Cell Physiology Test 1

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Cell Physiology Test 1
2015-02-09 12:35:48
Cell Physiology
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  1. Compartmentalization
    cells use different compartments to do different processes in order to be efficient
  2. Statistical Probablitity
    reactions occur when particles randomly bump into each other. Conditions can be changed in order to make the encounters more or less likely
  3. Unitary Structure
    components are linked together to make things which can be broken down into their components to be recycled
  4. Division of Labor
    different things have specific functions but can also perform more than one function simultaneously
  5. cell function is a result of their evolution
    processes follow a pathway that was determined by evolution and not necessarily because it was the best way to do it
  6. Homeobox genes
    determine the developmental plan, are highly conserved, and the position of the gene on the chromosome is similar to the point in the body plan it is coding for
  7. Why is Green Fluorescent protein better than Luciferin?
    GFP is always glowing while Luciferin only glows in the presence of oxygen
  8. What was PCR without Taq Polymerase like?
    very slow, because a new polymerase needed to be added after every cycle due to the denaturation at high temperatures
  9. What is Taq Polymerase?
    DNA Polymerase that was isolated from thermus aquaticus at yellowstone national park. Brock was interested in Taq, Chien and Trella isolated Taq Polymerase, Mullins used it, and LaRoche patented it
  10. Describe the steps of PCR
    • 1. Denaturation (95C) - separates the DNA 
    • 2. Annealing (60C) - primers stick to complementary areas
    • 3. Polymerization (70C) - Taq Polymerization adds the free nucleotides until it falls off the strands
    • Ingredients: DNA, free nucleotides, primers, and Taq Polymerase
  11. PCR does not require what?
    pure sample
  12. What is RNase?
    enzyme that chews up and degrades RNA from the 3' end
  13. Why and how do we convert mRNA to cDNA?
    • cDNA is more stable than mRNA. 
    • mRNA has a 3' poly A tail so an oligo-dT primer (poly T tail) to bind to the mRNA in an RNA sample and then using reverse transcriptase we convert to cDNA
  14. What is a cDNA library?
    Consists of all the RNA that a cell had at the time it was killed, when it is converted to cDNA it is a cDNA library
  15. How do you clone and convert a cDNA library?
    • ligate Gs to one side and Ts to the other
    • cut the DNA and the plasmid with the same restriction enzymes so that they are completely complementary and the cDNA can be added to the vector
  16. What is a polylinker?
    a site that can be recognized by restriction enzymes so that you can make them do what you want
  17. Why are plasmids ampicillin resistant?
    so that you can grow plasmids on ampicillin to ensure the survival of bacteria that has taken up the plasmid only.
  18. What is the SOS system?
    bacteria will suck up DNA from the environment if they are stressed. We use the sos system so that we can force the cells to take up the plasmids
  19. What stressors do we use to activate the SOS system?
    42 C and CaCl. These conditions kill most cells
  20. How can you harvest a plasmid?
    putting the bacteria in a centrifuge and popping them open with chemicals and DNA. This process will produce so much DNA that it can be seen with the naked eye
  21. what are the reasons for making cDNA?
    more stable, less mistakes than Taq polymerase, more DNA, can accomplish more with a vector than a PCR product
  22. Dideoxy
    missing 2 oxygens
  23. Sanger sequencing
    • label dideoxy nucleotides with a color
    • they randomly stop PCR and statistically if the products are sorted by size there should have been a dideoxy nucleotide at each bp
    • the sequence is recognized and determined by a laser
  24. Why is sanger sequencing used less?
    expensive, slow, and overshadowed by new techniques
  25. Ion Torrent
    uses a semi conductor chip which has wells that capture chemical information and translate it to base pairs
  26. What is a gel used for?
    to separate the molecules you are interested
  27. What is agarose gel used for?
    Agarose gel is used to transfer Nucleic Acids because they are big and negatively charged
  28. How do you read a gel?
    • Bigger molecules are at the top and the smaller ones are at the bottom because smaller molecules travel further
