1. PŘEDNÁŠKA 2013-14 Nucleic acids Historical view Emil Paleček Institute of Biophysics, Acad. Sci. CR v.v.i., 612 65 Brno Czech Republic G.J. Mendel 1866 The Road to DNA started in Brno F. Miescher Tübingen 1871 “NUCLEIN” Isolation “ELEMENTS OF HEREDITY” Chemical nature and spatial organization Biological FUNCTION STRUCTURE NUCLEIC ACIDS F. MIESCHER, TÜBINGEN G. J. MENDEL, BRNO 1871 1866 MENDEL C K MATHEWS , K E van HOLDE, BIOCHEMISTRY, 1990 MIESCHER MIESCHER Timeline of DNA 1865: Gregor Mendel discovers through breeding experiments with peas that traits are inherited based on specific laws (later to be termed “Mendel’s laws”). By mentioning Elements of Heredity he predicts DNA and genes (published 1866) 1866: Ernst Haeckel proposes that the nucleus contains the factors responsible for the transmission of hereditary traits. 1869: Friedrich Miescher isolates DNA/NUCLEIN for the first time. 1871: The first publications describing DNA (nuclein) by F Miescher, Felix Hoppe-Seyler, and P. Plosz are printed. 1882: Walther Flemming describes chromosomes and examines their behavior during cell division. 1884–1885: Oscar Hertwig, Albrecht von Kölliker, Eduard Strasburger, and August Weismann independently provide evidence that the cell’s nucleus contains the basis for inheritance. 1889: Richard Altmann renames nuclein to nucleic acid. 1900: Carl Correns, Hugo de Vries, and Erich von Tschermak rediscover Mendel’s Laws. 1902: T Boveri and W Sutton postulate that the heredity units (called genes as of 1909) are located on chromosomes. 1902–1909: A Garrod proposes that genetic defects result in the loss of enzymes and hereditary metabolic diseases. 1909: Wilhelm Johannsen uses the word gene to describe units of heredity. 1910: T H Morgan uses fruit flies (Drosophila) as a model to study heredity and finds the first mutant with white eyes. 1913: Alfred Sturtevant and Thomas Hunt Morgan produce the first genetic linkage map (for the fruit fly Drosophila). 1928: Frederick Griffith postulates that a transforming principle permits properties from one type of bacteria (heat-inactivated virulent Streptococcus pneumoniae) to be transferred to another (live nonvirulent Streptococcus pneumoniae). 1929: P Levene identifies the building blocks of DNA, incl. four bases adenine (A), cytosine (C), guanine (G), thymine (T) . 1941: George Beadle and Edward Tatum demonstrate that every gene is responsible for the production of an enzyme. 1944: Oswald T. Avery, Colin MacLeod, and Maclyn McCarty demonstrate that Griffith’s transforming principle is not a protein, but rather DNA, suggesting that DNA may function as the genetic material 1949: Colette and Roger Vendrely and A Boivin discover that the nuclei of germ cells contain half the amount of DNA that is found in somatic cells. This parallels the reduction in the number of chromosomes during gametogenesis and provides further evidence for the fact that DNA is the genetic material. 1949–1950: Erwin Chargaff finds that the DNA base composition varies between species but determines that the bases in DNA are always present in fixed ratios: the same number of A’s as T’s and the same number of C’s as G’s. 1952: Alfred Hershey and Martha Chase use viruses (bacteriophage T2) to confirm DNA as the genetic material by demonstrating that during infection viral DNA enters the bacteria while the viral proteins do not and that this DNA can be found in progeny virus particles. 1953: Rosalind Franklin and Maurice Wilkins use X-ray analyses to demonstrate that DNA has a regularly repeating helical structure. 1953: James Watson and Francis Crick discover the molecular structure of DNA: a double helix in which A always pairs with T, and C always with G. 1956: Arthur Kornberg discovers DNA polymerase, an enzyme that replicates DNA. 1957: Francis Crick proposes the central dogma (information in the DNA is translated into proteins through RNA) 1958: Matthew Meselson and Franklin Stahl describe how DNA replicates (semiconservative replication). 