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 the molecular biology discipline. • 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 prokaryotic 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. 2 Department of Experimental Biology MUNI SCI 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 the 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 Prokaryotic and Eukaryotic genomes • DNA replication, regulatory proteins and mechanism • Prokaryotic and Eukaryotic transcription, posttranscription modification of RNA •translation, cotranslation and posttranslational processes, selfassambly MUNI 4 Department of Experimental Biology r> r» t Studying sources • PowerPoint presentations • Literature 5 Department of Experimental Biology MUNI SCI Molecular Biology of THE CELL Sixth Edition i 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 Masaryk University Press, Brno, 2023 □ Slabý et al., Medicical Biology I. 2023 7 Department of Experimental Biology MUNI SCI MOLECULAR BIOLOGY Structure and Dynamics of ' Genomes and Proteomes 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 8 Department of Experimental Biology Jordanka Zlatanova Kensal E. van Holde GS MUNI SCI Exam requirements • Final Exam - written test and oral exam - 50% + 50% of final grade • Written Test • Oral Exam o 50 questions o 2 questions o 60 % to pass o Score ■ A-100-92 ■ B - 92 - 84 ■ C - 84 - 76 ■ D - 76 - 68 E - 68 - 60 9 Department of Experimental Biology MUNI SCI Lecture 1 •Definition and brief history of the Molecular Biology 10 Department of Experimental Biology MUNI SCI Aim of Molecular Biology • Clarify the relationship of the structure and interactions of biomacromolecules, in particular, the 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. 11 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. MUNI 12 Department of Experimental Biology _ _ T O 0 J. Molecular biology is not Biochemistry Biochemistry It studies chemical processes in living biological organisms. Description of nucleic acid and protein as well as organic molecules (lipids, sugars and carbohydrates). 13 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. 14 Department of Experimental Biology https://www.slideshare.net/inclraiay/riistory-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. 15 Department of Experimental Biology https://www.slideshare.net/inclraiay/riistory-of-molecular-bioloqy-134296287 MUNI SCI 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) 16 Department of Experimental Biology https://www.slideshare.net/indraiav/historv-of-molecular-biologv-134296287 Molecular biology: origin of the term, Science 170 (1970) 591-2. https://www.historyofinformation.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. Structuralist (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 17 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 MUNI SCI School of MB in Brno - prof. Stanislav Rosypal, DrSc. 18 Department of Experimental Biology I 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). MUNI 19 Department of Experimental Biology Jul 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 or principles"). • 1866: Ernst Haeckel proposes that the nucleus contains the factors responsible for the transmission of hereditary traits. • 1866: Felix Noppe-Sever - 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. 20 Department of Experimental Biology R. Dahm/ Developmental Biology 278 (2005) 274-288 MUNI SCI 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. • 1905: William Bateson as first person uses the term „ Genetics" in order to describe the study of heredity. • 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, deoxyribonucleic and ribonucleic acid, as well as four bases adenine (A), cvtosine (C), guanine (G), and thymine (T). The Tetra nucleotide hypothesis. MUNI 21 Department of Experimental Biology R. Dahm/ Developmental Biology 278 (2005) 274-288 n _ T 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 As as T's and the same number of C's as G's. MUNI 22 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 DNA as the genetic material. • 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. 