MUNI SCI Bi4025en Molecular Biology prof. RNDr. Jan Šmarda, CSc. Mgr. Jiří Kohoutek, Ph.D. 1 Department of Experimental Biology Content of the Course • 1. Definition and brief history o molecular biology. • 2. Nucleic acids: primary, secondary and tertiary structure of nucleic acids, conformation of DNA and RNA, different conformations of DNA and their significance for biological systems, genetic information and genetic code. • 3. Molecular structure and replication of procaryotic and eukaryotic genomes. • 4. Transcription of prokaryotic and eukaryotic genomes, posttranscriptional modifications and processing of RNA, mechanisms of RNA splicing and self-splicing. • 5. Translation of prokaryotic and eukaryotic mRNAs. MUNI 2 Department of Experimental Biology _ _ Content of the Course • 6. Posttranslational processing of proteins. • 7. Regulation of gene expression in prokaryotes and eukaryotes. • 8. Molecular mechanisms of mutagenesis and recombination. • 9. Molecular basis of cancerogenesis (oncogenes, antioncogenes). • 10. DNA Repair mechanisms. • 11 Mobile genetic elements, transposons and retrotransposons. • 12. Basic principles of genetic engineering. 3 Department of Experimental Biology MUNI SCI Content of the Course • the subject of study of molecular biology, its origin and the main stages of development, structure and function of macromolecules, nucleic acids and proteins • basic concepts of molecular biology: genetic information, genetic code, gene definition, types of genes • Characteristic of procaryotic and Eucaryotic genomes • DNA replication, regulatory proteins and mechanism • Procaryotic ane Eucaryotic transcription, posttranscription modification of RNA •Translation, cotranslation and posttranslational processes, selfassambly MUNI 4 Department of Experimental Biology _ _ T Studying sources • Audio recording ? • PowerPoint presentations • Literature 5 Department of Experimental Biology MUNI SCI Molecular Biology of THE CELL Sixth Edition ALBERTS JOHNSON LEWIS MORGAN RAFF ROBERTS WALTER W. W. Norton & Company, Inc., 500 Fifth Avenue, New York, New York 10110 □ Alberts et al.: Molecular biology of the cell. 2014 6 Department of Experimental Biology MUNI SCI Garland Science, Taylor & Francis Group: New York, USA and Abingdon, UK. □ Zlatanova and van Holde: Molecular Biology: Structure and Dynamics of Genomes and Proteomes 2016 7 Department of Experimental Biology MOLECULAR BIOLOGY Structure and Dynamics of Genomes and Proteomes y° Jordanka Zlatanova Kensal E. van Holde UNI SCI Exam rules • Final Exam • Written Test o 50 questions o 60 % to pass o Score ■ A-100-92 ■ B - 92 - 86 ■ C - 86 - 78 D - 78 - 60 8 Department of Experimental Biology MUNI SCI Lecture 1 •Definition and brief history of the Molecular Biology 9 Department of Experimental Biology MUNI SCI Aim of the Molecular Biology • Clarify the relationship of the structure and interactions of biomacromolecules, in particular informational biomacromolecules, on the functions and properties of living systems. • Explanation of functions and properties of living systems based on structure and interaction of their molecules. • Integration of physical, chemical, biological and bioinformatical approaches. • Knowledge of the processes that take place in the living systems at the molecular level in the realization of genetic information. 10 Department of Experimental Biology MUNI SCI Definition of Molecular Biology Study of the structure, interaction and function of biological macromolecules. Elucidation of the molecular properties of the life. Deciphering the molecular entity/constituency of the cell Elucidate the genetic information and the mechanisms of its impact on living organism. UNI 11 Department of Experimental Biology SCI Molecular biology is not biochemistry Biochemistry It studies chemical processes in living organisms. Description of nucleic acid and protein as well as organic molecules (lipids, sugars and carbohydrates). 12 Department of Experimental Biology MUNI SCI Origin of Molecular Biology • The history of the Molecular Biology begins in 1930s with the union of various, previously distinct biological disciplines, such as • Biochemistry • Genetics • Microbiology • Virology. • In the modern sense, molecular biology attempts to explain the phenomena of life starting from the macromolecule properties that generate them 13 Department of Experimental Biology https://www.slideshare.net/indrajay/history-of-molecular-bioloqy-134296287 MUNI SCI Origin of Molecular Biology • Molecular biologists focus primarily on two macromolecules • Nucleic acid • DNA- deoxyribonucleic acid propagating genes in time • RNA - ribonucleic acid - sustaining gene propagation • sncRNA, miRNA, piRNA... - regulatory function • Proteins • active agents of the life • Scope of the Molecular biology is to seek, characterize and interpret the structure, function and relationships between these types of macromolecules. Definition of Molecular Biology Director of the Natural Sciences Division of the Rockefeller Foundation Warren Weaver. In 1938 he coined the term „Molecular biology" to describe the use of techniques from the physical sciences (X-rays, radioisotopes, ultracentrifuges, mathematics, etc.) to study living matter. In the same year the Rockefeller Foundation awarded research grants to Linus Pauling for research on the structure of hemoglobin. Under Weaver's direction the Rockefeller Foundation became a primary funder of early research in molecular biology. Warren Weaver (1894-1978) 15 Department of Experimental Biology https://www.slideshare.net/indraiav/riistorv-of-molecular-bioloav-134296287 Molecular biology: origin of the term, Science 170 (1970) 591 -2. https://www.historyof information.com/detail.php?id=3962 MUNI SCI History of Molecular Biology Molecular biology arises in the form of molecular genetics synthesis