Following Molecules/Cells through TIME to Understand Processing and Processes

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Following Molecules/Cells through TIME to
Understand Processing and Processes
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An experimental strategy for investigating

• kinetics of synthesis or degradation of a
  molecule
• precursor/product relationships
• molecular mechanisms (e.g. DNA replication,
  signal transduction)
• which cells give rise to particular structures
  during development?

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Experimental Conditions I

• a means of differentially marking a population of
  molecules or cells
• a method for following them through time. Must
  distinguish labeled from unlabeled at various
  time points.

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Marking a Population of

• Molecules
• radioactivity (e.g. 35S, 32P)
• density
• fluorescence
• Cells
• enzyme expression
• morphology (e.g. chick versus quail)
• fluorescence

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Experimental Conditions II

• rapid labeling
• the label must be transparent to the process
• minimal redistribution of the label during the
  course of the experiment

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Pulse/Chase Is a Prototypical Example for
Molecules

• cells are initially grown in a medium deficient
  in a metabolite that will be subsequently used as
  a label, so that stores are depleted.
• add labeled metabolite for a discrete interval
  and then add an excess of unlabeled metabolite.

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Means of detecting population of marked molecules

• Dana and Nathans used polyacrylamide gel
  electrophoresis and autoradiography with
  quantitation of counts in bands.
• Schroeter et al. used immunoprecipitation,
  SDS-PAGE and autoradiography
• Meselson and Stahl used equilibrium density
  gradient centrifugation

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Meselson Stahla classic pulse chase experiment
Question What is the mechanism (process) by
which E. coli DNA is replicated?
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conservative
distributive
semi-conservative
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Equilibrium Density Gradient Sedimentation

• Pioneered by Meselson, Stahl and Vinograd
• Gradient of concentration of salt (CsCl in this
  case) set up by gravitational force field leading
  to a density gradient. This occurs relatively
  rapidly.
• DNA travels under the influence of gravitational
  force until it reaches a point where the density
  is equal to its own- can't go further into more
  dense material.
• Countervailing process is diffusion of DNA down
  its concentration gradient. The band width is
  inversely proportional to molecular weight of the
  substance because of diffusion.

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How would you answer this question today?
Look directly at the DNA molecule? Resolution is
an issue. Maybe atomic force microscopy What
about BrdU labeling? Resolution wouldnt be good
enough to distinguish strands.