1960-63: Julius Marmur and Paul Doty show separation of DNA strands and reformation of DNA double-helical structure – DNA renaturation/hybridization 1961–1966: Robert W. Holley, Har Gobind Khorana, Heinrich Matthaei, Marshall W. Nirenberg, and colleagues crack the genetic code. 1968–1970: Werner Arber, Hamilton Smith, and Daniel Nathans use restriction enzymes to cut DNA in specific places for the first time. 1972: Paul Berg uses restriction enzymes to create the first piece of recombinant DNA. 1977: Frederick Sanger, Allan Maxam, and Walter Gilbert develop methods to sequence DNA. 1982: The first drug (human insulin), based on recombinant DNA, on the market. 1983: Kary Mullis invents PCR as a method for amplifying DNA in vitro. 1990: Sequencing of the human genome begins. 1995: First complete sequence of the genome of a free-living organism (the bacterium Haemophilus influenzae) is published. 1996: The complete genome sequence of the first eukaryotic organism—the yeast S. cerevisiae—is published. 1998: Complete genome sequence of the first multicellular organism—the nematode worm Caenorhabditis elegans—is published. 1999: Sequence of the first human chromosome (22) is published. 2000: The complete sequences of the genomes of the fruit fly Drosophila and the first plant—Arabidopsis—are published. 2001: The complete sequence of the human genome is published. 2002: The complete genome sequence of the first mammalian model organism—the mouse—is published. Darwin C. 1859: Book - On the Origin of Species by Means of Natural Selection Mendel G. 1866 Miescher F. 1871 Charles Darwin - Important claims: A. Universal Common Descent - Tree of Life - the first one-celled organism, representing the root or trunk of the Tree, gradually developed and changed over many generations into new and more complex forms, representing the branches B. Natural Selection as a mechanism responsible for the branching pattern Variations in living forms arise at random Nature selects the adaptive ones Adaptive organism survive and reproduce Inherited adaptations may cause population changes Darwin understand neither how genetic traits were passed to the progeny nor how the variations arose. He is a founder of Evolution Biology At present: - Natural Selection as a mechanism for relatively simple processes is fully confirmed but also cooperation played a significant role - Universal Common Descent - Tree of Life and the role of natural selection in the origin of species are questioned papers Math & Biology, Vienna Univ. DIRECT RECIPROCITY Random distribution vs. non-uniform distribution of defectors and cooperators Horizontal gene transfer - cell conglomerate instead of single cell ancestor 13 Thus we regard as regrettable the conventional concatenation of Darwin’s name with evolution, because other modalities must also be considered EVOLUČNÍ BIOLOGIE - rychle se vyvíjející vědecká disciplina vedle ní existuje IDEOLOGIE EVOLUCIONISMU PODLE DARWINISTY M. RUSE NENÍ BOJ EVOLUCIONISMU S KREACIONISMEM BOJEM VĚDY S NÁBOŽENSTVÍM ALE BOJEM NÁBOŽENSTVÍ S NÁBOŽENSTVÍM M. Ruse, The Evolution-Creation Struggle HARVARD UNIVERSITY PRESS , 2005 JOHANN GREGOR MENDEL * 1822 in Hynčice (Moravia, Austro-Hungarian Empire) + 1884 in Brno (buried at Central Cemetery in Brno) In the 1950´s Mendelism declared to be a reactionary teaching (LYSENKO, LEPESHINSKAYA) Mendel statue removed and its destruction ordered Brno geneticist J. Kříženecký jailed His pupil V. Orel forced to work manually in industry 1964 attempts to rehabilitate Mendel Academicians B. Němec (biologist) and F. ŠORM (biochemist, President of the Czechoslovak Academy of Sciences) backed by Soviet Academicians. Dealing between N. Khrushtchov, A. Novotný (President of Czechoslovakia), F. Šorm and biologist J. Pospíšil (later the Party Secretary) resulted in the decision to organize an international conference in 1968 (100 anniversary of publication of Mendel´s paper) in Brno (F. Šorm warned by Novotný that his attempts may result in the end of his career if the action will get out of control). Beginning of Mendel´s Museum in Brno A milestone not only in the approach of Party and State to Mendel but also a beginning of rehabilitation of SCIENCE against the COMMUNIST IDEOLOGY discovered through breeding experiments with peas that traits are inherited based on specific laws (later to be termed “Mendel’s laws”). By mentioning Elements of Heredity he predicted DNA and genes (published 1866, lecture in Brno 