BMSC 210 Post Midterm 1

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jaz584
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263724
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BMSC 210 Post Midterm 1
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2014-02-26 21:01:21
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BMSC 210 Post Midterm
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BMSC 210 Post Midterm 1
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  1. Constitutive Proteins
    -proteins that are needed at the same levels in a cell at all times
  2. Why does mRNA have a short half life?
    -to prevent the production of unneeded proteins
  3. How do DNA binding proteins choose where to bind?
    • -specificity provided by AA side chain interactions with phosphate backbone of DNA
    • -major groove of DNA is main site of protein binding
    • -generally look for inverted repeats for regulatory proteins
  4. Homodimeric Proteins
    • -proteins composed of two identical polypeptides
    • -interact specifically with inverted repeats
  5. Helix-turn-Helix
    • -class of protein domain 
    • -first helix for recognition
    • -second for stabilization
  6. Zinc Finger Domain
    • -structure that binds zinc ion
    • -typically 2-3 zinc fingers to bind to DNA
  7. Leucine Zipper Domain
    • -spaced every 7 AA
    • -does not directly interact with DNA
  8. 3 Functions of DNA-Binding Protein
    • -catalyze specific reaction on DNA (transcription by RNA polymerase)
    • -binding to block transcription (negative regulation)
    • -binding to activate transcription (positive regulation)
  9. Negative Gene Expression Control in Bacteria
    • -greatly influenced by environment
    • -presence or absence of specific small molecules
    • -interactions between small molecules and DNA binding proteins
  10. Negative Control
    -regulatory mechanism that stops transcription
  11. Repression
    • -preventing the synthesis of an enzyme in response to a signal
    • -generally affects anabolic enzymes (arginine biosynthesis)
  12. Induction
    • -production of an enzyme in response to a signal
    • -typically affects catabolic enzymes (lac operon)
    • -enzymes only made when needed=no wasted energy
  13. Inducer
    -substance that induces enzyme synthesis
  14. Corepressor
    -substance that represses enzyme synthesis
  15. Effectors
    -collective term covering all inducers and repressors
  16. How do effectors work?
    • -effectors affect transcription indirectly 
    • -they bind to allosteric repressor proteins
    • -this activates them and they bind to area near DNA promoter called the operator
  17. Operon
    -cluster of genes arranged in a linear fashion whose expression is under the control of a single operator
  18. Where is the operator located?
    -downstream of the promoter
  19. Repressor role is ______ because it is what kind of control?
    -inhibitory, which makes it a negative control
  20. Positive Control
    • -a regulator protein that activates the binding of RNA polymerase to DNA
    • -maltose catabolism in E Coli
  21. Where do Activator proteins bind?
    • -at activator binding site
    • -NOT OPERATOR (like in negative control)
  22. What do activator proteins do?
    -help RNA polymerase recognize the promoter
  23. Regulon
    • -multiple operons controlled by the same regulatory protein
    • -exist for positive control (maltose)
    • -exist for negative control (arginine)
  24. Restriction Endonuclease Enzymes
    • -recognize specific DNA sequences and then cut there
    • -common in prokaryotes
    • -rare in eukaryotes
  25. Type 1 REase structure
    • -pentameric with separate restriction (R), methylation (M) and DNA sequence recognition (S) subunits
    • -cut DNA at RANDOM far from recognition sequences
  26. Type 1 REase function
    • -first REases to be discovered and purified 
    • -no lab use as they cut at random (unlike type 2)
    • -originally thought to be rare, now known as common
  27. Type 2 REase
    • -cleave DNA within their recognition sequence and most useful REase for specific DNA manipulation
    • -recognize inverted repeats (palindromes)
    • -sequence usually 4-8BP long
    • -form sticky or blunt ends
    • -protect cell from foreign DNA invasion (virus)
    • -used in gene cloning
  28. Type 3 REase
    • -large, combination and restriction enzyme
    • -cleave outside recognition sequence, 25 BP away
    • -require 2 sequences in opposite orientations within same DNA molecule to get cleavage, therefore rarely give complete digests
  29. Type 4 REase
    • -recognize modified, typically methylated DNA
    • -McrBC and Mrr systems in E Coli
  30. Modification Enzymes
    • -protect cell's DNA from REases
    • -usually a methylation of DNA
    • -type 1,2 and 3 have separate modification enzymes
  31. Gel Electrophoresis
    • -separates DNA molecules based on size
    • -gels usually agarose (a polysaccharide)
    • -Nucleic acids move through electrified gel to positive end(attract - DNA backbone)
    • -small molecules move farther
  32. Ethidium Bromide
    -can be used to stain gel in electrophoresis so DNA can be seen under UV light
  33. Restriction Map
    -map of the location of RE cuts on a segment of DNA
  34. Optical Mapping
    -allows creation of color coded gene map based on where RE cut and electrophoresis based on stained color movements
  35. Nucleic Acid Hybridization
    -base pairing of single strands of DNA or RNA from two different sources to give hybrid double helix
  36. Nucleic Acid Probe
    -segment of single stranded DNA used in hybridization with predetermined identity
  37. Souther Blot
    -hybridization where DNA is in the gel and probe is RNA or DNA
  38. Northern Blot
    -RNA is in the gel
  39. 3 Main Steps to Gene Cloning
    • 1-isolation and fragmentation of source DNA 
    • 2-Insertion of DNA fragment into cloning vector
    • 3-introduction of cloned DNA into host organism
  40. Step 1 of Gene Cloning
    • -isolation and fragmentation of source DNA
    • -source DNA can be genomic DNA, RNA or PCR amplified fragments
    • -genomic DNA must first be RE digested
  41. Step 2 of Gene Cloning
    • -insertion of DNA fragment into cloning vector
    • -most vectors derived from plasmids or viruses 
    • -DNA generally inserted in vitro
  42. DNA ligase
    • -enzyme that joins 2 DNA molecules
    • -works with sticky or blunt ends
  43. Step 3 of Gene Cloning
    • -introduction of cloned DNA into host organism
    • -transformation is often used to get recombinant DNA into host
  44. Gene Library
    -mixture of cells containing a variety of genes
  45. Shotgun Cloning
    -gene libraries made by cloning random genome fragments
  46. 2 Ways to detect the Correct Clone
    • 1-add radioactive DNA or RNA probes to lysed bacteria and denatured DNA then wash away anything not radioactive
    • 2-partially lyse cells then add antigen and attempt to detect blood serum antibodies
