gives one an idea of how a chemist or chemical biologist thinks. If one can use uric acid If from guano to make caffeine, why not use it to form the nitrogenous bases of DNA and inject it in people to induce desired genetic traits? desired Emil Fischer (1902) P-urine Emil “Currently the name "purines" is a generic term for a large class of nitrogenous organic compounds, some being certain animal excretions and others the active constituents of important stimulating beverages. The oldest member of the group is known by the rather unattractive name of uric acid and was discovered…as a constituent of… urine. …the enlightened farmer knows it to be a valuable constituent of guano.” “To this end I would ask you to glance at the accompanying set of formulae....” “It will readily be seen on comparing these formulae that they contain a common atomic group, a nucleus as it is called and which I have termed the purine nucleus.” “The simplest combination of this skeleton is … purine…., Replacing a hydrogen atom in purine by the amide NH, forms adenine, and if a further oxygen atom is added, the result is guanine. Lastly, the nitrogen atoms in vegetable products are also linked with the CH3 or methyl group.” “To determine the structure of the molecule the chemist proceeds in a similar way to the anatomist. By chemical actions he breaks the system down into its components and continues with this division until familiar substances emerge [and] the structure of the original system can be inferred from the decomposition products. Usually…the structure will only be finally elucidated by the reverse method, by building up the molecule from the decomposition products or similar substances, i.e. by what is termed synthesis [and] it has become possible to synthesize them all from the inexpensive uric acid….” “Among the purine substances there are two valued drugs, caffeine and theobromine, which so far had to be prepared by extraction from tea and cocoa…. Now that these drugs can be made synthetically from inexpensive uric acid the mind readily turns to exploiting the synthesis on an industrial scale….” “It is even possible to produce the true aroma of coffee or tea artificially, too, by synthesis; with the exercise of a little imagination the day can be foreseen when beans will no longer be required to make good coffee….The layman usually receives with skepticism such prophecies by the chemist and in this specific instance his skepticism will not be weakened by the knowledge that a constituent of guano would be used to prepare the synthetic drink.” “In our age, however, such prejudices on the part of the public do not normally persist for very long. Who, nowadays, still considers that the magnificent dyes colouring our garments and furnishings are derived from unsightly coal tar, or that sweet-tasting saccharin is prepared from the same material ? The point is that chemical transformations are so thorough that the endproduct retains nothing at all of the properties of the original substance. The manufacture of caffeine from uric acid is therefore nothing worse than the processes which occur when manure used to nourish plants is converted into appetizing fruits or into wonderfully scented flowers.” Twinkies: An Partially Synthesized Food Twinkies contain flour, sugar, salt, Twinkies baking soda, water and a trace of egg. baking THE FILLING also includes Partially hydrogenated vegetable oil is the main ingredient. Polysorbate 60 is a gooey substance that helps replace cream and eggs and is derived from corn, palm oil and petroleum. Cellulose gum gives the crème filling a smooth, slippery feel. Artificial vanillin is synthesized in petrochemical plants. petrochemical THE CAKE also includes Lecithin is an emulsifier made from soy. Diacetyl mimics the taste of butter. Yellow No. 5, Red No. 40 give the cake the golden look of eggs. Sorbic acid, the only actual preservative Sorbic in Twinkies, comes from petroleum. in Synthesizing Genes Synthesizing The possibility of testing the hypothesis that nucleic acids The were the genetic material seemed possible to Emil Fischer (1914). He wrote, “With the synthetic approaches to this group we are now With capable of obtaining numerous compounds that resemble, more or less, natural nucleic acids. How will they affect living organisms? Will they be rejected or metabolized or will they participate in the construction of the cell nucleus? Only the experiment will give us the answer. I am bold enough to hope that, given the right conditions, the latter may happen and that artificial nucleic acids may be assimilated without degradation of the molecule. Such assimilated incorporation should lead to profound changes of the organism, resembling perhaps permanent changes or mutations as they have been observed before in nature.” nature Organic Chemists Degraded Nucleic Acids in Order to Determine Their Structures Structures At the turn of the century, nucleic At