Oa SA ae gt Ua eee Une ee Presented by Vheejay B.Tampol, RND, MD Deparment cf Bacher School of Medicine Elo Agari Coleg Mania Tee iEAC_SASTE_BSBio (Cell & Molecular Biology LEC & LAB): March 2, 2021 (2021-03-01 at 22:35 GMT: LEARNING OBJECTIVES At the end of this lecture-discussion, you will be able to: Describe the units of measurement for cellular size Explain the rationale for the small size of most cells Characterize the field of Synthetic Biology Differentiate viruses and viroids from other infectious agents ‘Sides Prepared By Vhesjy B.Tompol RND, MD fee eee)CeO ac Oe eet a tO a ee ee ee) UNITS OF LINEAR MEASURE Units of Linear Measure used to describe internal cellular structures: MICROMETER (um): | um = 10 meters NANOMETER (nm): |_nm = 10°? meters ANGSTROM (A): | A = 1/10 of a nanometer = diameter of a H atom Commonly used by molecular biologists for atomic dimensions PeCeO ac Oe eet a tO a ee ee ee) UNITS OF LINEAR MEASURE Macromolecules are often described in either ‘Angstroms (A) Nanometers (nm) Examples: Myoglobin: = 4.5 nm x 3.5 nm x 2.5 nm Collagen or Myosin: = 100.nm in length DNA: = 2.0 nm in width Complexes of Macromolecules (Ex. Ribosomes, Microtubules, Microfilaments): between 5-25 nm in diameter ‘Sides Prepared By Vhesjy B.Tompol RND, MD Te ee)UNITS OF LINEAR MEASURE Cells & their organelles are more easily defined in micrometers (jim): Examples: Nuclei: = 5-10 um in diameter Mitochondria: < 21m in length Prokaryotic Cells: typically range in length from 1-5 um Eukaryotic Cells: 10-30 um in length Sides Prepared By: Vheejay B.Tampol RND, MDCeO ac Oe eet a tO a ee ee ee) WHY MOST CELLS ARE SMALL? Most eukaryotic cells possess a single nucleus that contains only 2 copies of most genes A cell can only produce a limited number of mRNAs in a given amount of time The greater a cell's cytoplasmic volume, the longer it will take to synthesize the number of mRNAs required by that cell ‘Sides Prepared By Vhesjy B.Tompol RND, MD PnSASTE_BSBio (Cell & Molecular Biology LEC & LAB): March 2, 2021 (2021-03-01 at 22:35 GMT) WHY MOST CELLS ARE SMALL? As a cell increases in size, the surface area/volume ratio decreases Ability of a cell to exchange substances with its environment is proportional to its surface area Ifa cell were to grow beyond a certain size, its surface would not be sufficient to take up the substances (Ex. oxygen, nutrients) needed to support its metabolic activities ‘Sides Prepared By Vhesjy B.Tompol RND, MDCRO ce nt Oa eee Ue eek) CELLULAR SIZE WHY MOST CELLS ARE SMALL? ul. In the case of cells specialized with absorption of solutes (Ex. Intestinal Epithelium) “Possess microvilli that greatly increases the surface area available for exchange eeWHY MOST CELLS ARE SMALL? Large Plant Cell Interior is typically filled by a large, fluid-filled vacuole Sides Prepared By-Vheejay B.Tarmpol RND, MDSASTE_BSBio (Cell & Molecular Biology LEC & LAB): March 2, 2021 (2021-03-01 at 22:35 GMT) WHY MOST CELLS ARE SMALL? A cell depends to a large degree on the random movement of molecules (diffusion) Time required for diffusion is proportional to the square of the distance to be traversed Ex. O, requires only 100 microseconds to diffuse a distance of | im, but requires(!0% times as long to diffuse a distance of | mm As a cell becomes larger & the distance from the surface to the interior becomes greater, the time required for diffusion to move substances in & out of a metabolically active cell becomes prohibitively long ‘Sides Prepared By Vhesjy B.Tompol RND, MD ee eeFAC_SASTE_BSBio (Cell & Molecular Biology LEC & LAB): March 2, 2021 (2021-03-01 at 22:35 GMT) CELLULAR SIZE EXCEPTIONALLY LARGE EUKARYOTIC CELLS