CSB351-Lecture 1-6 notes.docx

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Department
Cell and Systems Biology
Course
CSB351Y1
Professor
Mounir Abou Haidar
Semester
Fall

Description
CSB351 LECTURE 1 The germ theory of infectious diseases  1850 – Pasteur & Koch  Airtight flask + boiling = no microbial growth o Pasteurization removes spontaneous generation o Microbial growth in flask due to germs from outside, and not by spontaneous generation Ex; Tobacco virus  First viral infectious agent identified (transmitted by touch)  Yanofsky found germ to be unfilterable, therefore must be smaller than bacterium Koch’s postulates 1. Specific microbe must be demonstrated in all cases of the disease 2. Isolation and culture of germ in artificial medium 3. Pure culture can produce the original disease is inoculated to susceptible animals Filterable germ experiment  Porcelain filter able to filter out bacteria and fungus  Tobacco mosaic virus identified b/c sap from infected plant filtered o Still able to infect other plants o Infectious agent was not bacterium, yet was reproducing (ability to cause disease did not weaken after transfers + dilution  Must be a virus (Beijerinck 1890s)  Foot and mouth disease virus, yellow fever virus Definition of a virus  Attack all living organisms Lwoff:  Strictly intracellular, potentially pathogenic (disease-causing) entities with an infectious phase o Are absolute parasites  Possess only one type of nucleic acid  Multiply in the form of their genetic material  Unable to grow and undergo binary fission  Devoid of a Lipmann system o i.e., a system of enzymes for energy production o unlike bacteria o uses host energy Goodheart:  viruses have no metabolic systems, no intrinsic motility*, cannot respond to stimuli  ability to maintain genetic continuity (and mutations) is only basis for considering them to be “alive” o *bacteriophages are one exception to motility  has a contractile tail used to inject host Properties of viruses in comparison to bacteria  smaller  simpler o a nucleic acid + protein coat  unable to multiply outside a living cell o absolute parasitism: viruses use the host cell’s enzymatic system, tRNA’s, and ribosomes Life cycle 1. receptor binding 2. invagination into host cell 3. uncoating 4. transcription 5. protein synthesis 6. replication 7. assembly LECTURE 2 Composition of viruses Pleomorphic: viruses that have an adaptable, non-fixed shape  one type of nucleic acid + one or several types of protein o can also contain lipids or carbs (when host cell membrane used as envelope)  nucleic acids and proteins are NOT covalently linked o this allows for uncoating Nucleic acids  RNA has extra oxygen atom on 3’ position in sugar (ribose vs deoxyribose)  PHOSPHATE [ester bond] SUGAR [glycosidic bond] BASE o ATCGU  some viruses contain modified bases o Ex; majority of (+)ve RNA virues have a 5’ cap: guanine that is methylated on position 7  Others have a covalently linked 5’ protein called “VPG” o No (-)ve RNA viruses have a cap o All mRNA have a cap  Some plant virus RNAs have a structure at their 3’-OH end similar to that of tRNAs (clover leaf). o In 3’ uncoding region of TMV, the tRNA-like structure may fold into a "pseudoknot" structure. Nucleic acid enzymes DNA/RNA polymerases: proteins that can synthesize RNA and DNA from an existing template  Add nucleotide to 3’ end of strand  RNA-polymerases do NOT require a primer: a small segment of DNA OR RNA hybridized to template strand o DNA-polym DOES  Most viruses codes for their own polyms o Exception: HPV does not, requires cell division for polyms  All have helicase activity to unwind strands  DNA-dependent RNA polym: transcribes mRNA from DNA  RNA-dependent RNA polym: encoded by RNA viruses for their own replication; new RNA from RNA template o same idea for DNA-dependent DNA polym  RNA-dependent DNA polym: “reverse transcriptase”; DNA copy of RNA template o Only found in retroviruses  some viruses must take polymerases with them o ex; (-)RNA viruses or viruses