1965) Brno Augustinians 1860-62 Abbot C. Napp Abbot G. Mendel Teachers of Brno gymnasium (High School) Mendel’s Medal, Moravian Museum, Brno G J MENDEL, priest, teacher, scientist and abbot in BRNO In 1956 Mendel‘s Statue was ordered by the Regional Authorities to be destroyed. The workers who were supposed to the job decided not to do it because they believed that the statue was nice. Moreover it would be difficult to destroy it. Before the Symposium the Director of the Institute of Biophysics prof. F. Hercik was entrusted by the Academy to help with the organization of the Mendel International Meeting in Brno. To fulfill his duties he turned to the City Authorities asking to move the Mendel‘s Statue to the Abbey garden. As his request was ignored he asked his graduate students J. Koudelka and B. Janík to move the Statue from the Abbey yard to the garden. Both fellows were quite strong young men but they found the marble Statue too heavy. THE STATUE STORY In 1906 Dr. Hugo Iltis, the gymnasium professor in Brno organized an international collection to build the Mendel‘s Statue in Brno. Created by a French sculpturer T. Charlemont the Statue was errected at the Mendel Square in 1910 After February 1948 Soviet „Lysenkism“ (T. D. Lysenko 1896-1974) strongly affected biology in Czechoslovakia. After Stalin death (1953) attempts were made by soviet scientists (particularly by physists and chemists) to substitute Lysenko‘s „materialistic biology“ for normal science and by the end of 1950’s plans were made to organize in Brno International Mendel Memorial Symposium. In 1962 Lysenko‘s work was criticized by the Soviet Academy but still in September 1964 N.S. Khrushtchov raised objections against the Mendel Symposium in 1965 in Brno. During his visit in Prague he dealt with the President A. Novotny who finally agreed with the meeting organization after the President of the Academy F. Sorm personally guaranteed that the Symposium will not be politically misused. (F. Sorm was well informed about the activities of the influential Soviet scientist to rehabilitate fully the genetics - Soon after his visit of this country N.S. Khrushtchov was removed from his position). Fig. 1. Friedrich Miescher and his mentors. (A) Friedrich Miescher (1844–1895) as a young man. (B) Wilhelm His (1831–1904), Miescher’s uncle. His still is famous for his work on the fate of cells and tissues during embryonic development and for his insights into neuroembryology. He, for example, discovered neuroblasts and coined the term bdendriteQ (Finger, 1994; Shepherd, 1991). (C) Felix Hoppe-Seyler (1825–1895), one of the pioneers of physiological chemistry (now biochemistry). Hoppe-Seyler performed seminal work on the properties of proteins, most notably hemoglobin (which he named), introduced the term proteid (which later became protein), and worked extensively on fermentation and oxidation processes as well as lipid metabolism (Perutz, 1995). He was instrumental in founding Germany’s first independent institute for physiological chemistry (in 1884) and in 1877 founded and edited the first journal of biochemistry, the Zeitschrift fu¬r Physiologische Chemie, which still exists today as Biological Chemistry. (D) Adolf Strecker (1822–1871), a leading figure in chemistry in the mid-19th century and professor at the University of Tubingen from 1860 to 1870. Among other achievements, he was the first to synthesize amino acid (alanine from acetaldehyde via its condensation product with ammonia and hydrogen cyanide) in a reaction known today as Strecker synthesis (Strecker, 1850). (E) Carl Ludwig (1816–1895), a protagonist in the field of physiology in the second half of the 19th century. His focus was the physiology of the nervous system and its sensory organs. In 1869, he founded Leipzig’s Physiological Institute. F. Miescher W. His (FM’s uncle) F. Hoppe-Seyler (supervisor) A. STRECKER Hoppe-Seyler’s laboratory around 1879 F. Miescher’s laboratory Text Tübingen castle A, in Miescher’s time B, at present Before attempting the isolation of cells from the pus on surgical bandages, Miescher took great care to ensure that his source material was fresh and not contaminated. He painstakingly examined it and discarded everything that