    • Relative size is determined through the use of the ladder
    • Brightness and thickness is proportional to the amount
  29. What is a blot?
    A blot is a membrane that you transfer the target molecule to
  30. What is the purpose of a southern blot?
    The purpose of a southern blot is to determine if genes in a species are found in other animals by using a probe from one animal and DNA from another, copy number, and differences among genomes
  31. What is the method of band transfer for a southern blot?
    Diffusion because the membrane is thinner (nitrocellulose paper) and the bands are on the surface of the gel
  32. What are the steps of a southern blot?
    • Cleave the DNA with restriction enzymes
    • run electrophoresis
    • transfer the bands from the gel to the nitrocellulose paper via capillary action
    • hybridize with labeled DNA or RNA probes
  33. What is a northern blot used for?
  34. What can be determined using a northern blot?
    the size, presence of a sequence, and the amount of RNA
  35. What are the steps of a northern blot?
    similar to southern blot except you use RNA and you incubate the membrane with a radio-labeled probe
  36. What is replacing the northern blot?
    Real Time PCR (qPCR) and chips
  37. Why is qPCR used?
    qPCR is used in order to determine how much mRNA is made in condition A vs. condition B
  38. What are the drawbacks of qPCR?
    It doesnt tell us the size of the mRNA because only a small piece of cDNA can be amplified
  39. What is the advantage of using qPCR?
    It is easier than measuring the amount of protein that is made
  40. What is the process of qPCR?
    extract DNA, run many cycles of PCR, DNA is detected while it is being made using a fluorescent probe that sits in the middle of the strand and gets knocked off by the polymerase, the probe doesn't fluoresce when it is knocked off
  41. How do you present the data from qPCR?
    • X is the cycle number and Y is the amount of fluorescence 
    • How soon the signal amplifies is proportional to how much you started with
  42. Why is qPCR different from PCR?
    it is more sensitive because you measure the data while it is happening and you can see if there are different starting amounts rather than just the ending amount like in PCR
  43. How do you analyze qPCR?
    it is so sensitive that you can detect a single molecule, draw a line across the middle of the curves, determine what cycle it crossed the threshold and then use a formula to determine how different the two conditions are
  44. When do we use qPCR?
    diagnostic tests for genetic and non-genetic diseases
  45. The fear of vaccines and autism came from what?
    An incorrect qPCR study
  46. What is the western blot used for?
    Separating proteins, transferring them to a blot, and then incubating them with an Ab
  47. What can be determined from a western blot?
    the size, the presence, and the amount of protein
  48. What types of gels are used in Western Blot?
    • The gels used are usually acrylamide or resin based instead of agarose which are carbohydrate based
    • Acrylamide gels have smaller pores and allow you to look at smaller things
  49. How are bands transferred in a western blot?
    the gel is transferred onto a membrane using electricity because the membrane is too thick to transfer by capillary action, an Ab is used against particular protein and then a second Ab that is labeled is used in order to see where the protein is
  50. What are Western blots used for?
    used for AIDs testing (test against the presence of Ab against HIV)
  51. In a western blot what are proteins coated with and why?
    Proteins are coated with dodecylsulfate or soap becayse they do not have a uniform charge. the soap unfolds the protein and coats them with a uniform charge
  52. In a western blot what are proteins coated with and why?
    Proteins are coated with soap (dodecylsulfate) because they do not have a uniform charge and the soap causes them to unfold and coats them with a uniform charge
  53. What is a polyclonal Ab?
    Polyclonal Abs are more general and against multiple epitopes. Within a species. They are made in a limited supply through two antigen injections into an animal and then the harvesting of serum. The Ab cannot be remade when you run out of serum
  54. What is a monoclonal Ab?
    Monoclonal Abs are more specific and are made against a single epitope. They are made by injecting an animal with Ag, harvesting spleen cells, fusing the cells with tumor cells and growing them on HAT media, then testing to find the cell that is against the correct Ab and harvesting it. These cells, B cell hybridomas, are immortal and you will have the Ab for forever
  55. What is a primary Ab?
    a primary Ab is against the thing you are trying to detect and is usually not labeled
