Microbio Virus/Gene Engineering (10/11/12/13)

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  1. Microbio 10
  2. virus
    • an agent that is not itself alive (i.e. no metabolic machinery), but can reproduce itself within a living cell
    • a virus can exist as two different forms:
    • 1. virion
    • 2. replicative form
  3. virion
    • form of a virus found outside a cell that contains genomic material surrounded by a protective protein layer called a capsid
    • it may also sometimes have an envelope, an additional outer lipid layer derived from cell membrane
    • its purpose is to carry genetic information of the virus from one host cell and one host organism to another & to protect it from the depredations of the external environment
  4. replicative form of virus
    • exists in such a form when it is inside the cell
    • its purpose is to direct the cell to make more virions and to ensure that the replicated virus is further transmitted to other cells and other hosts
  5. eukaryotic mRNAs are monocistronic
    as a rule, only one protein can be translated from each mRNA
  6. ssRNA viral genomes
    • plus/sense: “normal” mRNA, usually with a polyA tail and can itself be used as a transcript for protein production
    • minus/antisense: complement to the sense strand, lacking a polyA tail & must be replicated in order for proteins to be made from it
    • replication of RNA requires enzymes not normally found inside host cells
  7. How does the viral genome migrate to the proper cellular compartment?
    • genome localization
    • it brings a genome to the nucleus for replication and transcription or causes the genome to associate with an intracellular membrane
  8. Why study viruses?
    • 1. they're important agents of human disease
    • 2. they're important models for cellular functions (esp. gene expression)
    • 3. they're important models for human disease, even if not caused by human viruses (animal models of virus induced cancers led to oncogene discovery)
    • 4. provide new tools for biotechnology & gene therapy
  9. viral polyprotein
    • the viral DNA is translated to make a single large polyprotein; the genome has one single coding region that results in a number of different proteins stuck together when initially translated
    • one protein in the polyprotein, usually a viral protease, can cleave different proteins out of the conglomerate
    • eg. poliovirus (picorna)
  10. How do viruses overcome the inability of eukaryotic cells to translate more than one polypeptide from a single mRNA?
    • 1. differential splicing of a pre-mRNA
    • 2. synthesis of a polyprotein followed by autoproteolysis to release individual polypeptides
    • 3. carriage of a segmented genome
  11. segmented viral genome
    • the viral genome is broken up into multiple parts which code for different mRNAs which can be translated into separate proteins
    • each segment of genome/the subsequent mRNA gives rise to distinct protein via translation
    • and together they make up the whole genome
    • eg. influenza (myxovirus)
  12. nested mRNA
    under viral control a single genome is preferentially transcribed to give separate mRNAs that code for individual proteins (the mRNAs come from a single genome though)
  13. differential splicing viral genome
    a phenomenon particularly present in DNA viruses, where it uses the host splicing machinery to differentiate mRNAs from a single DNA strand
  14. in vitro Translation Assay
    • the purified genome (genetic material ONLY) is added to a tube with ribosomes, tRNA, & buffer
    • PROTEIN can only be made by viruses who are + stranded RNA so they can be used as a template in their original form
    • 1. picornavirus
    • 2. retrovirus
  15. Enzymatic Activity Assay
    • the virion is added to ETHER, CHCl3, or a mild detergent that gently bursts the virion, so both it's genetic & proteinaceous contents remain intact
    • only thing added are dNTPs, NTPs, & buffer
    • NUCLEIC ACID TRANSCRIPTS (dna or rna) are only made if the virus carries ITS OWN transcription enzymes
    • 1. myxovirus: will make RNA *IF there is mRNA in the assay so it can steal their 5' caps
    • 2. retrovirus: will make DNA
  16. Transfection Assay
    • determines whether a purified genome is infectious
    • the virus must be able to use transcription materials (enzymes) from a host cell, and not need their own
    • 1. papilloma virus
    • 2. picornavirus
    • genomes are purified from a virus & introduced into cells
    • with viruses that do not require virion enzymes (DNA & + RNA) a complete infection cycle can be started in this way
  17. Microbio 11
  18. Picornaviruses
    • small viruses with an + strand (mRNA-like) RNA genome
    • they do not carry their own enzymes, but use host transcription enzymes
    • the capsid structure = icosahedral & they are NOT enveloped (nekkid)
    • they lyse cells
    • they make protein in an in vitro assay & are infectious (replicate) in a transfection assay but cannot replicate in an enzymatic activity assay
    • eg. Poliovirus, Hepatitis A, Foot-&-Mouth, Rhinovirus (common cold), Cardiovirus, Coxsackie, + other “enteroviruses” cause gastrointestinal discomfort + “stomach flu” symptoms