of the functionalist and structuralist "school,, in protein and nucleic acid research. Strukturalist (physicists, chemists) focus on structure of biomacromolecules (proteins, NK), not on function and inheritance Functionalists (biochemists, virologists, microbiologists, geneticists) focus on preservation and transfer of genetic information (bacteria and bacteriophages) W. T. Astbury J.D. Bernal L. Pauling E. Chargaff M.H.F. Wilkins F. H.C. Crick 16 Department of Experimental Biology M. Delbrück, E. Schrödinger G.W. Beadle, E.L. Tatum O.T.Avery, CM. MacLeod,M. McCarty, J. Lederberg A.D. Hershey J.D. Watson UNI SCI School of MB in Brno - prof. Stanislav Rosypal, DrSc. MUNI SCI History of Molecular Biology • Relatively young science. • The origin is established by many, but four fundamental discoveries: o Understanding the Structure and Function of Nucleic Acids (1944, 1953) o Deciphering the Genetic Code (1966) o Description and understanding of the processes by which genetic information is not only inherited but propagates in live (transcription, translation, regulation of gene expression) o Discovery, description, development and donation of approaches for gene editing (2011, 2013). 18 Department of Experimental Biology MUNI SCI History of Molecular Biology • 1865: Gregor Mendel discovers through breeding experiments with peas that traits are inherited based on specific laws (later to be termed „ Mendel's laws"). • 1866: Ernst Haeckel proposes that the nucleus contains the factors responsible for the transmission of hereditary traits. • 1866: Felix Noppe-Seyer - identifies hemoglobin and its ability to bound oxygen. • 1869: Friedrich Miescher isolates DNA for the first time. • 1871: The first publications describing DNA(nuclein) by Friedrich Miescher, Felix Hoppe-Seyler, and P. Plo'sz are printed. • 1882: Walther Flemming describes chromosomes and examines their behavior during cell division. • 1884 - 1885: Oscar Hertwig, Albrecht von Kflliker, 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. • 1885 - 1901: Albrecht Kossel describes pvrimidines and purines in nucleic acids. Tetranucleotides hypothesis. MUNI 19 Department of Experimental Biology R. Dahm / Developmental Biology 278 (2005) 274-288 _ _ History of Molecular Biology • 1900: Carl Correns, Hugo de Vries, and Erich von Tschermak rediscover Mendel's Laws. • 1902: Theodor Boveri and Walter Sutton postulate that the heredity units (called genes as of 1909) are located on chromosomes. • 1902-1909: Archibald 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: Thomas Hunt Morgan uses fruit flies (Drosophila) as a model to study heredity and finds the first mutant (white) 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: Phoebus Levene identifies the building blocks of DNA, including the four bases adenine (A), cvtosine (C), guanine (G), and thymine (T). Department of Experimental Biology R. Dahm / Developmental Biology 278 (2005) 274-288 MU SC N I History of Molecular Biology 1934: Caspersson and Hammersten determined that DNA is polymer. 1935: Max Delbrück, Nikolai V. Timofeeff-Ressovsky, and Karl G. Zimmer suggested that chromosomes are very large molecules, its structure can be changed by treatment with X-rays leading to changes of heritable characteristics. 1941: George Beadle and Edward Tatum demonstrated that every gene is responsible for the production of an enzyme. 1944: Oswald T. Avery, Colin MacLeod, and Maclyn McCarty demonstrated 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 Andre' 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 within a species 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. UNI 21 Department of Experimental Biology R. Dahm / Developmental Biology 278 (2005) 274-288 SCI History of Molecular Biology 1952: Alfred Hershey and Martha Chase use viruses (bacteriophage T2) to confirm DNAas 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) and speculates that three bases in the DNA always specify one amino acid in a protein. 1958: Matthew Meselson and Franklin Stahl describe how DNA replicates (semiconservative replication). 1960 - Jacob and Monod - determined the mRNA as a carrier of genetic information which is propagated in to the protein structure. 1961-1966: Robert W. Holley, Har Gobind Khorana, Heinrich Matthaei, Marshall W. Nirenberg, and colleagues crack the genetic code. UNI 22 Department of Experimental Biology R. Dahm / Developmental Biology 278 (2005) 274-288 SCI History of Molecular Biology • 1975: Sanger and Coulson the termination chain sequencing method. • 1977: Maxam and Gilbert the chemical method for sequencing. • 1986: Mullis established specific enzymatic amplification of DNA in vitro - polymerase chain reaction. • 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 Saccharomyces cerevisiae - is published. • 1998: Complete genome sequence of the first multicellular organism - the nematode worm. • 1998: Fire and Mello pull out RNA interference concept. • 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. • 2011 - 2012: Charpentier and Doubna introduce CRISPR editing approach to the science. • 2013: Zhang develops tools to edit genomic DNA in various organisms. 23 Department of Experimental Biology R. Dahm / Developmental Biology 278 (2005) 274-288 MUNI SCI Biochemistry foundation German physiologist and chemist, and the principal founder of the disciplines of biochemistry and moleular biology. He also recognized the binding of oxygen to erythrocytes as a function of hemoglobin, which in turn creates the compound oxyhemoglobin. Hoppe-Seyler was able to obtain hemoglobin in crystalline form, and confirmed that it contained iron. He performed important studies of chlorophyll. He is also credited with the isolation of several different proteins (which he referred to as proteids). In addition, he was the first scientist to purify lecithin and establish its composition. His students Friedrich Miescher and Nobel laureate Albrecht Kossel. Felix Hoppe - Seyler (1825- 1895) 24 Department of Experimental Biology https://en.wikipedia.org/wiki/Felix_Hoppe-Seyler UNI SCI Biochemistry foundation (A) Historic photography of Tubingen castle overlooking the old town. (B) Tubingen castle today. (C) Photograph of Felix Hoppe-Seyler's laboratory around 1879. Prior to becoming the chemical laboratory of Tubingen University in 1823. 