Virus Prokaryotes and Eukaryotes PDF

Experimental Models in Biomedicine Master’s Degree in Biomedicine Faculty of Medicine and Health Sciences, Universitat de Barcelona FROM PROKARYOTIC TO EUKARYOTIC EXPERIMENTAL MODELS Introductory session Judit Castillo Molecular Bi...

Experimental Models in Biomedicine Master’s Degree in Biomedicine Faculty of Medicine and Health Sciences, Universitat de Barcelona FROM PROKARYOTIC TO EUKARYOTIC EXPERIMENTAL MODELS Introductory session Judit Castillo Molecular Biology of Reproduction and Development Research Group Faculty of Medicine and Health Sciences, Universitat de Barcelona, Hospital Clínic and IDIBAPS  Types of experimental models 1.- Chemical, mechanical, mathematical, and computer simulations 2.- In vitro tests 3.- Non-Human organisms 4.- Human Studies Groups of non-human organisms Eukaryotes Viruses Prokaryotes Protists Fungi Plants Invertebrates Vertebrates VIRUSES – General facts - Structural and genetic simplicity - Replication process that includes DNA/RNA replication, transcription, translation, secondary modifications of proteins, and protein targeting processes - Many viral genomes are sequenced, and virus 3D structures established Findings using virus as model system: Understanding of eukaryotic DNA replication Structure and biosynthesis of mRNA: polyadenylation on the 3’ termini, RNA splicing, enhancer sequences Characterization of transcription factors  VIRUSES – General facts Non-pathogenic status and ease of laboratory cultivation Bacteriophage: virus that attacks bacteria Name Genome Laboratory host Useful properties Best understood model for modern functional genomics and dsDNA T4 Escherichia coli proteomics. Possesses eukaryote-like introns, high-speed DNA (169,000bp) copying and DNA repair mechanisms. Canonical temperate phage that has been the workhorse of dsDNA λ Escherichia coli molecular biology. Exceptionally well characterized lysis– (48,502bp) lysogenic switch. Lipid-containing phage highly similar to adenovirus; broad host dsDNA Salmonella PRD1 range, but plasmid-dependent. Valuable tool for membrane (14,927bp) typhimurium studies. dsDNA Φ29 Bacillus subtilis DNA polymerase (19,285bp) ssDNA First DNA genome sequenced; uses “antibiotic-like” proteins to ΦX174 Escherichia coli (5,386bp) lyse cells. Adapted from Dennehy 2009. Review evolutionary Microbiology, 17(10):450-7 VIRUSES – Bacteriophages Phi 29 DNA polymerase, 1985 Margarita The basis of the biotechnology Salas Adapted from Dennehy 2009. Review evolutionary Microbiology, 17(10):450-7 Phi X 174 Small genome size (5386 bp Sanger sequencing, 1977 encoding 11 genes) The basis of gene sequencing PROKARYOTES – General facts - “before the nucleus”: no presence of nuclei or other membrane-bound organelles - Prokaryotic chromosomes condensed in the nucleoid via DNA supercoiling and the binding of architectural proteins - Prokaryotic DNA can interact with the cytoplasm leading to simultaneous transcription and translation - Ideal models for studying many fundamental aspects of biochemistry and molecular biology Findings and applications of prokaryotes: Important advances in DNA Technology Topoisomerase and supercoiling Production of vitamins, antibiotics, hormones PROKARYOTES – Escherichia coli 1973: Birth of the genetic engineering Preferred host for gene cloning and protein production Advantages The most well-studied organism: entire genome sequenced and molecular functions of 63,7% of the total genes have been experimentally confirmed Fast and easy growth and manipulation (20-min doubling time in nutrient-rich conditions) High efficiency of introduction of DNA into cells Ability to express proteins at very high levels (except for large and complex proteins with disulfide bonds or modifications) PROKARYOTES – Escherichia coli Physiology or Medicine 1958: genes act by regulating definite chemical events Physiology or Medicine 1959: mechanisms in the biological synthesis of RNA and DNA Physiology or Medicine 1965: genetic control of enzyme and virus synthesis Physiology or Medicine 1968: interpretation of the genetic code and its function in protein synthesis Physiology or Medicine 1969: the replication mechanism