    • are transmitted fecal-orally
  19. Picornaviruses' + strand RNA have a polyA tail at the 3’ end and what at their 5' end?
    • a small terminal protein called VpG (Viral Protein G) covalently bound to the 5' end
    • besides capping the 5' end of the transcript it also serves as a primer for host DNA pol to replicate the virus
  20. What does the picornaviruses + strand RNA genome code for?
    • it contains one large open reading frame which gives rise to one polyprotein that is subsequently cleaved to make all the necessary viral proteins
    • this virus never enters the nucleus - stays in the host cell cytoplasm and can be translated by ribosomes there
  21. Picornavirus Adsorption
    • the virus infects cells via interaction with CD155, a SPECIFIC cell surface protein receptor
    • CD155 is normally used to establish intercellular adherens junctions between
  22. What limits the ability of the Picornavirus to infect some kinds of cells?
    the distribution of the receptor CD155, as well as the fact that only some species of animals (i.e. primates) have it
  23. How does the Picornavirus enter cells once bound to CD155?
    • it is endocytosed
    • the capsid fuses with the cell membrane, releasing the genome into the cell’s cytoplasm
    • there the genome associates with cellular polyribosomes, is translated, & gives rise to the viral polyprotein
  24. What part of the cell is not necessary for a picorna virus infection?
    • the cell's nucleus is not necessary for picornavirus replication
    • because the genome is + strand RNA there is no need for it to travel to the nucleus
    • studies have been done where the cell nucleus is removed and polio infection is still successful
  25. What happens to the viral polyprotein once it exists?
    • it's cleaved into 4 general sections
    • 1. replicase: copyies more viral genome
    • 2. accessory proteins: such as VpG
    • 3. protease: cleaves the polyprotein
    • 4. VPs (virion proteins): assembled as a precursor for an empty capsid, which is later made into a virion; there are 4 virion proteins (VP 1 -> 4) that are only cleaved from one another after assembly of the capsid
  26. What is the structure of the picornavirus capsid?
    • the capsid is an arrangement of 60 protomers in a tightly packed Icosahedral structure
    • each protomer consists of 4 polypeptides known as VP (viral protein)1, 2, 3 & 4
  27. picornavirus replication
    • VpG is the primer for replication so 1st it associates w/ the 3’ end of the + strand RNA template
    • replicase copies the entire plus strand, leading to production of a full length – strand RNA template w/ a poly(U) sequence at its 5’ end*
    • VpG then associates with the 3’ end of the – strand, & the same process is used to make another + strand
  28. What is absent from the – strand picornavirus RNA genome once it's been copied from an origininal + strand RNA template by replicase?
    • the minus strand lacks a polyA tail
    • instead has a complementary poly(U) sequence at its 5’ end
    • all of the RNA (- & +) have VpG at their 5’ end, but only + strand RNA have polyA tails
    • + strands: be used as mRNAs, templates for further replication, & as genomes
    • – minus strands: ONLY be used as templates for replication; are more efficient templates though, are better at directing synthesis of + strand RNA
    • Image Upload 1
  29. picornavirus assembly & release
    • when enough uncleaved virion proteins (VP 1 -> 4) are made they're assembled into capsids
    • capsids are stuffed w/ + strand RNA & the precursor
    • proteins are cleaved -> a very tight strong structure is cemented
    • the virion is released by lysis of the host cell (picornaviruses are naked, so budding is NOT the method of release)