acids were isolated from wheat germ, yeast and calf thymus in order to study their structures. These studies were based on the These perspectives of organic chemists, including Walter Jones and Phoebus Levene (→), who had a Phoebus ), reductionist approach. reductionist Based on their degradation Based studies, the organic chemists studies the considered nucleic acids to be simple macromolecules. Nucleic Acids Are Composed of Purines and Pyrimidines Purines When nucleic acids were When hydrolyzed, they were hydrolyzed they broken down into their component parts which turned out to be the purines, adenine and purines adenine guanine as well as a guanine new class of compounds known as pyrimidines, cytosine, thymine and cytosine, and uracil. uracil Emergent Properties Emergent The organic chemists made outstanding The contributions to understanding the components of nucleic acids. components However, they lost sight of the fact that the However, parent molecule was a macromolecule, macromolecule and may have a level of complexity that and complexity was greater than the complexity of its component parts. component That is, they did not have the prepared That minds to realize that the parent molecule minds may have emergent properties. emergent Limits to the Reductionist Perspective of Chemists Perspective The chemists developed extraction procedures The extraction that were suitable for degradation studies, but degradation but would not allow them to isolate the native molecule, and to reconstruct correctly the structure of the nucleic acids. Anatomists have the advantage of knowing a priori that their tissue priori slices come from a complex organism. slices The organic chemists did not even consider the The possibility that nucleic acids were complex selfcomplex perpetuating information bearing molecules. In In fact, due to the high number of acidic groups, the nucleic acids were naively considered by chemists to act as pH buffers in the nucleus. to Only Two Kinds of Nucleic Acids Only The nucleic acids seemed to be relatively simple The molecules. Consequently, the approach used to isolate nucleic acids, followed the approach used to isolate other simple materials, and work focused on isolating nucleic acids from a few convenient sources. There was one odd fact though: The nucleic acids of plants seemed to be composed of ribonucleosides while those of animals were composed of deoxyribonucleosides, and Walter Jones deoxyribonucleosides and (1920) concluded in his monograph on nucleic acids, that (1920) "...there are but two nucleic acids in nature, one obtainable from the nuclei of animal cells, and the other from the nuclei of plant cells." other Yeast Nucleic Acid and Thymonucleic Acid Yeast Thus plant nucleic acids were Thus known as either “yeast nucleic acid” or ribonucleic acid, and animal ribonucleic and nucleic acids were known as “thymonucleic acid” or desoxyribonucleic acid. desoxyribonucleic The chemist’s interpretations of the The simplicity of nucleic acids resulted from their preconceived notions preconceived of the simplicity of nucleic acids, and thus they developed inadequate extraction procedures. procedures Conclusion: Nucleic Acids were Simple Molecules Molecules Phoebus Levene (1921) Phoebus concluded that nucleic acids were composed of repeating units of four nucleotides. units According to the According tetranucleotide hypothesis, tetranucleotide the structure of thymonucleic acid was ATCGATCGATCG... acid The tetranucleotide The hypothesis, introduced in 1921 dominated the thinking of scientists for more than twenty years. The technique of X-ray diffraction was proposed by X-ray Max von Laue (1915) and developed by Lawrence and William Bragg (1922,1924) to determine the arrangement of atoms in molecules. William T. arrangement William Astbury (1938) first used X-ray diffraction to determine the structure of DNA. structure X-ray Diffraction of DNA X-ray The X-ray data obtained by The Astbury and Bell (1938) indicated regular repeats regular every 0.33-0.34 nm which they believed were due to the nucleotides, and a 2.7 nm repeat which they believed indicated that the DNA molecule was composed of a repeating sequence of 8 or 16 sequence nucleotides. Perfection of X-ray image Suggests that DNA is Too Simple to be the Genetic Material Material Astbury (1947) concluded: "It seems Astbury improbable, too, to judge by the degree of perfection of the X-ray fibre diagram that these four different kinds of nucleotides are distributed simply at random.