Stentor coeruleus Free-living single celled organism Lives in freshwater ponds Grows to be more than a millimeter long ee ee)PRO ao At aod CELLULAR SIZE EXCEPTIONALLY LARGE EUKARYOTIC CELLS Acetabularia Giant single-celled green alga More than 10 cm long Caulerpa Giant single-celled green alga Can grow to a length of several meters Contains millions of nuclei in a common cytoplasm Te ee)FAC_SASTE_BSBio (Cell & Molecular Biology LEC & LAB): March 2, 2021 (2021-03-01 at 22:35 GMT) EXCEPTIONALLY LARGE CELLULAR STRUCTURES Neurons: send out extremely long processes Example: Motor Neurons (Spinal Cord): send out axons that can be a meter long Te ei)CeO ac Oe eet a tO a ee ee ee) A research field inspired by the fact that cells are collections of nanomachines Goal: To create some minimal type of living cell in the laboratory out of the same types of component parts found in a real cell To demonstrate that life at the cellular level emerges spontaneously when the proper constituents are brought together from chemically synthesized materials To develop novel life forms, using existing organisms as a starting point, that have a unique value in medicine and industry, or in cleaning up the environment ‘Slides Prepared By Vheejy B.TampolRND, MD Tee ee)CeO ac Oe eet a tO a ee ee ee) Uses the molecules, molecular complexes & organelles of a cell as building blocks Where one type of cell is directed into the formation of an entirely different type of cell ‘Sides Prepared By Vhesjy B.Tompol RND, MD © 1949/10009SASTE_BSBio (Cell & Molecular Biology LEC & LAB): March 2, 2021 (2021-03-01 at 22:35 GMT) In 2007: J. Craig Venter & colleagues Successfully replaced the genome of one bacterium with a genome isolated from a closely related species Effectively transformed one species into the other haa ‘Sides Prepared By Vhesjy B.Tompol RND, MD eeeCeO ac Oe eet a tO a ee ee ee) In 2010: J. Craig Venter & colleagues Using a copy of a bacterial genome that had been assembled (inside of a yeast cell) from fragments of DNA that had been chemically synthesized in the laboratory Produce a synthetic copy of the donor genome approx. |.1 million bp of DNA that contained a number of modifications Modified copy of the genome (from M .nfycoides) was transplanted into a cell of a closely related Bacterial species (M. pricolum), where it replaced the host's original genome ‘Sides Prepared By Vhesjy B.Tompol RND, MDEAC_SASTE_BSBio (Cell & Molecular Biology LEC & LAB): March 2, 2021 (2021-03-01 at 22:35 GMT: In 2010: ). Craig Venter & colleagues After genome transplantation: Recipient cell rapidly took on the characteristics of the species from which the donor DNA has been derived Researchers have produced cells containing a “genetic skeleton” to which they can add combinations of new genes taken from other organisms. ‘Sides Prepared By Vhesjy B.Tompol RND, MDCeO ac Oe eet a tO a ee ee ee) WHY GENETICALLY ENGINEER ORGANISMS? For organisms to possess metabolic pathways capable of producing pharmaceuticals, hydrocarbon-based fuel molecules & other useful chemicals from cheap, simple precursors Examples: Genetically-engineered cyanobacteria capable of producing diesel fuel from sunlight, water, & CO, Genetically-engineered E, coli to ferment the complex polysaccharides present in seaweed into the’biofuel ethanol ‘Sides Prepared By Vheejy B.Tompol RND, MDEAC_SASTE_BSBio (Cell & Molecular Biology LEC & LAB): March 2, 2021 (2021-03-01 at 22:35 GMT: 0 Presented by Vheejay B.Tampol, RND, MD Deparment of Bacher School of Medicine Ei Agaric Coleg = ManiaSASTE_BSBio (Cell & Molecular Biology LEC & LAB): March 2, 2021 (2021-03-01 at 22:35 GMT) BRIEF HISTORY End of 