  30. Which type of viruses are the only group to lyse host cells?
  31. Myxovirus
    • virus whose genome is minus (-) strand RNA
    • the only test in which myxoviruses will show a positive result is an enzymatic activity assay *as long as mRNA are thrown into the mixture so the virus can steal their caps
    • carry transcriptase in their helical nucleocapsid
    • they are have an envelope
    • eg. influenza, fowl plague
  32. What is the only virus that has a helical nucleocapsid structure?
    • the Myxovirus
    • all others (papilloma, picorna, & retro) have a icosahedral shaped capsid
  33. Myxovirus
    • a very large group of viruses that all cause influenza-like disease
    • it's not naturally a human virus; found predominantly in wild waterfowl populations & occasionally makes its way into domestic animals or humans
    • eg. 1918 influenza epidemic (worst pandemic in the history of the world # of deaths-wise) or the recent H1N1 pandemic
    • myxo in Greek = “slime”; myxoviruses are often found in slimy environments
    • Image Upload 2
  34. Myxovirus Structure
    • an enveloped viruses containing single, - strand RNA
    • on the envelope are 2 glycoproteins, HA & NA
    • within the envelope are 8 helical capsids, or nucleocapsids, that each contain a – RNA genome segment, transcriptase, & nucleoprotein (NP) protein
    • a myxovirus genome is segmented amongst the 8 nucleocapsids
    • the virion contains also contains Matrix Protein
  35. Hemagglutinin (HA)
    • protein that binds sialic acid (N-acetyl neuraminic acid), a carbohydrate often found as the terminal sugar on glycoproteins
    • sialic acid is the receptor for myxovirus (how they enter cells)
    • HA can cause hemagglutination, aggregation of RBCs b/c RBCs contain sialic acid on their cell surfaces
  36. Agglutination Assay
    • as assay that can detect a myxovirus
    • mix virus with a preparation of RBCs; these have sialic acid on their surface
    • if myxovirions they will interact with RBC surface and give rise to a clump/precipitate of RBCs = agglutination reaction
    • agglutination reaction can be blocked by adding an antibody for the virus --> Ig binds virus, making it unable to bind RBCs' sialic acid, won't see clump
    • can be used as asimple way of testing peoples' serum to see if they've been exposed to the same virus
  37. Neuraminidase (NA)
    • an enzyme that destroys the sialic acid cell receptor so that newly made virions can break free from agglutination
    • also prevents other viruses from entering the host cell, as myxoviruses work a little slowly
  38. What is within the 8 nucleocapsids of a myxovirus?
    • 1. a segment of the – strand RNA genome (the entire genome is segmented amongst the 8 helical capsids)
    • 2. Transcriptase
    • 3. Nucleoprotein (NP)
  39. Transcriptase/Replicase
    • an enzyme that catalyzes the synthesis of a complementary RNA molecule using an RNA template
    • doesn't exist in eukaryotic cells
  40. Nucleoprotein (NP)
    • a component of nucleocapsids that always stays attached to a viral genome/template
    • it initiates the switch from transcribing mRNA from the - strand RNA genome to making + strand RNA from which more - strand RNA genome can be made for packaging and viral release
  41. Matrix Protein (M1)
    links the outer envelope to the inner nucleocapsids
  42. Myxovirus Adsorption
    • the virus infects cells via HA interacting with the terminal sialic acid found on the end of many cell surface proteins
    • it acts as a receptor, and once bound, the virus is endocytosed after a drop in pH
    • once the virus is inside the 8 helical nucleocapsids move to the nucleus
  43. What initiates the fusion of the myxoviral envelope and the cell membrane?
    a drop in pH
  44. How and where is myxoviral mRNA synthesized?
    • viral transcriptase reads the – strand RNA genome as a template inside the nucleus to produce complementary viral mRNA
    • 5' caps are stolen from newly made host cellular mRNA as primers
    • the PolyA tails are added using a “stuttering” mechanism
    • the newly synthesized viral mRNA is transported back to the cytoplasm where translation of the necessary viral proteins begins
  45. Which viral proteins are synthesized on MEMBRANE-bound polyribosomes and which are synthesized on FREE polyribosomes?