25 Department of Experimental Biology Biol. 2005 Feb 15;278(2):274-88. doi: 10.1016/j.ydbio.2004.11.028. MUNI SCI Discovery of Nuclein Swiss naturalist and physician. Miesher works as the doctoral student in the lab of prof. Hoppe-Seyer. He isolates leukocytes from pus (on bandages), breaks down nuclear proteins by pepsin (a proteolytic enzyme isolated from the stomach of pigs) in order to disrupt the structure of cells and to describe released ingredients. He subjected the purified nuclei to an alkaline extraction followed by Johannes Friderich Miescher acidification, resulting in the formation of a precipitate that Miescher called (1844 - 1895) nuclein, which is resistant to proteases and lipases The function of the nuclein remains unclear for a long time, but Miescher proves,that it is present in the nuclei of all cells and suggests that it could playrole in inheritance. 26 Department of Experimental Biology Biol. 2005 Feb 15;278(2):274-88. doi: 10.1016/j.ydbio.2004.11.028. UNI SCI Discovery of Nuclein (A) Glass vial containing nuclein isolated from salmon sperm by Friedrich Miescher while working at the University of Basel. (B) The laboratory in the former kitchen of the castle in Tubingen as it was in 1879. It was in this room that Miescher had discovered DNA 10 years earlier. The equipment and fixtures available to Miescher at the time would have been very similar, with a large distillation apparatus in the far corner of the room and several smaller utensils, such as glass alembics and a glass distillation column on the side board. Biol. 2005 Feb 15;278(2):274-88. doi: 10.1016/j.ydbio.2004.11.028. UNI SCI Nuclein is Nucleic Acid • German pathologist and histologist. • 1889 named Miescher's term „ nuclein" by the term „ nucleic acid", when he demonstrated that nuclein was acidic. • He i also recognized for observation of filaments in the nearly all cell types, developed from granules. He named the granules „ bioblasts". • He explained them as the elementary living units, having metabolic and genetic autonomy, it is believed he decribed the mitochondria. Richard Altmann 1852- 1900 28 Department of Experimental Biology https://alchetron.com/Richard-Altmann MUNI SCI Nucleic Acid contains Nucleobases German biochemist, who studied under Felix Hoppe-Seyer. He described chemical composition of nucleic acids having pyrimidines and purines. Between 1885 - 1901, he was able to isolate and name its five constituent organic compounds: adenine, cytosine, guanine, thymine, and uracil. These compounds are now known collectively as nucleobases, and they provide the molecular structure necessary in the formation of stable DNA and RNA molecules. 1910 - Nobel Prize for Physiology or Medicine. Department of Experimental Biology https://alchetron.com/Albrecrit-Kossel Nucleic Acid has two Forms • In 1909, Levene and Walter Jacobs recognised D-ribose as a natural product and an essential component of nucleic acids. • In 1929 Levene also discover the D-deoxyribose in nucleic acid. • He identified components within the nucleic acids and showed that were linked together in the order phosphate-sugar-base to form units. He called each of these units a nucleotide, and stated that the DNA molecule consisted of a string of nucleotide units linked together through the phosphate groups, which are the (1869 - 1940)6 backbone of the molecule. 30 Department of Experimental Biology https://en.wikipedia.org/wiki/Phoebus_Levene MUNI SCI Nucleic Acid has two Forms Levene's Tetranucleotide Hypothesis (1910) dGMP dCMP N O Y ii s TL /^z Y z dAMP • He called the phosphate - sugar - base unit a nucleotide. • Note that adjacent sugar molecules are connected by a 3'-5' phospho-diester linkage, and bases are attached to the 1'-C of the sugar, just as in the Watson-Crick model. However, each four-nucleotide component is a separate molecule, and the bases are directed to the outside. • The simplicity of this structure implied that nucleic acids were too uniform to contribute to complex genetic variation. Attention thereafter focused on protein as the probable hereditary substance. 31 Department of Experimental Biology https://www.mun.ca/bioloav/scarr/Tetranucleotide Hypothesis.html UNI SCI Nucleic Acid is a polymer - macromolecules Swedish biochemist Einar Hammersten, conducting investigations into the molecular mass of DNA (deoxyribonucleic acid). This research led to their discovery that DNA was a polymer, or macromolecule, made up of small, repeating units. In the 1934 he and Einar Hammarsten showed that DNA was a polymer. Previous theories suggested that each molecule was only ten nucleotides long. 32 Department of Experimental Biology https://www.jbc.org/article/S0021 -9258(19)60918-X/pdf Tjorborn Caspersson (1910-1997) Einar Hammersten li UN i (1889 - 1968) SCI Chromosomes are macromolecules and carry heritable traits Max Delbrück • Max Delbrück, Nikolai V. Timofeeff-Ressovsky, and Karl G. Zimmer published results in 1935 suggesting that chromosomes are very large molecules. NV Timofeeff-Ressovsky •The structure of chromosomes can changed by treatment with X-rays. be KG Zimmer Wpit|| Alteration of chromosome's structure led to change of the heritable characteristics governed by those chromosomes. It was thought as a major advance in understanding the C34c-dedf-48ce-8318-7ad4a3eae585 33 Department of Experimental Biology https://alchetron.com/Max-Delbruck https://alchetron.com/Nikolay-Timofeev-Ressovsky https://www.mdc-berlin.de/karl-guenther-zimmer UNI SCI Nucleic Acid has regular structure William Astbury was an English physicist and molecular biologist who made pioneering X-ray diffraction studies of biological molecules. 