19° century, first identified in the studies of: Tobacco mosaic disease in tobacco plants (Ex. sap from a diseased tobacco plant could transmit mosaic disease toa healthy plant, even when the sap showed no evidence of bacteria in the light microscope) Hoof-and-mouth disease in cattle ‘Sides Prepared By Vhesjy B.Tampol RND, MD ee eonEAC_SASTE_BSBio (Cell & Molecular Biology LEC & LAB): March 2, 2021 (2021-03-01 at 22:35 GMT: BRIEF HISTORY Dmitri lvanovsky Russian biologist Forced the sap from a diseased tobacco plant through filters whose pores were so small that they retarded the passage of the smallest known bacterium But his filtrate was still infective He concluded in 1892 that certain diseases were caused by pathogens that were even smaller & presumably simpler than the smallest known bacteria ‘Sides Prepared By Vhesjy B.Tompol RND, MD fee en)CeO ac Oe eet a tO a ee ee ee) BRIEF HISTORY Wendell Stanley (1935) Biologist from the Rockefeller Institute Reported that the virus responsible for tobacco mosaic disease could be crystallized & that the crystals were infective He mistakenly concluded that tobacco. mosaic yirus (TMV) was a protein ‘Sides Prepared 8 Veja 8. Tampal RND. MDFAC_SASTE_BSBio (Cell & Molecular Biology LEC & LAB): March 2, 2021 (2021-03-01 at 22:35 GMT) BRIEF HISTORY Tobacco Mosaic Virus (TMV) Rod-shaped particle Approx. 300.nm long & 18 nm in diameter Consisted of a single molecule of RNA surrounded by a helical shell composed of protein subunits ee een)EAC_SASTE_BSBio (Cell & Molecular Biology LEC & LAB): March 2, 2021 (2021-03-01 at 22:35 GMT: OVERVIEW Responsible for dozens of Occur in a wide variety of very human diseases: different shapes, sizes, & AIDs constructions Polio All share certain common Influenza Properties Ebola Not considered to be organisms & are not described as being alive Measles Few types of cancer ‘Sides Prepared By Vhesj B.Tompol RND, MDEAC_SASTE_BSBio (Cell & Molecular Biology LEC & LAB): March 2, 2021 (2021-03-01 at 22:35 GMT: OVERVIEW All are obligatory intracellular parasites Cannot reproduce unless present within a host cell: Plant Cell ‘Animal Cell Bacterial Cell ‘Sides Prepared By Vhesjy B.Tompol RND, MD Tee ee)FAC_SASTE_BSBio (Cell & Molecular Biology LEC & LAB): March 2, 2021 (2021-03-01 at 22:35 GMT) VIRUSES OVERVIEW Potynearat Sprencat = @D Neo "eon Yo — maz vos acento, ’ poe suerte suomi sades Prepared By eB Tal AND. ND © 2519 /10009EAC_SASTE_BSBio (Cell & Molecular Biology LEC & LAB): March 2, 2021 (2021-03-01 at 22:35 GMT: OVERVIEW VIRION Name of a virus when it exists outside of a living cell Macromolecular aggregates, inanimate particles that by themselves are unable to reproduce, metabolize, or carry on any of the other activities associated with life Contains a small amount of genetic material: ssRNA or dsDNA No. of Genes: 3,to several hundred ‘Sides Prepared By Vhesjy B.Tompol RND, MDSa ert Ome e a Pea neces) VIRUSES _OVERVIEW CAPSID Protein capsule that surrounds the genetic material of the virion Generally made up of a specific number of subunits Many of the subunits are organized into a polyhedron, a structure having planar faces ADENOYIRUS: a common cause of * 20:sided icosahedron: common —_respiratory infections in mammals polyhedral shape of many viruses ‘Sides Prepared By Vhesjy B.Tombol RND, MD Pe)CeO ac Oe eet a tO a ee ee ee) VIRUSES OVERVIEW CAPSID = In many animal viruses (Ex. HIV) ~ Protein capsid is surrounded by a lipid- containing outer envelope that is derived from the modified plasma membrane of the host cell as the virus buds from the host-cell surface ese HUMAN IMMUNODEFICIENCY VIRUS (HIV): causative agent for AIDS ‘Sides Prepared By Vhesjy