    • membrane-bound: HA + NA
    • free: M1, NP, transcriptase, & other internal proteins
    • HA, NA, & M1 are sent to the cellular membrane, while all other internal VPs are sent to the nucleus to aid in replication
  46. What does accumulation of enough synthesized nucleoprotein do?
    • it signals a switch from making mRNA (w/ stolen caps & a poly-A tail) to making EXACT plus strand copies
    • *Nucleoprotein (NP protein) initiates & signals for this switch to occur
    • these are then used as templates to make MORE minus strand genome
    • mRNA --> + strand RNA --> - strand RNA genome
  47. What happens when the myxovirus is ready to assemble itself and be released from the host cell?
    • NP protein & transcriptase have been associated w/ the genome during replication all along in the nucleus
    • complete nucleocapsids (genome, NP, transcriptase) are transported to cytoplasm
    • in cytoplasm they associate with M1 protein which itself is partnered w/ cytoplasmic ends of HA and NA inside the cell membrane
    • *the virus is released by budding, resulting in an ENVELOPED virus & a perfectly intact host cell
  48. Microbio 12
  49. What made HeLa cells so aggressively malignant?
    that they contained – and continuously expressed – several copies of a partial DNA molecule from human papillomavirus (HPV) type 18, the cause of the cancer and death of Henrietta Lack some 30 years previously
  50. papilloma
    • a benign, protruding, epithelial tumor (eg. wart)
    • papilla = a nipple-like protrusion
  51. Papillomaviruses
    • associated with warts on many parts of the body
    • are many types of papillomaviruses
    • almost all cause only common, benign, self-limiting infections of epidermal tissue
    • a few (eg. HPV 16 & 18) are the major causes of cervical, penile, & throat cancer
    • vaccines are now available & are quite effective at preventing infection and cancer if administered prior to encounter
  52. Papillomaviruses Structure
    • are naked, double-stranded DNA viruses with an icosahedral capsid made up mostly of L1 protein in association with a few molecules of L2 (coded for by genes of the same name respectively)
    • its genome is 8 kb of circular double stranded DNA (~picornavirus genome)
    • it contains early (E1-7) & late (L1-2) genes
    • Image Upload 3
  53. What are the ONLY types of cells HPV can infect?
    • basal cells of the skin & mucosal tissue (epithelia)
    • its replication is tightly coupled to the development of these cells as they divide, move, differentiate, & eventually slough off
    • unlike picorna or mixo, HPV replication is very restricted
  54. What 'receptor' does HPV use to get into host cells in the basal layer of the epidermis?
    • 1. heparan sulfate proteoglycan: a nonspecific component of the cell membrane & extracellular matrix)
    • 2. another, more specific receptor, that is unknown
  55. By what mechanism does the papillomavirus enter the cell and which protein is required for the entry process?
    • after binding to the proteoglycan heparan sulfate, HPV enters the cell via ENDOCYTOSIS & once inside the cell passes through the endosomal membrane
    • entry requires participation of the L2 PROTEIN
    • once inside the cell the dsDNA virus travels to the nucleus where it uses HOST cellular machinery to aid in transcription & DNA replication
  56. What is the goal of early HPV gene expression?
    • to make the gene products (viral proteins) necessary for genome replication
    • viral genome is originally transcribed by cellular RNA polymerase from the early promoter
    • the transcript is differentially spliced & translated to make a number of early gene products: E1 --> E7
  57. E1
    • forms a complex at the origin of DNA replication that, along with E2, recruits the host cell’s DNA polymerase machinery
    • E1 also serves as a HEL1CASE that unwinds the DNA, making it accessible to the replication complex
  58. E2
    • the master regulator of both transcription & DNA replication
    • it interacts w/ both promoters, the origin of replication, & the cell’s RNA synthesis machinery
  59. E3 & E4
    interact with various cellular components (including the cytoskeleton) to modify the cell structure and make it amenable to virus replication & release
  60. E5, E6 & E7
    critical viral proteins for altering the growth & DNA replication patterns of the infected cell
  61. E5
    • stabilizes (therefore activates) the receptor for epidermal growth factor
    • this constitutively stimulates the cell to divide
  62. E6
    • interacts w/ & causes the degradation of p53, preventing the infected cell from recognizing the viral DNA as foreign and thereby preventing apoptosis
    • [E6 prevents the cell from apoptosing itself]
    • think p5 … E6 … 3, 5 --> 6
  63. E7
    • binds to Rb, a central regulator of cell division, stimulating entry into S-phase DNA synthesis
    • excessive division of the infected cell not only leads to warts, but also helps to propagate the virus & to cause the cell to maintain high levels of DNA replication components (both enzymes & nucleotides) needed for viral genome synthesis
  64. HPV DNA Replication
    • the double-stranded DNA genome is replicated using the normal cellular machinery, directed to the HPV replication origin by E1 & E2
    • HPV genome replication is coupled to division of the infected cell (unlike other viruses whose genomes replicate exponentially)
    • Image Upload 4
  65. As basal epithelial cells divide, differentiate, & migrate toward the epidermal surface, why does the pace of viral DNA synthesis greatly increase?