showed signs of decomposition, either in terms of smell, appearance under the microscope, or by having turned acidic. A great deal of the material he could obtain did not meet these strict requirements (Miescher, 1871d). Those samples that did were subsequently used to isolate leucocytes. In a first step, Miescher separated the leucocytes from the bandaging material and the serum (Miescher, 1869a, 1871d). This separation posed a problem for Miescher. Solutions of NaCl or a variety of alkaline or alkaline earth salt solutions used to wash the pus resulted in a “slimy swelling” of the cells, which was impossible to process further (His, 1897b). (This “slimy swelling” of the cells was presumably due to high-molecular-weight DNA, which had been extracted from cells that had been damaged.) Only when Miescher tried a dilute solution of sodium sulfate [a mixture of one part cold saturated Glauber’s salt (Na2SO4d 10 H2O) solution and nine parts water] to wash the bandages did he manage to successfully isolate distinct leucocytes, which could be filtered out through a sheet to remove the cotton fibers of the bandaging. Miescher subsequently let the washing solution stand for 1–2 h to allow the cells to sediment and inspected the leucocytes microscopically to confirm that they did not show any signs of damage. Having isolated the cells, Miescher next had to separate the nuclei from the cytoplasm. This had never been achieved before and Miescher had to develop new protocols. He washed the cells by rinsing them several (6–10) times with fresh solutions of diluted (1:1000) hydrochloric acid over a period of several weeks at “wintry temperatures” (which were important to avoid degradation). This procedure removed most of the cells’ bprotoplasm,Q leaving behind the nuclei. The residue from this treatment consisted in part of isolated nuclei and of nuclei with only little fragments of cytoplasm left attached. Miescher showed that these nuclei could no longer be stained yellow by iodine solutions, a method commonly used at the time for detecting cytoplasm (Arnold, 1898; Kiernan, 2001). He then vigorously shook the nuclei for an extended period of time with a mixture of water and ether. This caused the lipids to dissolve in the ether while those nuclei, still attached to cytoplasm, collected at the water/ether interface. By contrast, the clean nuclei without contaminating cytoplasm were retained in the water phase. Miescher filtered these nuclei and examined them under a microscope. He noticed that in this way he could obtain completely pure nuclei with a smooth contour, homogeneous content, sharply defined nucleolus, somewhat smaller in comparison to their original volumes (Miescher, 1871d). Miescher subsequently extracted the isolated nuclei with alkaline solutions. When adding highly diluted (1:100,000) sodium carbonate to the nuclei, he noticed that they would swell significantly and become translucent. Miescher then isolated a yellow solution of a substance from these nuclei. By adding acetic acid or hydrochloric acid in excess, he could obtain an insoluble, flocculent precipitate (DNA). Miescher noted that he could dissolve the precipitate again by adding alkaline solutions. Although this protocol allowed Miescher for the first time to isolate nuclein in appreciable purity and quantities, it was still too little and not pure enough for his subsequent analyses. He consequently improved on this protocol until he established the protocol detailed in Box 2, which enabled him to purify sufficient amounts of nuclein for his first set of experiments on its elementary composition. Box 1 FIRST PROTOCOL A key concern of Miescher’s was to get rid of contaminating proteins, which would have skewed his analyses of the novel substance. “I therefore turned to an agent that was already being used in chemistry with albumin molecules on account of its strong protein-dissolving action, namely, pepsin solutions (Miescher, 1871d). Pepsin is a proteolytic enzyme present in the stomach for digesting proteins. Miescher used it to separate the DNA from the proteins of the cells’ cytoplasm. He extracted the pepsin for his experiments from pig stomachs by washing the stomachs with a mixture of 10 cc of fuming hydrochloric