with double stranded DNA Methylases: transfers –CH3 group from adenosylmethionine to nitrogenous base  Responsible for 5’ guanine cap of (+)RNA viruses and mRNAs o Ribosomal recognition o Prevents exonuclease degredation o Has structure: m7GpppGp Nucleases: cleave phosphodiester bonds b/w nucleotides in RNA and DNA  Deoxyribonucleases only cut DNA o ENDO: restriction enzymes (oligo insertion) o EXO: E.coli exonucleases  Ribonucleases only cut RNA o ENDO: RNase A (cuts 3’ side of pyrimidine’s) o EXO: snake venom phosphodiesterase Ligases: catalyze joining of 2 molecules through the formation of a new chemical bond  Important in UV irradiation repair, and making circular DNA from linear Terminal transferases: adds a few nucleotides (ATCG) to 3’ end of DNA/RNA LECTURE 3 Viral proteins  Structural (capsid or envelope proteins) or enzymatic o Most enzymatic proteins are left in host cell after use  Some viruses (ex; HIV) take lots of proteins o Others take none (ex; TMV)  Helical and icosahedral coats are quaternary structures  Functions: o Protective coating for genetic material o Receptor on surface of virus that interacts with host cell surface  Not in plant viruses o SPECIFIC attachment to receptor site on host  Coat specifies host range o When introduced into a vertebrate animal: as antigens to elicit the production of antibodies (antigenic sites)  b/c coat more antigenic than nucleic acid o In plants viruses:  Capsid protein involved in replication and transport (movement proteins), and life cycle +symptom development regulation  RNA alone is infectious; host specificity is not affected by presence or absence of coat protein o Some viruses contain enzymes used to degrade cell membrane  Ex; phage enzyme Glycoproteins  Highly antigenic; immunological responses  Some involved in protein folding, some have enzymatic activity, some protect against degradation by nucleases/proteases (ex; T-even phages)  In ortho and paramyxoviruses (ex; influenza and measles), glycoprotein spikes (containing haemagglutinin and neuraminidase) are involved in attachment, penetration, and exit o HA – trimeric glycoprotein, functions in attachment o NA - spikes  Glycosylation takes place in cell’s ER and GA o O-linked – Ser, Thr, Tyr o N-linked – Asn Viral lipids  Some viruses (mostly animal) contain lipids  Enveloped viruses are generally released by budding (rather than lysis)  Lipids + cholesterol originate from host CM Ex; sphingomyelin, phosphotidylcholine, phosphotidylserine  b/c lipid bilayers are expensive  contribute to envelope stability  interact with viral proteins (non-covalent) [lipoproteins]  good protection against cellular antibdies Metals and polyamines Polyamines: (+)ve charge; neutralize viral Nacid Ex; spermine, spermidine, putrescine (phages T2/T4) [highly basic, found in plants]  Nacids polar b/c of (-)ve phosphate groups o Viruses will also contain cations which are ionically bound to Nacids Ex; Fe, Ca, Mg, N LECTURE 4 Centrifugation Force applied to molecules (F) = friction coefficient (f) * velocity of molecules  Friction coefficient depends on shape and viscosity of medium In centrifugal field: F = mass (M) * angular velocity (w) * radius (r)  M = weight (W)/ gravitational constant (g) On earth, relative centrifugal force: F = (r w )/g  Where w= 2pi/60 x RPM Sedimentation coefficient: constant for each particle, proportional to the molecular weight  Unit is Svedberg (S): 10-13seconds  So S is proportional to molecular weight o Angular velocity is irrelevant  In practice: o cell debris has highest S, will sink first o viruses next, then small protein (ribosomes) o size of virus can be identified by differentiating the speed at which they separate Ex; brome mosaic virus (~80S)  in plants, same S as ribosomes o both will sediment together at bottom  EDTA used to split ribosome into 2 subunits: 30S and 50S Centrifugation techniques Differential: alternating cycles of high and low speed centrifugation
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