    • so that large numbers of virions can be present in terminally differentiated keratinocytes that make up the outermost layer --> when they die naturally lots of virions will be released
    • up until the final stages of terminal differentiation the amount of viral DNA is maintained at about 100 copies per cell
  66. What is the goal of late gene expression in the papillomavirus?
    • to make the L1 and L2 proteins necessary for virion assembly
    • similar to early gene expression, late gene expression also uses differential splicing to make mRNAs for both proteins from a SINGLE primary transcript
  67. At what cellular stage are HPV virions assembled and how are they released?
    • virions are assembled in the infected cells in the granular layer immediately below the outermost epithelium
    • they are released when dead, cornified cells containing lots of virions are sloughed off
    • (the virus can survive for a long time on surfaces, ready to be picked up by & infect the next person)
  68. How can papillomaviruses be oncogenic/cause malignancy?
    • by a rare events during which a portion of the viral DNA including E1 & E6, but NOT E2, integrates into the host cell genome
    • in these cells, continued expression of the early genes will enable the cell to divide continuously out of control & become a cancer cell (the genetic change becomes heritable)
    • HPV, particularly high risk types (16, 18, + a few others) is a major cause of naturally occurring human cancer, particularly at genital sites
  69. What happens if E2 doesn't integrate along with the rest of the viral genome? What happens if it does integrate into the host cell chromosome?
    • if part of the viral genome integrates without E2 (but say with E6 & E7 in a way they're expressed) there's NO control over the early genes promoter --> the cell replicates uncontrollably --> cancer
    • if the WHOLE genome integrates, E2 is present & still regulates the virus' expression --> wart but not cancer
    • [E2 is the MASTER regulator of transcription]
  70. Retroviruses
    • viruses whose information flows in the reverse of the usual pattern: RNA --> DNA (eg. HIV)
    • are enveloped, + strand RNA viruses w/ an icosahedral capsid
    • the envelope is derived from the host cell membrane modified by Env, a viral-encoded protein
    • contain MA, a matrix protein that connects the outer envelope to the inner capsid
  71. Env is structurally similar to what other viral protein?
    HA protein of myxoviruses (influenza)
  72. What does a retroviral capsid contain?
    • TWO copies of the + strand RNA genome
    • the enzymes reverse transcriptase (RT), integrase (IN), and protease (PR)
    • the nucleocapsid (NC) protein
  73. retroviral genome
    • its capped, poly-adenylated, and has a tRNA base-paired near the 5’ end
    • tRNA acts as the primer for reverse transcription
    • the genome contains a minimum of 4 genes (gag, pro, pol, env) & has short, repeated sequences (R) at both ends
    • it has a unique sequence at the 5’ end (U5) & a different unique sequence at the 3’ end (U3)
  74. How do retroviruses get into host cells?
    • they interact with host cells via the Env protein (found modifying the viral envelope) & a specific cellular receptor (CD4 on CD4+ T cells w/ HIV)
    • the virus enters the cell either by direct membrane fusion or receptor-mediated endocytosis, depending on the type of virus
    • Image Upload 5
  75. Where does reverse transcription take place once the retrovirus has entered the host cell via membrane fusion or RME?