  47. Synthetic DNA
    • -De Novo synth of 100 BP oligonucleotides
    • -multiple oligonucleotides can be ligated together
    • -can be used for primers, probes and in-site directed mutagenesis
  48. Problem with conventional mutagens?
    -produce mutations at random
  49. Site-Directed Mutagenesis
    • -performed in vitro
    • -used to access specific AA in protein
  50. Cassette Mutagenesis
    -process of cutting DNA fragment out and replacing it with synthetic DNA fragment
  51. Gene Disruption
    • -when cassettes are inserted into the middle of the gene
    • -causes knockout mutations
  52. Reporter Genes
    • -proteins that are easy to detect and assay
    • -LacZ, Luciferase, GFP
  53. Benefits of Plasmids as Cloning Vectors
    • -small size
    • -easy to isolate DNA
    • -independent ORI
    • -get multiple copies of cloned gene per cell
    • -selectable markers
  54. pUC19
    • -modified ColE1 plasmid 
    • -common cloning vector
    • -contains ampicillin resistance and LacZ genes
    • -contains polylinker within LacZ gene
  55. Blue/White Screening
    • -white colonies have foreign DNA, blue do not
    • -LacZ is inactivated by foreign DNA, inactivated LacZ cannot process Xgal=blue does not develop
  56. Virus
    -genetic element that cannot replicate independently of a living host
  57. Virion
    • -virus particle
    • -extracellular form of a virus
    • -exists outside host
    • -facilitates transmission from one host cell to another 
    • -contain nucleic acid genome surrounded by protein coat and in some cases other layers of material
  58. How are viruses classified?
    -on the basis of the hosts they infect
  59. Capsid
    • -protein shell that surrounds the genome of a virus particle
    • -made up of a highly repetitive and precise pattern around nucleic acids
  60. Capsomere
    • -subunit of the capsid
    • -smallest morphological unit visible with electron microscope
  61. Nucleocapsid
    -complete complex of nucleic acid and protein packed in the virion
  62. Enveloped Virus
    • -virus that contains additional layers around the nucleocapsid
    • -lipid bilayer with embedded proteins 
    • -envelope makes initial contact with host cell
    • -
  63. Nucleocapsid Structure
    • -helical (rod shaped)
    • -icosahedral (spherical)
  64. Helical Symmetry
    • -rod shaped viruses 
    • -length of virus determined by length of nucleic acid 
    • -width of virus determined by size and packaging of protein subunits
    • -tobacco mosaic virus
  65. Icosahedral Symmetry
    • -spherical viruses 
    • -most efficient arrangement of subunits in a closed shell
    • -human papillomavirus
  66. Influenza Virus
    • -contains rigid spikes of haemagglutinin and neuraminidase that form characteristic halo projections around negatively stained virus particles
    • -binds sialic acid on host membranes
  67. Complex Viruses
    • -virions composed of several parts, each with separate shapes and symmetries
    • -bacterial viruses contain complicated structures (icosahedral heads and helical tails)
  68. 3 Types of Enzymes in Viruses
    • -lysozyme
    • -nucleic acid polymerases 
    • -neuraminidases
  69. Function of Lysozyme
    • -make hole in cell wall
    • -lyses bacterial cell
  70. Neuraminidase
    • -enzyme that cleaves glycosidic bonds
    • -allows liberation of viruses from cell
  71. Titer
    -number of infections units per volume of fluid
  72. Plaque Assay
    • -analogous to the bacterial colony 
    • -one way to measure virus infectivity
  73. Plaques
    • -clear zones that develop on lawns of host cells
    • -lawn can be bacterial or tissue culture
    • -each plaque results from infection by a single virus particle
  74. Efficiency of Plating
    • -quantitative virology 
    • -number of plaque forming units almost always lower than direct counts by electron microscopy due to inactive virions and conditions not appropriate for infectivity
  75. Phases of Viral Replication
    • 1.attachment (adsorption)
    • 2.entry(penetration)
    • 3.synthesis
    • 4.assembly
    • 5.release
  76. Phases of Viral Growth Curve
    • -one step growth
    • -latent period (eclipse and maturation)
    • -burst size (number of virions released)
  77. Permissive Cell
    host cell that allows the complete replication cycle of a virus to occur
  78. Attachment and Penetration for T4
    • -very complex
    • -virions attach to cells via tail fibers that interact with polysaccharides on E. Coli cell envelope
    • -tail fibers retract
    • -tail core makes contact with E. Coli cell wall
    • -lysozyme like enzyme forms small pore in peptidoglycan 
    • -tail sheath contracts and viral DNA passes into cytoplasm

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