“ distributed A nonuniform arrangement of nucleotides would be necessary if the nucleic acids were to function as the genetic material. genetic DNA as the Genetic Material? DNA There was little reason There to even think that nucleic acid was the genetic material based on studies by organic chemists. In fact, Wendell Stanley In (1935) thought that he thought crystallized the hereditary component of the tobacco mosaic virus when he crystallized its protein. Feulgen Stain Shows Plants Also Have DNA DNA But, cytologists were obtaining data that But, conflicted with those of the organic chemists. Using a stain now known as Feulgen’s reagent, Feulgen (1924) discovered that DNA occurred in the nuclei of plants and animals, and not just in those of animal cells. He wrote: just “This gave us great surprise, for the This nuclei of the wheat embryo gave the nucleal reaction more intensively than we have ever seen in any animal tissues…. The old dualism of yeast and thymonucleic acids is thus set aside....” aside....” DNA Content of Nuclei in a Given Organism is Constant Conclusions about the importance of Conclusions DNA based on Feulgen staining were not looked upon favorably because the staining of various cells in the same organism seemed to vary in an unreasonable manner. Approximately two decades later Approximately Alfred Mirsky and Hans Ris (1948,1949) determined that the DNA content of nuclei from somatic cells of the same somatic organism is constant, and organism and variations in the ratio of DNA to protein in the nucleus accounted for the variations in the staining. the A Return to Thinking about DNA as an Information-Bearing Molecule an The chemistry of nucleic acids The went through a revolution when Erwin Chargaff (→), “a licensed Erwin ), biochemist” became aware of the genetic evidence that DNA was the hereditary material. Chargaff was instantly Chargaff captivated by the experiments performed by Oswald Avery, Colin MacLeod and Maclyn McCarty in 1944, which indicated that DNA was the genetic material. genetic Fred Griffith’s Transformation Experiment Experiment Avery’s experiments were Avery’s based on the observations of Fred Griffith, who died in his Fred who laboratory when London was bombed during WWII. Griffith (1928) discovered Griffith that when mice were injected with virulent pneumococci (smooth) that had been heat (smooth) killed, along with live avirulent pneumococci (rough), the mice died of (rough), pneumonia and their blood pneumonia contained living virulent pneumococci. pneumococci. Pneumococci and Pneumonia Pneumococci Smooth bacteria kill mice. They are virulent. Rough bacteria do not. They are avirulent. Heat-killed Heat-killed smooth (virulent) smooth bacteria do not kill mice. kill Live rough Live rough (avirulent) bacteria and dead smooth smooth (virulent) bacteria mixed together do kill mice. do Transformation Transformation Something from virulent bacteria Something transforms avirulent bacteria and makes them virulent. them Dawson and Sia (1931) found that the Dawson transformation of avirulent to virulent transformation pneumatococci could also take place in a test tube outside of a mouse. test Avery et al. (1944) interpreted all of these results Avery to mean that there was a transfer of genetic material between the virulent and avirulent material bacteria during transformation. Avery et al. (1944) wrote: wrote: “Biologists have long attempted by chemical Biologists means to induce in higher organisms predictable and specific changes which…could be transmitted…as hereditary characters. Among microorganisms the most striking example of inheritable and specific alterations…that can be experimentally induced and are reproducible under well defined and adequately controlled conditions is the transformation of… conditions Pneumatococcus.” Transformation: A Transfer of Genetic Material Genetic Chemical Fractionation Shows that the Transforming Factor is DNA the Avery et al. (1944) Avery fractionated the pneumatocci and meticulously isolated the various fractions and found that the transforming principle was none principle other than the proteinproteinfree DNA fraction. Erwin Chargaff: Prepared Mind Erwin Hearing of Avery’s results and believing that DNA Hearing was the genetic material, Chargaff began isolating DNA in earnest. He developed the techniques necessary to isolate gently intact macromolecules of DNA and do quantitative work. Chargaff (1950) “...started in our work from the Chargaff assumption that nucleic acids were complicated and intricate high-polymers, comparable in this respect to the proteins, and that the determination of their structures and their structural differences would require the development of methods suitable for the precise analysis of all constituents of nucleic acids prepared from a large number of different cell types." different Erwin Chargaff: DNA is Complicated Enough to Carry Hereditary Information Carry I cannot stress enough how important it cannot was to approach DNA from the mind set that it may be a complicated, information containing molecule. Chargaff showed that contrary to the Chargaff prediction of the tetranucleotide hypothesis, adenine, guanine, cytosine and thymine were not present in equimolar amounts in DNA, and the proportions of nucleotides d...
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