1937 he studied the structure for DNA. Tjorborn Capersson prepared DNA for his first srudies. r William Astbury (1898- 1961) The patterns showed that DNA had a regular structure and therefore it might be possible to deduce what this structure was. X-ray diffraction photographs taken by Elwyn Beighton in Astburv's laboratory of B-form sodium thymonucleate fibres on (i) 28th May 1951 and (ii) 1 st June 1951. 34 Department of Experimental Biology Studies in History and Philosophy of Biological and Biomedical Sciences 42 (2011) 119-128 UNI SCI Johann Gregor Mendel principles Pure breeding round, yellow seeds Dominance Segregation RRYY Pure breeding wrinkled, green seeds 0 Independent assortment 0 —> 0 rryy 0 0 © 0 0 RRYY Ú RrYY RRYy J RrYy Round, yellow . seeds Self- {^J pollination RrYY rr YY Ú RrYy rrYy ^ RrYy RRYy Ú RrYy • R R y y • Rryy 0 RrYy rrYy • Rryy • rryy First generation (Fi) 9 Yellow, round 3 Green, round 3 Yellow, wrinkled 1 Green, wrinkled Second generation (Fj) Department of Experimental Biology https://www.sciencelearn.org.nz/resources/2000-mendel-s-principles-of-inheritance UNI SCI Johann Gregor Mendel principles y? Gametes from parent of unknown genotype Y ? The Test Cross c oj S <5 y vi $ n - Si to E Q) „ 10 y " O 2? yy yy yy yy A test cross resulting in all dominant offspring indicates that the parent is homozygous dominant. Gametes from parent of unknown genotype y ? c E S 2 §. f/) j B oj E ši a O) > Yy yy Yy yy A test cross resulting in a 1:1 ratio of yellow to green offspring indicates that the parent is heterozygous. • Mendel also came up with a way to figure out whether an organism with a dominant phenotype was a heterozygote (Yy) or a homozygote (YY). • Test cross is an experimental cross of an individual organism of dominant phenotype but unknown genotype and an organism with a homozygous recessive genotype (and phenotype). • Test cross is still used by plant and animal breeders today. Johann Gregor Mendel (1822-1884) 36 Department of Experimental Biology https://www.khanacademy.org/science/ap-biology/heredity/rn ■■■ U N I of-segregation ^ Q Rediscovery of Mendel s principles • 1990 - Huge de vries, Carl Correns, Erich von Tschermak rediscovered Mendel'sprincipals of heridity. • 1901 - Hugo de Vries - introduce term „ Mutation? H. De Vries C. Corens E. Von Tschermak (1848 - 1935) (1864-1933) (1871 - 1968) 37 Department of Experimental Biology https://www.eucarpia.eu/tschermak-seysenegg Rediscovery of Mendels principles • First person to use the term „ Genetics" in order to describe the study of heredity. • Based on Mendel's findings, he said, we can develop a new theory that is the correct way to study heredity and will further shed light on the nature of evolution. Wilhelm Johansen (1857-1927) William Bateson (1861 - 1926) 1909 - plant physiologist, and geneticist. He is best known for coining the terms gene, phenotype and genotype. Gene - unit of heriditary material. 38 Department of Experimental Biology https://mendel-genetics.cz/ UNI SCI Chromosomes carry heritable traits Boveri-Sutton chromosome theory, also known as the chromosome theory of inheritance is a fundamental theory of genetics proposing that the behavior of chromosomes during meiosis can explain Mendel's laws of inheritance and identifies chromosomes as the carriers of genetic material. Boveri studed sea urchins - all the chromosomes had to be present for proper embryonic development to take place. Sutton's work with grasshoppers showed that chromosomes occur in matched pairs of maternal and paternal chromosomes which separate during meiosis and "may constitute the physical basis of the Mendelian law of heredity". Watler Sutton Theodor Boveri (1877- 1916) (1862- 1915) 39 Department of Experimental Biology https://en.wikipedia.org/wiki/Boveri%E2%80%93Sutton_chromosome_trieory https://www.khanacademy.org/science/ap-biology/heredity/chromosomal-inheritance- ap/a/discovery-of-the-chromosomal-basis-of-inheritance UNI SCI Chromosomes carry heritable traits 1910 Morgan noticed a white-eyed mutant male among the red-eyed wild types. 1911, he concluded that: o(1) some traits, white-eye, were sex-linked, o(2) the trait was probably carried on one of the sex chromosomes, o(3) other genes were probably carried on specific chromosomes as well. Thomas Hunt Morgan (1866- 1945) 40 Department of Experimental Biology He and his collegues combined Mendelism and the chromosome theory of inheritance. They established the Mendelian genetics - the inheritance patterns may be generally explained by assuming that genes are located in specific sites on chromosomes. https://www.mun.ca/biology/scarr/4270_Sex-linkage_in_Drosophila.ritml UNI SCI Discovery of bacterial Transformation • Frederick Griffith - English bacteriologist. • In the 20s of the 20th century, he examines the bacterium Streptococcus pneumoniae, as a consequence of the Spanish flu in 1918,often accompanied by pneumonia caused by this bacterium. • The Ministry of Health requires research on S. pneumoniae and the Creation Of a vaccine. Frederick Griffith (1877- 1941) • In January 1928 he reported his work, what is now known as Griffith's Experiment, the first widely accepted demonstrations of bacterial transformation, whereby a bacterium distinctly changes its form and function. 