Download LOCALIZATION OF A MOLECULE

Following Molecules/Cells through
TIME to Understand Processing and
Processes
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?
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.
Marking a Population of
– Molecules
– radioactivity (e.g. 35S, 32P)
– density
– fluorescence
– Cells
– enzyme expression
– morphology (e.g. chick versus quail)
– fluorescence
Experimental Conditions II
–rapid labeling
–the label must be transparent to the
process
–minimal redistribution of the label during
the course of the experiment
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.
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
Meselson & Stahl:
a classic pulse chase experiment
Question:
What is the mechanism (process) by
which E. coli DNA is replicated?
conservative
distributive
semi-conservative
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.
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 wouldn’t be good
enough to distinguish strands.