23 Department of Experimental Biology R. Dahm/ Developmental Biology 278 (2005) 274-288 MUNI 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 seguence 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. 24 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 molecular 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 on chlorophyll. Felix Hoppe - Seyler (1825-1895) • 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. 25 Department of Experimental Biology https://en.wikipedia.org/wiki/Felix_Hoppe-Seyler MUNI SCI b 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. 26 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-Seyler. 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 play a role in inheritance. MUNI Department of Experimental Biology Biol. 2005 Feb 15;278(2):274-88. doi: 10.1016/j.ydbio.2004.11.028. p> o t 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. MUNI SCI Biol. 2005 Feb 15;278(2):274-88. doi: 10.1016/j.ydbio.2004.11.028. 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 is 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 29 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. Albrecht Kossel •1910- Nobel Prize for Physiology or Medicine. 1852 -1927 30 Department of Experimental Biology https://alchetron.com/Albrecht-Kossel MUNI SCI 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 backbone of the molecule. Phoebus Levene (1869-1940) 31 Department of Experimental Biology https://en.wikipedia.org/wiki/Phoebus_Levene MUNI SCI Nucleic Acid has two Forms Levene's Tetranucleotide Hypothesis (1910) dGMP hi dCMP N r-y-0-?-0-UoJ 0 0-ti=o 1 0 1 ° TL dTMP ° V1 32 Department of Experimental Biology • 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. MUNI https://www.mun.ca/biology/scarr/Tetranucleotide Hypothesis.html O 0 J. dAMP Nucleic Acid is a polymer - macromolecules Swedish biochemist Einar Hammersten, conducted investigations into the molecular mass of DNA (deoxyribonucleic acid). This research led to the 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. 33 Department of Experimental Biology https://www.jbc.org/article/S0021-9258(19)60918-X/pdf Tjorborn uaspersson (1910- 1997) Einar Hammersten MUNI (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 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 nature of gene mutation and gene structure. 34 Department of Experimental Biology https://alchetron.com/Max-Delbruck https://alchetron.com/Nikolay-Timofeev-Ressovsky https://www.mdc-berlin.de/karl-guenther-zimmer MUNI 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 studies. 1 William Astbury (1898-1961) 35 Department of Experimental Biology • 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) 1st June 1951. MUNI SCI Studies in History and Philosophy of Biological and Biomedical Sciences 42 (2011) 119-128 Johann Gregor Mendel principles Pure breeding round, yellow seeds 1. Dominance 2. Segregation RRYY Pure breeding crinkled, green seeds 0 3. Independent assortment Q —*0 rryy 0 0 0 0 0 RRYY Ú RrYY J R R Yy RrYy Round, yellow ^"v . seeds Self-pollination J RrYY rrYY J RrYy rrYy ' RrYy ffTyJ R R Yy J RrYy • R R y y • Rryy 0 3 RrYy rrYy • Rryy # rryy First generation (Ft) 9 Yellow, round 3 Green, round 3 Yellow, wrinkled 1 Green, wrinkled Second generation (Fj) 36 Department of Experimental Biology https://www.sciencelearn.org.nz/resources/2000-mendel-s-principles-of-inheritance MUNI SCI Johann Gregor Mendel principles The Test Cross Gametes from parení of unknown genotype Y ? £ Q- in Si CD > Si 1/3 e (ľ ľ) ľ Vy 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 £= OJ = £ y 