41 Department of Experimental Biology https://www.mun.ca/biology/scarr/4270_Sex-linkage_in_Drosophila.ritml MUNI SCI Discovery of bacterial Transformation • There are 2 related strains S. pneumoniae, which differ morphologically and the degree of pathogenicity: o the R strain forms rough colonies, avirulent, not lethal o the S strain forms smooth colonies, virulent, after injection kills experimental mice. host's immune system) host's immune system) UNI 42 Department of Experimental Biology httDs://slideDlaver.com/slide/4955288/ DUI Discovery of bacterial Transformation • The R strain forms rough colonies, avirulent, not lethal. • The S strain forms smooth colonies, virulent, after injection kills experimental mice. R-Cells S-Cells No cells S-Cellsand R-Cells from healthy mouse from dead mouse from healthy mouse from dead mouse 43 Department of Experimental Biology https://www.sciencephoto.com/media/717359/view/griffitri-s-experiment-illustration MUNI SCI Results of Griffith's Experiments • There is a chemical compound capable of transmitting hereditary instructions between organisms „ gene molecule". Restrained Griffith delays the publication of this revolutionary conclusion. • In January 1928 under pressure from friends he publishes its experiments in unknown journal „ Journal of Hygiene". • Article written in an remorseful style for the turmoil, which it causes to the genetics. Volume xxvtt January, 192s No. 2 THE SIGNIFICANCE OF/pNEUMOCOCCAL TYPES. Ry FREft. GRIFFITH, H.B. {A Medical Oßcer of tke Ministry of Health.) {from the MiHfutrif's Pathvlvtjical Laboratory.) fOXTKNTS, I. OlHKSVAtlůSÍ DM LUHJCML fl1*TEKIAr. .... 113 Typ™ in Lohar Pnriijm.jiin I li Variety -I Typta in sputum Fnim (lie MimtW .114 A Rwigh Virulent Stnln....... .117 A Strain ■jgfntinatnig «pedlk«llj «iili hro dUTcmit Group IV Ser» 1 IB II, BX PUBIHldTAL MODIFICATTOH 120 Attrftuatfon in ťLiIluřf ......... ISO [I] OnrtBt&in Immaat&mtn....... 120 ;■>) tinnvth DA Hoitit Metiiu........ 121 (3) tXfftrvxtrbcivtvH Individ^&md $ wlwiiw 12% \{>-wcHiiin íiťin Eiiiiifili tri Suiíinth ....... JÍ5 A. Origin nfifk H Slraina umt ...... LÍB lí. Passage nf R 1/ Strain*........ 126 c. Ha&xiir /Jarajf ^/í ....... 121} InucdkUťM rf living it und kLUod S culture*..... 12» Frebtaůtdfy .ftzfWrnuHtb ........ 129 ffrmpíFSeaíJBW+fi/ajwí//....... \tt Tgpe l S orď4(i*+ H // ana- í 131 Tspt SI! 3 culture +Ji 1 anri JI....... 1+1 Type II 8 t.tilH.it + R 1........,144 Ttjjies [ mní II &ctitturt/r + )ir;rauii IV..... 146 1 niicujiitkm of living and dead li cultures ..... 147 III. lJiKcirsMdK . ,.......... 148 IV. Si-mmabv............ 167 t. Observations oh Clinical ""»1 ■■ 11: ■«- ■■ i. Sincr communicating my report1 on the distribution of pneumococcal types in a series of 150 casea. of lobar pneumonia occurring in Uis period from ApriL 1920 to January, 1022, I have not made any special investigation of this Subject-. In tlw; course, however, of other inquiries und of the routine examination of sputum during the period from the end ol January, 1922, to March, 19^7, some further data have been accumulated1. Table 1 gives the results iu two aeries and, fur comparison,those previously published. 1 fteporU Public HiaMt and Jferfiwl 0w$«*r (IKZj, Nft ti * I owemurj lb«"lrSaiHhnii:k , Joraendirj ma m*riy a]i('t:]nif'ne ((Villi etaet or \uhu ipuismiKmia. Joun. of Hyg. iiyu B 44 Department of Experimental Biology MUNI SCI Genes and enzymatic activity • The were using mold Neurospora crassa model, new to the molecular biologists. • They x-rays Nerospora creassa and induced mutations. • In a series of experiments, 1941, they showed that these mutations caused changes in specific enzymes involved in metabolic pathways. George Beadle (1903-1989) Edward Tatu m (1909-1975) The implementation and exploiation of novel model to the Molecular Biology becomes a recurring theme. UNI 45 Department of Experimental Biology SCI Genes and enzymatic activity • 1941 - The direct link between genes and enzymatic reactions leading to postulation of One gene-one enzyme hypothesis. (a) X rays Mulagenized conidia Wild type (b) Complete medium (C) Minimal medium Crossed with wild type No growth r (d) fi Minimal I (control) I n ,• Complete Minimal (control) + amino It 1 acids i í i r Ti Í l \ (e) nnnn Q> (DO) U O o < 3 1 o - FT & rx c 8 CT 46 Department of Experimental Biology https://www.mun.ca/biology/scarr/Beadle_&_Tatum_Experiment.html UNI SCI DNA harbors the genetic information Confirmation of Griffith's experiment. DNA is responsible for the transformation of Streptococcus pneumoniae bacteria, 1944. Adding purified DNA to bacteria changes their properties (shape of colonies, ability to cause disease, etc.). Acquired properties are transferred to subsequent generations. Oswald Avery Colin MacLeod (1877- 1955) (1909-1972) Maclyn McCarty (1911 -2005) STUDIES ON THE CHEMICAL NATURE OF THE SUBSTANCE INDUCING TRANSFORMATION OF PNEUMOCOCCAL TYPES Induction or Transformation by a Desoxyrtbonucleic Acid Fraction Isolated from Pneumococcus Type HI By OSWALD T. AVERY, M.d., COLIN M. MacLEOD, M.d., and maclyn Mccarty,* m.d. (From the Hospital of The Rockefeller Institute for Medical Research) Plate 1 (Received for publication, November 1, 1943) 47 Department of Experimental Biology https://www.mun.ca/biology/scarr/Beadle_&_Tatum_Experiment.html UNI SCI DNA harbors the genetic information Oswald Avery's Isolation of the Transforming Substance Centrifuge Heat-kill^ Homogenize cells » Recover IMS filtrate MIS cells spun to bottom of tube HIS cells in liquid culture medium Treat with deoxyribonuclease Treat with ribonuclease m I a Assay for Transformatlonj MR cells + DNase-treated MIS filtrate MR cells + RNase-treated i IIIS filtrate Extract carbohydrates, lipids, and proteins Treat with protease 1 MR cells + Protease-treated IMS filtrate MR cells + MIS filtrate No transformation occurs ♦ Transformation occurs Transformation occurs ♦ Transformation occurs Mixture of SStrain digests DNA y -»B ->B ■*B -»B DNAso R strain \ RNAm + •••* •* • \ Lipas* o o . o Protease ••• \ Other + ••• \ -> <-Z& Alive! y*& Dead -> Dead ->J*^*> Doad -> Dead no transformation! ONAIttht ' (ramtofmlnc principle!! Only MR v cells Conclusion: Active factor is DNA MR cells IMS cells Conclusion: Active factor is not RNA 48 Department of Experimental Biology MR cells + IMS cells Conclusion: Active factor is not protein IIR cells mm ms ceiis Control: MIS contains active factor UNI SCI Hereditary genetic information is carried by DNA Phage DNA and proteins are separable. The phages inject their DNA into the host bacteria. The phages inject their DNA into the bacteria and then the DNA serves as as replicating element genetic element of phages. HERSHEY- CHASE EXPERIMENT VS- NUCLEIC ACIDS RADIOACTIVE SULFUR 35 RADIOACTIVE PHOSPHOROUS il Q&T) INFECTION BLENDING Martha Chase and Alfred Hersey 1952 CSHLUSA RADIOACTIVE « CENTTSIFUGATION c i RADIOACTIVE I Wlinwiu ^ pour^ ARE THE MOLECULE OF HEREDITY 49 Department of Experimental Biology https://www.cshl.edu/from-phages-to-faces/ https://twitter.com/bogobiology/status/947161622019280898 MUNI SCI Hereditary genetic information is carried by DNA £/**L_ O Mix radioactively £ß labeled phages with infect the bacterial cells, from the cells and their contents. Phage Host cell (a) DNA (E coll) Phage■ oi , m Bacterium- Waring blender experiment. Batch 1: Phages grown with radioactive sulfur ("S) 35S Q Agitate in a blender to 0 Centrifuge the separate phages mixture so bacteria bacteria. The phages outside the bacteria form a pellet at the bottom of the test tube. — Empty protein shell <5T «°—Phage mK y DNA Radioactive I Pr°tein Batch 2: Phages grown with radioactive phosphorus (J2P) 32p / Q Measure the radioactivity in the pellet and the liquid. Radioactivity in liquid % Pellet Radioactivity in pellet 50 Department of Experimental Biology Pellet (b) The experiment showed that T2 proteins remain outside the host cell during infection, while T2 DNA enters the cell https://embryo.asu.edu/pages/hershey-chase-experiments-1952-alfred-hershey-and-martria-criase https://i.pinimg.com/originals/c9/62/82/c96282125ff929437ae46adb23bc0301.jpg UNI SCI Mutation in the DNA causes disease • He applied principles of quantum mechanics in chemistry and also participates in on the study of the spatial structure of proteins. • He formulates a hypothesis, that the cause of sickle cell anemia could be abnormal form of hemoglobin. • The hypothesis successfully verified by electrophoretic techniques, 1949. • For the firts time he linked specific genetic mutation with the sickle cell disease to a demonstrated change in an individual protein, the hemoglobin in the erytrocytes of impacted individuals. • Nobel Prize in Chemistry in 1954. 51 Department of Experimental Biology Hunt for the structure of DNA In the 1950s, three groups made it their goal to determine the structure of DNA. The first group to start was at King College London and was led by Maurice Wilkins and was later joined by Rosalind Franklin. Another group consisting of Francis Crick and James D. Watson was at Cambridge. A third group was at Caltech and was led by Linus Pauling. The fourth group was in Leeds led by William Astbury and Elwyn Beighton Department of Experimental Biology Hunt for the structure of DNA • 1948 Pauling discovered that many proteins included helical shapes. Pauling had deduced this structure from X-rav patterns and from attempts to physically model the structures. • There remained the questions of how many strands came together, whether this number of bases was the same for every alfa-helix, whether the bases pointed toward the helical axis or away, and ultimately what were the explicit angles and coordinates of all the bonds and atoms. Such questions motivated the modeling efforts of Watson and Crick. 53 Department of Experimental Biology MUNI SCI Hunt for the structure of DNA Watson and Crick restricted themselves to what they saw as chemically and biologically reasonable. A breakthrough occurred in 1952, when Erwin Chargaff visited Cambridge and inspired Crick with a description of experiments Chargaff had published in 1947. Chargaff had observed that the proportions of the four nucleotides vary between one DNA sample and the next, but that for particular pairs of nucleotides adenine and thymine guanine and cvtosine guanine cytosine • the two nucleotides are always present in equal proportions. 54 Department of Experimental Biology UNI SCI Discovery of DNA structure • 1953: James Watson a Francis Crick derive the structure of DNA on the basis of the following data: • Chemical data: Erwin Chargaff principles: • the concentration of thymine and adenine is the same • the concentration of cytozine and guanine is the same • Physical data: Maurice Wilkins a Rosalind Franklin after exposure of purified DNA molecules to X-rays, there is a characteristic scattering of rays that signal method of arranging DNA components into a helix. Department of Experimental Biology > o 2 a> t_ o dz o 'co 0 Hydrogen • Oxygen • Nitrogen G Carbon O Phosphorus Discovery of DNA structure Rosalinda Franklin James Watson a Francis Crick Pyrimidines Purines £1 Photo 51 56 Department of Experimental Biology https://commons.wikimedia.Org/wiki/File:DNA_Structure%2BKey%2BLabelled.pn_NoBB.png https://www.sciencephoto.com/media/222783/view https://en.wikipedia.org/wiki/Rosalind_Franklin UNI SCI Discovery of DNA polymerase • American biochemists. • In 1956 he isolated, for the first time the DNA-polymerase from E. coli. • Function osynthesis of short sections of DNA (filling in the gaps between Okazaki fragments) ocomponent of reparation mechanisms Arthur Kornberg omain function: removal of RNA-primers. (1918-2007) • 1959 - Nobel Prize in Physiology or Medicine. 