acid and one liter of water and filtering the resulting solution until it was clear. In contrast to his earlier protocol, Miescher first washed the pus cells (leucocytes) three or four times with warm alcohol to remove lipids. He then let the residual material digest with the pepsin solution between 18 and 24 h at 37–45 C. After only a few hours, a fine gray powdery sediment of isolated nuclei separated from a yellow liquid. Miescher continued the digestion process, changing the pepsin solution twice. After this procedure, a precipitate of nuclei without any attached cytoplasm formed. He shook the sediment several times with ether in order to remove the remaining lipids. Afterwards, he filtered the nuclei and washed them with water until there was no longer any trace of proteins. He described the nuclei isolated in this way as naked. The contours were smooth in some cases or slightly eaten away in others (Miescher, 1871d). Miescher washed the nuclei again several times with warm alcohol and noted that the nuclear mass cleaned in this way exhibited the same chemical behavior as the nuclei isolated with hydrochloric acid. Miescher subsequently extracted the isolated nuclei using the same alkaline extraction protocol he had previously employed on the intact cells (see Box 1) and, when adding an excess of acetic acid or hydrochloric acid to the solution, again obtained a precipitate of nuclein. M. SECOND PROTOCOL TO ISOLATE DNA Fig. 5. Glass vial containing nuclein isolated from salmon sperm by Friedrich Miescher while working at the University of Basel. The faded label reads Nuclein aus Lachssperma, F. Miescher (Nuclein from salmon sperm, F. Miescher). Possession of the Interfakult-res Institut fqr Biochemie (Interfacultary Institute for Biochemistry), University of Tubingen, Germany; photography by Alfons Renz, University of Tubingen. Fig. 6. This picture of Friedrich Miescher in his later years is the frontispiece on the inside cover of the two volume collection of Miescher’s scientific publications, his letters, lecture manuscripts, and papers published posthumously by Wilhelm His and others (His et al., 1897a,b). (a) 1944: Oswald T. Avery, Colin MacLeod, and Maclyn McCarty demonstrate that Griffith’s transforming principle is not a protein, but rather DNA, suggesting that DNA may function as the genetic material (b) 1952: Alfred Hershey and Martha Chase use viruses (bacteriophage T2) to confirm DNA as the genetic material by demonstrating that during infection viral DNA enters the bacteria while the viral proteins do not and that this DNA can be found in progeny virus particles. A, B and left-handed Z-DNA as we know them now How did we arrive to them ? 30 21st Anniversary: The DNA Double Helix Comes of Age 31 DNA is a polyanionic biomacromolecule with bases in its interior and sugar-phosphate backbone on the surface. At neutral pH it carries one negative charge per nucleotide. Below pH 5 and and above pH 9 ionization of bases become important 34 Parameters of DNA structures A B Z DNA structures from X-ray crystal analysis DNA double helix is polymorphic depending on the nucleotide sequence 1953 A paragraph dealing with nucleic acids from a text book of Organic Chemistry (in Czech) is shown. Briefly, it says nucleic acids (NA‘s) form complexes with proteins which are the building blocks of plant and animal viruses and of cell nucleus. Total hydrolysis of NA‘s proceeds according to the following scheme: alkaline hydrolysis enzym. digestion Polynucleotide mononucleotide uracil or purine bases Considering that uracil and adenine were discovered in 1885 and G in 1844 while C in 1894 and T in 1900, our lectures on NA‘s were up-todate in 1885 but not in 1894 In courses of Marxism-Leninism (obligatory to all students) we were tought that G. Mendel was a burgeois reactionary pseudoscientist. Interestingly there was not a single chemist among us who believed it. To my surprise there were some biologists who took this nonsense seriously SCIENTOMETRIE / BIBLIOMETRIE 304 papers 11227 times cited 7990 without self 4110 citing articles 3837 articles without self 36,9 average per item 59 h-index Research Professor 36 741 564 382 20.58 18 Assoc. Prof. Candidate 18 19. 18x 14x h-index 18