    • the genome remains in the cytoplasm for reverse transcription
    • Image Upload 6
    • the process initiates downstream of the 5’ end of the genome where the tRNA primer is located
    • RT moves TOWARD the 5' cap (to the left) and once it hits the end, the small piece of DNA made “JUMPS” to the 3’ end of the RNA, its RU5 sequence annealing to the U3R sequence
    • reverse transcription now proceeds toward the 5’ end again, completing the strand of “minus” DNA (complementary to the viral RNA genome)
    • a plus strand of DNA is made using the minus strand as a template --> dsDNA made
  76. What is the structure of the final double-stranded retroviral DNA that is produced by reverse transcriptase in the cytoplasm?
    • U3RU5-gag-pro-pol-env-U3RU5
    • the U3RU5 sequence = LTR (long terminal repeat)
    • the completed dsDNA is transported to the nucleus as part of the preintegration complex
  77. How does the viral dsDNA integrate into the cellular DNA?
    • using the viral integrase enzyme
    • integration is irreversible
    • the genome is integrated at random sites, forming a provirus
    • the provirus is replicated w/ cellular DNA & passed on to daughter cells via mitosis
  78. What transcribed the provirus?
    • CELLULAR RNA polymerase II, directed by a promoter located within the 5’ U3 region
    • transcripts are poly-adenylated after the 3’ LTR
    • if the mRNA is spliced, Env protein is translated on membrane-bound polyribosomes & sent to the cell surface
    • if the mRNA is NOT spliced, internal viral proteins (Gag, Pro, Pol) are translated by free ribosomes in the cytosol as polyproteins (Gag, Gag-Pro, Gag-Pro-Pol)
  79. Retroviral Assembly & Release
    • polyproteins associate w/ the viral genome, then move to the inner cell membrane
    • as the capsids assemble, Env proteins incorporate on the outside of the membrane, & an immature capsid buds from the membrane
    • ONCE BUDDED, viral protease cleaves the viral polyproteins to produce individual Gag, Pro, Pol, changing the virion from an immature, noninfectious agent to a mature and infections virus
  80. What do the individual Gag, Pro, and Pol proteins do?
    they provide the virion w/ all the necessary replication enzymes (reverse transcriptase, integrase, & protease)
  81. What do all protooncogenes encode even thought they're all very different from each other?
    • they all encode proteins critical for regulation of cell division
    • can be activated by:
    • 1. retroviruses integrating into the genome adjacent to a protooncogene, causing it to be expressed under control of the viral LTR
    • 2. protooncogenes becomming part of the viral genome; such viruses can transform all the cells they infect & cause cancer rapidly
    • 3. means other than virus infection – chromosomal rearrangement & mutation
  82. What are some targets of AIDS prevention or treatment regimens, as well as just general antiretroviral treatment?
    • 1. inhibitors of viral DNA synthesis, re: reverse transcriptase
    • 2. integrase inhibitors
    • 3. Protease inhibitors
  83. Simple Retrovirus
    • retroviruses whose genes (not counting oncogenes) code for virion proteins & their interaction with cellular functions (like transcription and splicing) is entirely PASSIVE
    • simple retroviruses can only infect, replicate, & cause disease in newborn or immunosuppressed individuals
  84. Complex Retrovirus
    • retroviruses that encode additional proteins that interact w/ cellular proteins & participate directly in regulation of viral expression and inhibit functions critical to virus replication
    • eg. HIV
  85. Tat
    • a complex retroviral protein that activates transcription by interacting with TAR, a region near the 5’ end of the nascent HIV RNA transcript
    • this interaction leads to alteration of RNA polymerase by phosphorylation allowing continuation of transcription to the end of the provirus
  86. Rev
    • a complex retroviral protein that regulates expression of genes for virion proteins by mediating export from the nucleus of incompletely spliced (gag-pro-pol & env) mRNAs
    • without Rev, only smaller, completely spliced mRNAs encoding Tat, Rev, & several accessory proteins are translated
  87. Vif, Vpu, Vpr, Nef
    • bind to & neutralizing host cell proteins that can block virus replication at various stages of replication
    • they are accessory proteins that aren't essential for replication of HIV in cell culture, but play important roles in enhancing the efficacy of the virus
  88. What is the overall function of Tat, Rev, & the accessory proteins?
    they allow HIV to evade host adaptive and innate immune responses & replicate efficiently in immunoCOMPETENT adults