B.Tompol RND, MD ee er)CeO ac Oe eet a tO a ee ee ee) OVERVIEW BACTERIOPHAGES aka Bacterial Viruses One of the most complex viruses & one of the most abundant biological entities on Earth ‘Sides Prepared By Vhesjy B.Tompol RND, MD @ 4004/1000CeO ac Oe eet a tO a ee ee ee) OVERVIEW T.BACTERIOPHAGE Consists of a: Polyhedral head containing DNA Cylindrical stalk through which the DNA is injected into the bacterial cell Tail fibers that cause the particle to resemble a landing module for the moon ‘Sides Prepared By Vhesjy B.Tompol RND, MDCeO ac Oe eet a tO a ee ee ee) VIRUSES OVERVIEW SURFACE PROTEIN —; » Able to bind to a particular surface ‘component of its host cell » Ex. gp12Qon the surface of HIV particle ¥ Glycoprotein of molecular mass 120,000 daltons (| dalton is equivalent to | unit of atomic mass, the mass of a single hydrogen atom) ~ Interacts with’CD4 Gn the surface of certain white blood itating entry of the virus into its host cell ‘Slides Prepared By Vhejay B.Tompot RND, MDSa ert Ome e a Pea neces) OVERVIEW HOST CELL SPECIFICITY Determined by the interaction between viral & host proteins Some viruses have a wide host range, being able to infect cells from a variety of different organs or host species Bx: RabiesVirus: infect many different types of mammalian hosts (dogs, bats & humans) Most viruses have a relatively narrow host range Ex: Human Cold & Influenza viruses: infect only the respiratory epithelial cells of human hosts ‘Slides Prepared By Vhesjy B.Tompol RND, MDCeO ac Oe eet a tO a ee ee ee) OVERVIEW CHANGE in HOST CELL SPECIFICITY Dramatically illustrated by the 1918 influenza pandemic, which killed >30 million people worldwide Influenza virus was especially lethal in young adults, who do not normally become infected Viral Genome: 8 separate RNA molecules encoding for || proteins Determined from the preserved tissues of victims who had died from the infection 90 years earlier Best preserved samples were obtained from a Native American woman who had been buried n the Alaskan.permafrost ‘Sides Prepared By Vhesjy B.Tompol RND, MD © 4626/1000OVERVIEW CHANGE in HOST CELL SPECIFICITY Dramatically illustrated by the 1918 influenza pandemic, which killed >30 million people worldwide Influenza virus was especially lethal in young adults, who do not normally become infected Viral Genome: 8 separate RNA molecules encoding for |! proteins Determined from the preserved tissues of victims who had died from the infection 90 years earlier Best preserved samples were obtained from a Native American woman who had been buried n the Alaskan.permafrost Sides Prepared By: Vheejay B.Tampol RND, MDCeO ac Oe eet a tO a ee ee ee) fee en)CeO ac Oe eet a tO a ee ee ee) OVERVIEW _ CHANGE in HOST CELL SPECIFICITY Sequence of the 1918 Influenza Virus Suggested that the pathogen had jumped from birds to humans Accumulated significant number of mutations to adapt in a mammalian host Had never exchanged genetic material with that of a human influenza virus as had been thought likely ‘Sides Prepared By Vheejy B.Tompol RND, MD Pee)Sa ert Ome e a Pea neces) TYPES OF VIRAL INFECTION MAJORITY (LYTIC INFECTION) Virus arrests the normal synthetic activities of the host Redirects the cell to use its available materials to manufacture viral nucleic acids & proteins, which assemble into new yirions Assembled from components directly into the mature-sized virions Ultimately, the infected cell ruptures (Iyses).