  56. What is a secondary Ab?
    Ab against the primary Ab and is usually labeled because you can make more of it while the primary Ab is in limited supply
  57. What is immunohistochemistry?
    A western blot of a tissue sample that is looking for protein concentrations
  58. What is in situ hybridization?
    a northern blot that is searching for RNA and DNA in a tissue slice
  59. What is epigenetics?
    modification of the genome without changing the actual base pairs via methylation and covalent modification
  60. What was the study that we discussed about epigenetics?
    23 volunteers that did bike training on one leg only and submitted to muscle biopsies before and after in both legs.
  61. How was RNAseq used in the epigenetics study?
    • RNAseq was used in order to count the experimental myoglobin mRNA and the control myoglobin mRNA 
    • It is better than a microarray
  62. Which types of mRNA went up with exercise?
    contraction, myofibril bundles, sarcomeres, regulation of glycolysis, regulation of adipose deposition
  63. How many genes were cataloged that changed with exercise?
  64. What is a microarray used for?
    Microarray is used to look at a bunch of DNA at the same time as well as compare samples by labeling the different treatments different colors.
  65. How do you run microarray?
    Isoltate the total RNA from each sample, reverse transcribe each into cDNA, label the different treatments with different colors (green or red), hybridize to a glass slide with oligos that are stuck to the slide, the color will indicate where the cDNA sticks and then you can look up which gene it is
  66. Does microarray require labeling, a library, or both?
    Just requires labeling.
  67. What is RNAseq?
    RNAseq is similar to microarray but it is cheaper, more sensitive, and more quantitative.
  68. How do you run RNAseq?
    collect RNA from your 2 conditions, make cDNA, make a library and sequence all of the cDNA (this will result in sequencing the same type of cDNA thousands of times), send the cDNA to a company who will send the data back to you
  69. In RNAseq, do you need labels, a library, or both? and why?
    RNAseq requires a library and does not need labeling as a result. The library is made so tht you only have to use one primer to sequence everything.
  70. What is important about the RNA transcriptase for RNAseq?
    The RNA transcriptase must be good at both short and rare or abundant and long sequences, and in order to publish your results you must use one of the RNA transcriptase molecules that is determined good.
  71. What are the disadvantages of RNAseq?
    you have so much data that you may not even know what the results are. There is likely to be about 30-40% of data that you do not understand
  72. What is bioinformatics?
    A developing field that analyzes mass quantities of data such as the results of RNAseq
  73. What is a candidate gene?
    A candidate gene is a gene that goes way up in a disease compared to the control
  74. What can you do with candidate genes to determine their function?
    Knock outs or knock downs
  75. What is a knock out experiment?
    A knock out experiment is when there is no expression of the gene of interest, there are traditional methods as well as CRISPR, you would typically raise the animal to an adult to see how the lack of gene effects everything, and a lot of times there is no obvious answer to what the gene actually does
  76. What is CRISPR?
    A knockout experiment that uses RNA and the Cas9 enzyme
  77. How do you run CRISPR?
    A strand of RNA is used as a guide which hybridizes to a specific spot in DNA which will lead to a double stranded break, the cell has repair mechanisms which repair the break and typically makes a mistake resulting in changes in the gene that can result in the termination of protein synthesis. The last step is to screen the organism to see if the mutation actually occured
  78. What is Cas9?
    Cas9 is an enzyme that is used in CRISPR, it comes from bacteria that recognize bacteriophage and chop them up
  79. Leptin Mice
    Big Mouse has no leptin production due to a lack of the leptin gene, if you connect its circulatory system to a normal mouse or give it leptin it will go to normal size
  80. What is a knock down experiment?
    A knockdown experiment is when you reduce the amount of mRNA that is available to be translated
  81. What is RNAi?
    RNAi is a knockdown experiment that lasts for about 2-3 days
  82. How do you run RNAi?
    introduce mRNA that is complementary to the mRNA strand that you want to knock down, the mRNA will become double stranded and then will be degraded. Can be done in mammals and C.elegans