and the genetic structure of viruses Physiology or Medicine 1978: restriction enzymes and their application to problems of molecular genetics Chemistry 1980: determination of base sequences in nucleic acids Chemistry 1989: catalytic properties of RNA Chemistry 1997: enzymatic mechanism underlying the synthesis of adenosine triphosphate (ATP) Physiology or Medicine 1999: proteins have intrinsic signals that govern their transport and localization in the cell. Chemistry 2008: discovery and development of the green fluorescent protein, GFP Chemistry 2015: mechanistic studies of DNA repair  EUKARYOTES Organisms whose cells have a nucleus enclosed within a nuclear envelope. Although representing a minority of the number of living organisms, their collective worldwide biomass is comparable to that of prokaryotes Protists Fungi Plants Invertebrates Vertebrates Peng et al., 2014. Development, 141:4042-4054 PROTISTS – General facts - Non-plant, non-animal, non-fungi eukaryotes - Unicellular - Rich evolutionary diversity - Responsible for the 40% of the earth’s photosynthesis and almost all marine productivity - Vital roles in ecosystems - Rapid generation times - Protocols for genetic modification only available for a small number of species - Can cause disease - Can be parasites PROTISTS – Tetrahymena thermophila Advantages Short generation time Cheap culture conditions Two cell nuclei, one of which is a macronucleus with high quantities of minichromosomes: Gene expression, integrity and function First insights on cell cycle regulatory mechanisms  PROTISTS – Tetrahymena thermophila Telomerase builds telomere DNA Telomere DNA protects de chromosomes https://www.nobelprize.org/prizes/medicine/2009/illustrated-information/ FUNGI – General facts - Easy cultivation - Short generation times - Easy accessibility towards molecular and classical genetics - Haploid genomes amenable to mutation - Genome sequence availability - More related to animals than to plants - Study of microbodies: peroxisomes, glyoxysomes and hydrogenoosomes FUNGI – Saccharomyces cerevisiae Advantages Easy cultivation and manipulation Study of cell Fast growth processes and diseases Small genome size Connections Typical features of eukaryotic cells between genes and proteins Complete genome sequenced “Baker’s yeast” Nobel Prizes awarded for work in yeast: 2001 Nobel Prize in Physiology or Medicine: key regulators of the cell cycle. 2006 Nobel Prize in Chemistry: molecular basis of eukaryotic transcription. 2009 Nobel Prize in Physiology or Medicine: how chromosomes are protected by telomeres and the enzyme telomerase. 2013 Nobel Prize in Physiology or Medicine: machinery regulating vesicle traffic. 2016 Nobel Prize in Physiology or Medicine: mechanisms of autophagy FUNGI – Neurospora crassa Advantages Easy to propagate Fast growth Simple genetic manipulation Entire genome sequenced DNA methylation, polycomb and recessive Red bread mold mutations Non-pathogenic “One gene – One enzyme” FUNGI – Neurospora crassa Aim: To demonstrate the connection between genes and enzymes https://courses.lumenlearning.com/microbiology/chapter/using-microbiology-to-discover-the-secrets-of-life/ FUNGI – Neurospora crassa Each mutant differed from the original by only one gene “One gene – One enzyme” One gene – One polypeptide https://courses.lumenlearning.com/microbiology/chapter/using-microbiology-to-discover-the-secrets-of-life/ ANIMALS – General facts - The most used kingdom as model organism - Anatomical and physiological similarities with humans: similar diseases - ~ 90% of Nobel Prize research in Physiology or Medicine - Ethical concerns: The principle of the 3 R’s  ANIMALS – General facts Application in experimental studies: Sheep, cows, pigs, large Dogs, cats Fish, mammals Non- amphibians, human reptiles, primates birds Rodents ANIMALS – General facts Divergence time in million of years Weeler & Brändi, 2009. Dev Dyn 238:1287-1308 ANIMALS – General facts Invertebrates Dr Marta Morey: Drosophila (September 27, 2024) Dr Cristina Gonzalez: Planarians (September 30, 2024) Vertebrates Josep Maria Marimon: Laboratory animal science (October 1, 2024) Dr Francesc Piferrer: Zebrafish (September 