0 cd ' ui gj m > 1 S3 i 8 y (3 Si 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) 37 Department of Experimental Biology https://www.khanacademy.org/science/ap-biology/heredity/mendelian-genetics-ap/a/the-law-of-segregation MUNI SCI Rediscovery of Menders principles • 1990 - Huge de vries, Carl Correns, Erich von Tschermak rediscovered Mendel's principals of heridity. • 1901 - Hugo de Vries - introduce term „ Mutation? H. De Vries (1848- 1935) 38 Department of Experimental Biology C. Corens (1864-1933) https://www.eucarpia.eu/tschermak-seysenegg E. Von Tschermak (1871 -1968) MUNI SCI Rediscovery of Menders 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. William Bateson (1861 -1926) Wilhelm Johansen (1857-1927) 1909 - plant physiologist, and geneticist. He is best known for coining the terms gene, phenotype and genotype. Gene - unit of heriditary material. 39 Department of Experimental Biology https://mendel-genetics.cz/ MUNI 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 Menders laws of inheritance and identifies chromosomes as the carriers of genetic material. • Boveri studied sea urchins - all the chromosomes had watier Sutton Tneodor Boveri to be present for proper embryonic development to take 0877 -1916) (1862 -1915) 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". .«.. T An - ,x 1 o- 1 https://en.wikipedia.org/wiki/Boveri%E2%80%93Sutton chromosome theory U 111 1 40 Department of Experimental Biology — — ■ https://www.khanacademy.org/science/ap-biology/heredity/chromosomal-inheritance- C P ap/a/discovery-of-the-chromosomal-basis-of-inheritance O O ± 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) He and his colleagues 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. 41 Department of Experimental Biology https://www.mun.ca/biology/scarr/4270_Sex-linkage_in_Drosophila.html MUNI 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. • 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. 42 Department of Experimental Biology https://www.mun.ca/biology/scarr/4270_Sex-linkage_in_Drosophila.html 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) , rH uamta MUNI 43 Department of Experimental Biology https://slideplaver.com/shde/4955288/ O 0 J. Discovery of bacterial Transformation Living R-Cells Living Heat-killed S-Cells R-Cells from healthy mouse 5-Cells from dead mouse No cells from healthy mouse Living R-Cells + - Heat-killed .Z S Cd s S-CellsandR-Cells from dead mouse • The R strain forms rough colonies, avirulent, not lethal • The S strain forms smooth colonies, virulent, after injection kills experimental mice. 44 Department of Experimental Biology https://www.sciencephoto.com/media/717359/view/griffith-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 XXVil JANUARY, li>28 No, 2 THE SIGNIFICANCE OlV^NEUMOCOCCAL TYPES. By FRFJJ. CKIFFITH. M.B. {A Medical Officer oj the Ministry of Health.) [Frtuii the Mut\i||.i=M lr.i|| lln «mr I W . [11 A KinijjFi Vimlrnl strain..... [IT A Slrnili a^liilinntiiiK atidily mill Iwu ilifiVr. nt Vmuy iv Sern LID |[. EsrnuionL Modification........ 130 AtK-niiiktinn in : 'ulluiv......... 130 fl| f.'sovfit r* l>MM tmum...... 130 C'l drouth m txiitt J/*rfii..... 121 \Sl liifferenrrir bcfwrrn !m!ii-iilind ft tmd 8 taiiitti/x . Ill Rnrninji from HihirIi Iij Sni<«ilh....... 12.1 A. tiritjl* of Ike It gitatH, ma!....... 12fl I). i'aanogr »/ RSI WratHx........ 12« I". Jiff«fn itawjr u:ith an....... 121J Inoculation i>E living K and killnl K nJtum..... 12» Prftiminarif Hijitrimexta........ 129 OmptrStutturt^Jtlndli....... 132 Tgpil Stxliiire + im a*d I....... 134 Tgpt ttl 6emhan +61 and it....... 14L T»!X II 5 trfm-k-JIJ......... IU Tfpa } and il H ctilMrt* + H flrasp IV..... uc J :. at lirinp and . : i: cnttuirs..... in Dncvttmc............ 148 iv. Bdmmm............ Ifl7 I. (5fl.StRVAT[UXS ON CLINICAL MATfcKlAL. Sinte communicating my report' on. the distribution of pneumococcal types in a scries of 150 cases of lobar pneumonia occurring in the period from April. -'.'J" to January, 1022, I have not made nay special investigation of this subject. In the course, however, of other inquiries and of the routine examination of sputum during the period from the end of January, 1922, to March, 1927, some further data have been accumulated1. Tabic I gives the results in two series ahd, foruoniparis-uu,those previously published. 1 HtporU an Pubt2Jt. Ko. 13. * Inueinanylhanba lulli J. fell Ferguson, formerly Mniicai Ul>icrr if tfralth lur Siruihwi. k , for wndirg me many fljivfliiHf n» fr-rni cuci ol lobar pneumonia. of I !;■■.-. lim t 45 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 Tatum (1909-1975) The implementation and exploitation of novel model to the Molecular Biology becomes a recurring theme. 46 Department of Experimental Biology MUNI SCI Genes and enzymatic activity • 1941 - The direct link between genes and enzymatic reactions leading to postulation of „ One gene - one enzyme hypothesis". 47 Department of Experimental Biology x rays Wild type (b) Complete medium (c) Minimal medium Crossed with wild !ype [ No grawlh :— i m H Minimal I (control) 1 Complete .'.'Minimal (eon( rol) :. + amino acids í I I I Í nfinnnt + 4-44 5 3 £ * 1 I J i I äfc||.E'| i I " I https://www.mun.ca/biology/scarr/Beadle_&_Tatum_Expe riment.html MUNI 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 of Transformation by a Desoxyrtbontjcleic Acid Fraction Isolated from P.neumococcos Type III 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) 48 Department of Experimental Biology https://www.mun.ca/biology/scarr/Beadle_&_Tatum_Experiment.html MUNI SCI DNA harbors the genetic information Oswald Avery's Isolation of the Transforming Substance I Centrifuge - Heat-kill Homogenize cells * Recover IMS filtrate HIS cells spur) to bottom of tube HIS cells In liquid culture medium Treat with deoxyribonuclease Treat with ribonucleasc - i 1 Extract carbohydrates, lipids, and proteins Treat with protease I I JAssay for Tran sformatlonl fo * MR cells + DNase-treated HIS filtrate IIP cells + RNase-t rested i HIS filtrate IIA cells + Protease-treated HIS filtrate llfl cells + HIS filtrate No transformation occurs Transformation occurs Transformation occurs ♦ Transformation occurs ♦ Mixtur* of SStraln haetfila digests DNA —*b fi strain +«•0* x Protease -*-* Th» Other ^ +*J*. > \ no h, i nsl<..■', 1 m.-i I ioi 11 ->j-^'r Doad -> ■ '.' dead -^J*/* Dead -i-Dead ■f i-:'iivi:r; crln[lpjg His Arg U Ll-u Pro His Arg C Leu Pra__ gin Aug A Leu Prp cín Arg "q" A IIb Thr A*n icr u lie. Thř Abn Scr c He Ttir Afg A Met rhi in Arg C c Vat Ala Cly u Val Ala fAp ty t Vil Ab Clu m A Val Ala Clu Cly Marshall Nirenberg assembled a team of about 10 researchers and technicians who discovered the chart above — the genetic codes describing 20 amino acids. 63 Department of Experimental Biology https://www.acs.org/content/acs/en/education/whatischemistry/landmarks/geneticcode.html MUNI 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 DNA strands, 1975. Gilbert and Sanger were awarded by the Nobel Prize in Chemistry in 1980. Tn—m—m—rr AC GTAAATCGATAC 1-TT G T A A T G C A T T I I I I- TACCTATGCATT —L___J_I_I_I_L Template DNA Strand separation TTI-TTT G+A I I I I I I - Chemical treatment "*Ť+c c TT Reactions T i i—i i i—m—i—n—rr ACGTAAATCGATACGTAA _I I '_I_I_I_I_ j_l J_L J_I_L I I ♦ Electrophoresis G+A T+C C B Tn—m—m—TTT ACGTAAATCGATACGTAA ■P"P:-p <+- Reactions y *t~i i—m—m—m—nr ACGTAAATCGATACGTAA Extension Products ddA, ddAi i i i ri::Al + Electrophoresis á& ^ J? 64 Department of Experimental Biology Horticultural Science and Technology. 31 October 2019. 