  89. What is the only type of virus that can enter the cell via something other than endocytosis?
    • the retrovirus: it can enter by direct membrane fusion OR receptor mediated endocytosis
    • eg. HIV enters by direct membrane fusion
  90. Which two types of viruses are enveloped and therefore do not lyse cells?
    • 1. myxovirus
    • 2. retrovirus
    • both contain a contain a matrix protein that connects the outer envelope to the inner capsid
    • myxoviruses: M1
    • retroviruses: MA
  91. Which kinds of cells are most often affected by which viruses?
    • HPV (papilloma): basal epithelial cells
    • Polio (picorna): cells w/ CD155 (eg. neural/epithelial)
    • Influenza (myxo): gastrointestinal/respiratory
    • HIV (retro): CD4+ T cells
  92. Microbio 13
  93. Genetic Engineering
    • the alteration of the genetic make-up of living organisms (bacteria, plants, animals, humans) to treat or cure genetic disorders or to endow organisms with traits they lack
    • purposes -
    • 1. research: isolation and study of specific genes
    • 2. large-scale production of useful products (protein replacement therapy)
    • 3. genetic analysis (screening for genetic defects)
    • 4. gene replacement therapy
  94. Recombinant DNA Technology
    the collection of techniques used to rearrange & re-assort portions of DNA molecules in order to create new combinations of genes, promoters, etc.
  95. What are 3 regulated promoters you could use to make a bacterial construct that would express a certain protein you could harvest but would INHIBIT expression until you had a robust cell culture?
    • 1. λ phage PL (left promoter)
    • 2. Plac (lac operon promoter)
    • 3. tcdR promoter
    • keep the promoter repressed until culture reaches saturation
    • then add whatever removes the repressor from the promoter
    • cells will die, but only after producing lots of insulin
  96. λ phage PL
    • the bacteriophage λ has a left promoter normally bound & inhibited by a repressor
    • adding N protein allows allows transcription to proceed through PL
  97. Plac
    • the promoter which controls the lac operon under normal circumstances has a repressor bound to it --> lacI
    • in the present of lactose, allolactose, or an analog IPTG, the repressor is bound by one of those 3 molecules and cannot bind Plac
    • transcription is free to proceed
  98. tcdR promoter
    • can be repressed when CodY & certain amino acids are added to the culture (leucine, isoleucine & valine (VILe)
    • CodY binds to the TcdR promoter region & prevents RNA pol from binding to the promoter/transcribing gene
    • can remove CodY/amino acids and tdcR promoter will work --> transcribing the constructed gene of interest
  99. What are a few markers (antibiotic resistance genes) you could construct in a plasmid to make sure it picked up the gene construct of interest?
    • penR: penicillin
    • kanR: kanamycin
    • tetR: tetracycline
    • AmpR: ampicillin
    • sulf: sulfonamide
    • neoR: neomycin
    • all need promoters
  100. What should be present in a good plasmid construct for permanently correcting a mutated insulin gene (in this order):
    • 1. protein promoter or LTR
    • 2. cDNA gene or human gene (might be too big)
    • 3. LTR --> rep then oriR
    • 4. promoter for marker/resistance --> resistance gene
    • 4. integrase
  101. What should be present in a good plasmid construct for growing a protein missing in someone with a mutated gene?
    • 1. Plac --> cDNA protein gene
    • 2. LTR --> rep + oriR
    • 3. Pmarker --> marker (antibiotic resistance)
    • 4. T7 PE --> lac repressor (or you can just add in the protein?)
  102. PTC124
    • drug that acts only to prevent nonsense codons not REAL codons; can target because there are multiple controls when tRNA and a ribosome correctly stops translating a protein
    • helps prevent loss of dystrophin in muscular dystrophy due to a mutation that causes an incorrect stop codon in the mRNA sequence
Card Set
Microbio Virus/Gene Engineering (10/11/12/13)
Exam 4
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