& releases a new generation of viral particles capable of infecting neighboring cells ‘Sides Prepared By Vhesjy B.Tompol RND, MDCrea cee net a tea eee amen cat Ks) TYPES OF VIRAL INFECTION MINOR (PROVIRUS FORMATION): Infecting virus does not lead to the death of the host cell Virus inserts (integrates) its DNA,into the DNA of the host cell's chromosomes Integrated viral DNA is referred to as a provirusFAC_SASTE_BSBio (Cell & Molecular Biology LEC & LAB): March 2, 2021 (2021-03-01 at 22:35 GMT) Phage nA " ° = = f \ f : Tex vA meyer nn © “bacterial chromosome by recombination, Sonne nosepeCRO ce nt Oa eee Ue eek) TYPES OF VIRAL INFECTIONCrea cee net a tea eee amen cat Ks) TYPES OF VIRAL INFECTION EFFECTS of an INTEGRATED PROVIRUS Bacterial Cells Behave normally until exposed to a stimulus (Ex. UY radiation) Activates the dormant viral DNA Leads to the lysis of the cell Release of viral progenyCrea cee net a tea eee amen cat Ks) TYPES OF VIRAL INFECTION EFFECTS of an INTEGRATED PROVIRUS Animal Cells Produce new viral.progeny that bud at the cell surface without lysing the infected cell (Ex. HIV) Infected cell may remain alive for a period, acting as a factory for the production of new virionsCeO ac Oe eet a tO a ee ee ee) TYPES OF VIRAL INFECTION EFFECTS of an INTEGRATED PROVIRUS Animal Cells Become malignant: lose control over their own growth & division Readily studied in the laboratory by infecting cultured cells with the appropriate tumor virus ‘Sides Prepared By Vhesjy B.Tompol RND, MDSASTE_BSBio (Cell & Molecular Biology LEC & LAB): March 2, 2021 (2021-03-01 at 22:35 GMT) APPLICATIONS Used in studying the mechanism of DNA replication & gene expression in complex hosts Used as a means to introduce foreign genes into human cells (One of the Bases of Gene Therapy) Insect & bacteria-killing viruses may play an increasing role in the war against insect pests & bacterial pathogens ‘Slides Prepared By Vhesjy B.Tompol RND, MD Tee en)CeO ac Oe eet a tO a ee ee ee) OVERVIEW Discovered in 1971 Named by Theodor Otto Diener of the US. Department of Agriculture Reported that potato spindle-tuber disease, which causes potatoes to become gnarled & cracked, is caused by an infectious agent consisting of a small circular RNA molecule that totally lacks a protein coat ‘Sides Prepared By Viesjy B.Tampol RND, MDCeO ac Oe eet a tO a ee ee ee) Potato Spindle Tuber Viroid tora + Small single strand infectious (3BNA circular genome, self-complementary (forms dsRNA rod Structure) + Genomes of 250-360 nucleotides + Capable of autonomous replication + Appear to encode no proteins + Genomes all contain 5 regions called domainsCeO ac Oe eet a tO a ee ee ee) OVERVIEW Size of RNAs: range from about 240 to 600 nucleotides (|/10 the size of the smaller viruses.) No evidence has been found that the naked viroid RNA encodes any proteins Any biochemical activities they take place using host-cell proteins Example: Duplication of the viroid RNA within an infected cell utilizes the host's RNA polymerase II that normally transcribes the host's DNA into mRNAs. ‘Slides Prepared By Vhesjy B.Tompol RND, MD Pere)CeO ac Oe eet a tO a ee ee ee) VIROIDS OVERVIEW Thought to cause disease by interfering with the cell's normal path of gene expression Effects are devastating for crops Examples: Cadang-Cadang (Philippines): coconut palm groves Chrysanthemum industry in © 10601 10009SASTE_BSBio (Cell & Molecular Biology LEC & LAB): March 2, 2021 (2021-03-01 at 22:35 GMT) VIROIDS OVERVIEW CSV¢ infected CChMV¢ infected chrysanthemum ev. Secret chrysanthemum cv, Yellow Pink SVa infected chrysanthemum cv. Borami Jrsease smn onthe se Chreanthonaon moritaion clivars infcie with CSV or CCMA ‘Sides Prepared By Vhesjy B.Tampol RND, MD ee ee)EAC_SASTE_BSBio (Cell & Molecular Biology LEC & LAB): March 2, 2021 (2021-03-01 at 22:3 REFERENCE Iwasa, J.and Marshall, W. 2016. 8th edition. USA: Wiley PRC ee eon)