26, 2024) ANIMALS - Invertebrates – Caenorhabditis elegans Advantages Transparent Cheap Easy and fast growth Rapid embryonic development Nematode Easy to genetically manipulate 1 mm in length 70 µm in diameter Eggs can be stored Diploid Small genome: 100 Mb, 20,000 genes, 5 + 1-2 chromosomes Genetics and development First animal genome sequenced (1998)  ANIMALS - Invertebrates – Caenorhabditis elegans Genetic regulation of organ development and programmed cell death (1982) Brenner: model organism Sulston: mapping the cell lineage Horvitz: identification of “death genes” The corresponding genes exist also in higher https://www.nobelprize.org/prizes/medicine/200 animals, including human 2/press-release/  ANIMALS - Invertebrates – Caenorhabditis elegans RNA interference-gene silencing by double-stranded RNA (1991-1998) https://www.nobelprize.org/prizes/medicine/2006/press-release/  ANIMALS - Invertebrates – Xenopus Advantages Large-size eggs and embryos easy to manipulate External embryo development All developmental stages are accessible and African clawed frogs known: target gene knock-out, knockdown and overexpression studies Embryology and development Genetic analysis (X. tropicalis) Immune tolerance on tissue transplantation (X. laevis) ANIMALS - Invertebrates – Xenopus Hypothesis: a specialized cell might still contain all the information needed to drive its development into all the different cell types of an organism 1962 1962 https://www.nobelprize.org/prizes/medicine/2012/press-release/ ANIMALS - Invertebrates – Aplysia Advantages Simple nervous system Giant neurons: 600,000 copies of a haploid genome in a single R2 neuron Sea slug ANIMALS - Vertebrates – Chicken Advantages Comparable developmental process with humans Large size and accessible embryo Semitransparent embryo External rapid development Inexpensive 1860s: First lab-derived attenuated vaccine (Louis Pasteur) 1910: Discovery of the first cancer causing virus: Rous sarcoma virus (The Nobel Prize in Physiology or Medicine 1966: Peyton Rous) ANIMALS - Vertebrates – Mice and rats Mouse: The most used experimental animal (75%) BALB/c Wistar C57BL/6 Athymic nude rat Nu/J Mouse advantages Rat advantages Similar genome size and organization Physiological and genetic similarity with than humans (98%) humans Short life span and fast reproductive rate Larger size than mouse WT, mutant and transgenic strains Disease-linked human genes homologs Easy genetic manipulation Genetically modified rat models Fundamental research, disease models, biology, medicine, surgery, vaccines  ANIMALS - Vertebrates – Naked mole-rat Advantages Longevity (>28,3 years): aging research Relationship life span and mass in rodents Heterocephalus glaber Mouse-size rodent East Africa Underground habitat Lacks pain sensitivity in the skin, low metabolic and respiratory rates Buffenstein 2005, The Journals of Gerontology: Series A, 60:1369-1377 Resistance to age-associated chronic diseases and cancer Low metabolic and respiratory rates ANIMALS - Vertebrates – Non-human primates But… Advantages Genetic variation Similarity with humans Long generation times and small litter sizes Viral replication Increased ethical considerations No genetic edition Reshus macaque Housing and maintenance Anderson, 2008. Sourcebook of models for Biomedical Research. Humana Press Inc., Totowa, NJ ANIMALS - Vertebrates – Non-human primates Estes et al., 2018, Nature Reviews Immunology, 18:390-404 What experimental model should I use for my research?  What experimental model should I use for my research? ‘The animal that I use is dependent on the question that I’m asking’ Kathy Grant, Oregon National Primate Research Center Alternatives to an animal model: - non-animal organism - organoids, cell lines, bioprinting, simulations. Dietrich et al., 2020. Studies in History and Phylosy of Biol & Biomed Sci, 80:101227 Not just one model organism… Obesity and diabetes mellitus: Kleinert et al., 2018. Nature Reviews Endocrinology. 14: 140-162 Not just one model organism… Aging: Holtze et al., 2021. Front Mol Biosci. Not just one model organism… Neurodegeneration: Drummond and Wisniewski, Acta Neuropathol. 2017 Feb; 133(2): 155–175