549-558 MUNI SCI DNA sequencing Maxam-Gilbert technique label Sanger method i i i i Singlu sívíinth-d DNA template Odivu ai specific bases Separate by gel electrophoresis and deBeet labels 1 PCR with fluorescent, chain-terminating ddNTPs — — -c — T — — — — — C — T I □ I Mixture of dNTPs & fluorescently- labelled ddNTPs Size separation by capillary gel electrophoresis Laser excitation & detection by sequencing machine Large fragments Small fragments Original DNA sequence, PCR amplified 81 denatured Fluorescently-labelled oligonucleotides Photomultiplic Output chromatogram i i i i i i i 1 i ACAATGCGT Department of Experimental Biology https://www.researchgate.net/publication/268048875 Strategies for de novo DNA segue ., .. .,. T ncing/figures?lo=1 M U 111 1 https://www.sigmaaldrich.com/CZ/en/technical-documents/protocol/genomics/sequencing/sanger- C P T sequencing o o ± 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. 66 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 (§05) i^to^t) @> @) @> £j5§) (353) (555) Soaking in dsRNAs 67 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. Sense RNA •taa__ Parent Antisense RNA 1 Double-stranded RNA i .0^ No effect No effect Twitching Loss of target mRHA Fire and Mello injected RNA (mex-3 RNA) into the gonads of the worm C, eiegans and studied the effect on the corresponding rnRNA. They found that double-stranded RNA, but not single-stranded RNA, eliminated the target rnRNA. A four-cell embryo from C. elegans. U n i nj e cte d RNA (stained black) is abundant in the early embryo, Antisense RNA {..... i Double-stranded RNA Injection of antisense RNA reduced the content of rnRNA to some extent, The target nnRNA was eliminated after injection □f double-stranded RNA, _ ._. Nature. 1998 Feb 19;391(6669):806-11. doi: 10.1038/35888. MUNI 68 Department of Experimental Biology _ _ T https://bastiani.biology.utah.edu/courses/3230/db%20lecture/lectures/wormrnai.html ^ f CRISPR method for genomic DNA editing • Nobel Prize for Chemistry in 2020. Emmanuelle Charpentier 69 Department of Experimental Biology Jennifer A. Doudna 17 august 2012 vol 337 science muni SCI CRISPR method for genomic DNA editing (Clustered Regularly Interspaced Short Palindromic Repeats) • 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 Protospacer tracrRNA Short palindromic repeats pre-crRNA 5 Target recognition miinniiiuiiiiiiiiiimiuiUHi Target sequence Protospacer adjacent (Protospacer) motif (RAM) ^ DNA unwound at target sequence pre-crRNA processing iiiiiiiiii/^TT^Tm^^^ i i DNA cleavage (Double-strand break) Individual Cas9:crRNA complexes iniiniiniiiiii umu https://wvw.broadinstitute.org/what-broad/areas-focus/project-spotlight/crispr-time MUNI 70 Department of Experimental Biology r\ r\ -r https://www.addgene.org/crispr/history/ k 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. They demonstrated targeted genome cleavage in human and mouse cells. • (i) could be programmed to target multiple genomic loci, • (ii) could drive homology-directed repair. gRIMA Scaffold + Complex formation and target binding Spacer ^ Target+PAM 1111111:1 Non-homologous end joining (NHEJ) Target cleavage (DSB formation) .........imnn .....t.....nf Homology directed repair (HDR] riiiiiiiiriiirrniuiMiii WT Insertion ......■......... Deletion ...........IHIIHIir iiniiiimmin ***.....*........M* Repair template with homology arms, desired genomic edit and PAHfl mutation Frameshift ......i................1 Precise edit LLU. 11 him in in phihihi 71 Department of Experimental Biology https://www.broadinstitute.org/what-broad/areas-focus/project-spotlight/crispr-timeline https://www.addgene.org/crispr/history/ MUNI 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. 72 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 73 Department of Experimental Biology _ _ T o 0 J. THANK YOU FOR YOUR ATTENTION. ^ ,_ , , https://www.dreamstimexom/center-molecular-qenetics-center-molecular-qenet MUNI 74 Department of Experimental Biology . ,r . . Hnooo^rr ^ ^ -r r r infographics-imagel 18834055 Q P T