57 Department of Experimental Biology https://www.jbc.org/article/S0021-9258(20)59088-1/fulltext MUNI SCI Discovery of DNA polymerase The two reports describing "DNA polymerase," reaction were declined by the Journal of Biological Chemistry when submitted in the Fall of 1957. Among the critical comments were: "It is very doubtful that the authors are entitled to speak of the enzymatic synthesis of DNA"; "Polymerase is a poor name"; "Perhaps as important as the elimination of certain banalities..." etc. V 4 dNTPs PP) ssDNA Template Salts Cell extract Incubate @ 37°C If there's DNA pol activity, then radioactive polymers generated Gel Autoradiogram r a o r n Separate by gel electrophoresis followed by Southern transfer *" Unlabelled polymers (i.e. template) no visualized on autoradiogram 58 Department of Experimental Biology https://slideplayer.eom/slide/14061307/ UNI SCI Evidence based on the study of DNA density after marking with heavy nitrogen 15N. Matthew Meselson (1930 - Franklin Stahl (1929 - DNA replication is semiconservative process In 1958 they proved the validity of semi-conservative model of the proposed Watson and Crick in 1953. (a) Grow bacteria in 15N l| Centrifuge DNA in CsCI n "Heavy-^M DNA (15N) Original DNA (b) Transfer cells to 14N, grow for one generation IF Centrifuge DNA in CsCI y Hybrid (15N/I4N) (c) Grow for second generation in 14N "Light" DNA (14N) First-generation DNAs A A Centrifuge DNA in CsCI Hybrid DNA KJ (15N/14N) Second-generation DNAs 59 Department of Experimental Biology https://www.nature.com/scitable/topicpage/semi-conservative-dna-replication-meselson-and-stahl-421/ UNI SCI There is intermediate between DNA and protein At the beginning of 1960, Jacob and Monod observed regulatory proteins at the edges of the genes and control the transcription of these genes into messenger RNA, in other words they direct transition of these genes. Two concepts of utmost importance came out of those experiments in 1961: o that of messenger RNA o the operon. Jacques Monod Francois Jacob (1910- 1976) (1920-2013) Replication /dna-dnw (~) Transcription /dna-rna/ I " ^\/\/ Translation /una - Protein/ J Protein 60 Department of Experimental Biology Research in Microbiology Volume 161, Issue 2, March 2010, Pages 68-73 https://mubi.com/cast/francois-jacob https://www.thoughtco.com/dna-transcription-373398 UNI SCI Cracking the genetic code Work by Crick and coworkers showed that the genetic code was based on non-overlapping triplets of bases, called codons. H. G. Khorana, R. Holley and M. Nirenberg and others deciphered the encoding the meaning of all codons in 1966. if / H. G. Khorana R. Holley M. Nirenberg In 1968H.G. Khorana R. Holley M. Nirenberg were awarded by the Nobel Prize in Physiology or Medicine. GTGCATCTGACTCCTGAGGAGAAG CACGTAGACTGAGGACTCCTCTTC i GUGCAUCUGACUCCUGAGGAGAAG VHLTPEEK DNA (transcription) RNA (translation) protein 61 Department of Experimental Biology https://www.researchgate.net/publication/282279062_Computational_Statistics_for_trie _ldentification_of_Transcriptional_Gene_Regulatory_Networks UNI SCI Cracking the DNA code • Khorana synthesis of polynucleotides with defined sequence of nucleotides, repeated mostly —► in vitro transcription —► in vitro translation —► polypeptide analysis. The technique works only for DNA, not for RNA. • 1961 - Nirenberg and Matthaei discovered that poly-U RNA nucleotide makes phenylalanine polypeptide chain in vitro. • Team of researchers workd to identify meaning for all 64 codons. • 1966 - The complete Geneitc Code was deciphered. U c A G u Phe Ser Tyr Cys U Phe Ser Tyr Cys c Leu Ser STOP STOP A Leu Ser STOP Trp G c Leu Pro His Arg U Leu Pro His Arg c Leu Pro Gin Arg A Leu Pro Cln Arg G A lie Thr Asn Ser U lie Thr Asn Ser c He Thr lys Arg A Met Thr lys Arg G G Val Ala Asp Gly U Val Ala Asp Cly C Val Ala Clu Cly A Val Ala Glu Cly G Marshall Nirenberg assembled a team of about 10 researchers and technicians who discovered the chart above — the genetic codes describing 20 amino acids. 62 Department of Experimental Biology https://www.acs.org/content/acs/en/education/whatischemistry/landmarks/geneticcode.html UNI SCI DNA sequencing Maxam-Gilbert technique depends on the relative chemical liability of different nucleotide bonds, 1977. Sanger method interrupts elongation of DNA sequences by incorporating dideoxynucleotides into the sequences, 1975. Gilbert and Sanger were awarded by the Nobel Prize in Chemistry in 1980. tn—m—m—m—nr AC GTAAATCGATACGTAA TGCATTTACCTATGCATT I III_|_1_|_I_I I I Template DNA i i i m ▼ Strand separation i I I TT - Chemical treatment G G+A ^T+C C TT Ö Reactions 3 'I I I-m-1—I—I-m--i—r ACGTAAATCGATACGTAA l-l I I_I_I_I_I_I_I_I_ J—l—L _L J_ J_L _I__L _I__I__L _ ' ' i _ Electrophoresis G G+A T+C C B Tn—m—m—m—tt Temp|ate DNA AC GTAAAT CGA TACGTAA C A T T Primer DNA Polymerase ! - - G t+C Single stranded DNA template Cleave at specific bases 1 PCR with fluorescent, chain-terminating ddNTPs Size separation by capillary gel electrophoresis Laser excitation & detection by sequencing machine OA A>G t+C Separate by gel electrophoresis and detect labels Large fragments — — C — T "VeS — Vj^S -C sr's' Mixture of dNTPs & fluorescently- labelled ddNTPs Origins DNA sequence, PCR amplified & denatured Fluorescently-labelled oligonucleotides Small fragments Photo multiplier Output chromatograrn i i i i A C A A I I I G C G Department of Experimental Biology UNI https://www.researchgate.net/publication/268048875 Strategies for de novo DNA segue ncing/figures?lo=1 https://www.sigmaaldrich.com/CZ/en/technical-documents/protocol/genomics/sequencing/sanger- C P sequencing Polymerase Chain Reaction - PCR • 1979 Cetus Corporation hired Kary Mullis to synthesize oligonucleotides. • May 1983 Mullis synthesized oligonucleotide probes for a project to analyze a sickle cell anemia mutation. • In the spring of 1985 the development group began to apply the PCR technique to other targets. • Early in 1985, the group began using a thermostable DNA polymerase (the enzyme used in the original reaction is destroyed at each heating step). • Nobel Prize in Chemistry 1993. 