  83. How do you do RNAi in C.elegans?
    Place the RNA vector in bacteria and then feed it to the worms
  84. How do you use morpholino oligonucleotides?
    Morpholino oligonucleotides bind to the RNA of interest and don't allow the translation of mRNA for about 4 days. Can be done in zebrafish
  85. What are the components of a gene?
    UTR (untranslated region), exons, and introns
  86. Why are cDNA and mRNA not considered a gene?
    Because components, such as introns, are missing
  87. What is the structure of a gene?
    chromosomes must be unwound to be transcribed, long range regulatory elements, cis-regulatory elements (part of the DNA sequence which regulates how much you make), trans-regulatory elements (outside of the DNA that binds and regulates), and transcription factors (assemble on the promoter)
  88. How do you clone a gene?
    Isolate RNA, make cDNA, run PCR, label the PCR product, use the probe to screen a genomic library
  89. What is the proton motive force?
    Separation of charges that results from the pumping of protons across the membrane as electrons travel through the electron transport chain
  90. What is different about the proton motive force in mitochondria?
    The direction that the protons move
  91. What are mitochondria?
    Double membrane bound organelles that were once free living single celled organisms that were engulfed by higher organisms
  92. What are common misconceptions about mitochondria?
    • Kidney bean shape (comes from the fact that they are fixed in EMS, actually more like a lava lamp)
    • A cell has a specific number of mitochondria (cant be counted in a living cell because of fusion and fission)
  93. How can you test for purity in an isolated sample of mitochondria?
    Test for glycolytic enzymes, if they are not present, the sample is pure
  94. What is the primary component of the mitochondrial inner membrane?
    Protein (as much as 90% by weight)
  95. What are some differences between chloroplasts and mitochondria?
    • Chloroplasts have 3 membranes instead of 2
    • can use energy from light to move protons
  96. Why were plants not allowed in the emergency room?
    Originally because they have mitochondria and they take oxygen from the environment at night. Now it is because of the potential for infection
  97. What is different about the F1F0 channel in mitochondria?
    The F1F0 channel has a different orientation. In chloroplasts it uses light to excite and move electrons. There is also a different terminal electron acceptor
  98. What is photorespiration?
    Photorespiration is the process through which carbon dioxide is released instead of fixing the carbon into a 3-carbon molecule. It is catalyzed by rubisco which causes it to be inefficient as well as the low affinity for carbon dioxide
  99. What makes C4 plants better at photorespiration?
    C4 plants are better at photorespiration because of compartmentalism.  They can concentrate carbon dioxide in one tissue and then move it to the tissues where photorespiration occurs
  100. What is the composition of cytochrome oxidase?
    13 subunits, 10 by the nuclear genome and 3 by the mitochondrial genome
  101. What problems can result from incorrect folding of cytochrome oxidase?
    miss folded proteins will be placed in vessicles, the cell will die, and lesions result
  102. What is the F1 domain of the F1F0 complex and how does it work?
    The catalytic domain, protein rotates and the F1 domain changes shape allowing the addition of ADP and P to make ATP. It has 3 conformations: open, low, and tight. B subunits stabilize the head and keep it from moving
  103. What is the F0 domain and how does it work?
    F0 is the channel domain, it is a half channel because the proton binds to Arg-210 causing a conformational change and the proton leaves on the other side.
  104. What causes the conformational changes and rotation in the F1F0 complex?
    Asp 61 on each C subunit binds protons.
  105. What is an uncoupling protein (UCP)?
    a channel that destroys the proton gradient because it allows the passage of protons without the generation of ATP. These channels generate heat instead of storing energy in the form of ATP.
  106. What demographic has a high number of UCPs?
    Infants have UCPs in their scapular fat which they use to regulate their body temperatures.
  107. What is an example of a photosynthetic animal and how does it harness light energy?
    Sea slug that eats chloroplasts from algae and instead of digesting them keeps them intact and puts them on its mantle in order to photosynthesize and feed itself.