65 Department of Experimental Biology Dig Dis Sci. 2015 Aug; 60(8): 2 Kary Banks Mullis (1944-2019) Saiki RK et al."Enzymatic Amplification of/3-globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia" Science vol. 230 pp. 1350-54(1985). MUNI RNA interference - non-coding RNA In 1998, Fire and Mello demonstrated that they could efficiently and selectively dial down the expression of various genes in the worm Caenorhabiditis elegans by injecting small quantities of short interfering RNA (siRNA) molecules, which comprise paired strands of RNA. Discovery of additional biologically active RNA followed: o siRNA o piRNA o sncRNA Nobel Prize in Physiology or Medicine in 2006. Andrew Z. Fire dsRNA injection into body cavity Craig C. Mello dsRNA injection into syncytial female gonad Feeding with bacteria expressing dsRNAs <_j) <§3><@) (_*)<^^) @> (^g) Soaking in dsRNAs 66 Department of Experimental Biology https://jbiol.biomedcentral.com/articles/10.1186/jbiol97 MUNI SCI RNA interference - non-coding RNA Gene silencing Fire and Mello injected RNA corresponding to a gene important for muscle function in the worm C. eiegans. Single-stranded RNA (sense or antisense) had no effect. But double-stranded RNA caused the worm to twitch in a similar way to worms that lack a functional gene for the muscle protein, Antisense RNA Sense RNA Parent i Offspring Double-stranded RNA No effect No effect Twitching Loss of target mRIIA Fire and Hello injected RNA (mex-3 RNA) into the gonads of the worm C. elegans and studied the effect on the corresponding mRNA. They found that double-stranded RNA, but not single-stranded RNA, eliminated the target mRNA. A four-cell embryo from C. elegans. mex-3 RNA (stained black) is abundant in the early embryo. Antisense RNA Double-stranded RNA 1 Injection of antisense RNA reduced the content of mRNA to some extent. The target mRNA was eliminated after injection of double-stranded RNA. UNI r^ ^ fC lo. Nature. 1998 Feb 19;391(6669):806-11. doi: 10.1038/35888. 67 Department of Experimental Biology _ _ https://bastiani.biology.utah.edu/courses/3230/db%20lecture/lectures/wormrnai.html ^ CRISPR method for genomic DNA editing • Nobel Prize for Chemistry in 2020. Emmanuelle Charpentier 68 Department of Experimental Biology Jennifer A. Doubna 17 august 2012 vol 337 science MUNI SCI CRISPR method for genomic DNA editing • 2011 — Emmanuelle Charpentier, showed that tracrRNA forms a duplex with crRNA, and that it is this duplex that guides Cas9 to its targets. • 2012 — Charpentier and Jennifer Doudna reported that the crRNA and the tracrRNA could be fused together to create a single, synthetic guide, further simplifying the system. CRISPR array ProtQS pacer 1 tracrRNA Short palindromic repeats pre-crRNA 5 Target recognition '....."■"............. ............ Target sequence Protospacer adjacent (Protospacer) motif (PAM) i DNA unwound at target sequence pre-crRNA processing 6 i Mllllllll^iüm^f I DNA cleavage {Double-strand break) Individual Cas9xrRNA complexes .......Illlllllll TTTTTTT 69 Department of Experimental Biology https://www.broadinstitute.org/what-broad/areas-focus/project-spotlight/crispr-timeline https://www.addgene.org/crispr/history/ UNI SCI CRISPR method for genomic DNA editing • 2013 - Zhang was first to successfully adapt CRISPR-Cas9 for genome editing in eukaryotic cells. • They engineered two different Cas9 orthologs (S. pyogenes and S. thermophilus) demonstrated targeted genome cleavage in human and mouse cells. • (i) could be programmed to target multiple genomic loci • (ii) could drive homology-directed repair. gRNA Scaffold + Complex formation and target binding Spacer u__Jl uuuuu^gi' llllllllll / Target+PAM ■■■■■■in Non-homologous end joining (NHEJ) Target cleavage (DSB formation) IIIIHIIIIllIMM 111 ill 11111III Homology directed repair (HDR) ""»"«......... LULU TTTTT WT Insertion Deletion Frameshift I......ilHHIHIIIHIimiMH IIIIIHÉ.......Í.....umiim IIIIITT 1111111 niiniiiinniiiui Repair template with homology arms, desired genomic edit and PAM mutation iiiiimiiiiiiiiiii i iiiiiiiiiiiiiiii IN..... Precise edit lllllllllllll IIIIIIMT lllllllll 70 Department of Experimental Biology https://www.broadinstitute.org/what-broad/areas-focus/project-spotlight/crispr-timeline https://www.addgene.org/crispr/history/ UNI SCI Current age of Molecuar biology • Research area • New separate disciplines within molecular biology: • Transcriptomics, metabolomics, exposomics, microbiomics, secretomics, kinomics a"... omics". • Study of regulation of gene expression and cell differentiation processes (cell cycle, signaling pathways, regulatory disorders, stem cell research). • Neurobiology. • Use of the molecular methodology in a number of fields: molecular microbiology,virology, immunology, physiology, anthropology, evolution. 71 Department of Experimental Biology MUNI SCI Current age of Molecuar biology • Practical applications • Gene engineering - overlaps into agriculture, pharmacy, medicine. • Modern biotechnology - preparation of transgenic and genetic modified organisms and new substances by targeted gene repurchase. • Genome editing - targeted changes in genomes in vivo, CRISPR/Cas. • Molecular diagnostics of infectious, hereditary and cancerous diseases, new ways of their treatment (detection of latent pathogens, prenatal diagnostics). • Pharmacogenomics - drugs "tailored" to the individual genetic constitution (allergies, susceptibility...). • gene therapy - treatment of genetic diseases (beginning in the 80s,but not yet too widespread, big risks). MUNI 72 Department of Experimental Biology SCI -,n ~ r- . i o1 i https://vvww.dreamstime.com/center-molecular-genetics-center-m .UNI 73 Department of Experimental Biology . , .. ^oon^cc ^ ~ T ^ K a/ mfographics-imagel 18834055 Q P T