Following Molecules/Cells through TIME to Understand Processing and Processes

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?

– 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.

Experimental Conditions I

– Molecules

– radioactivity (e.g. 35S, 32P)

– density

– fluorescence

– Cells

– enzyme expression

– morphology (e.g. chick versus quail)

– fluorescence

Marking a Population of

–rapid labeling

–the label must be transparenttransparent to the process

–minimal redistribution of the label during the course of the experiment

Experimental Conditions II

– 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.

Pulse/Chase Is a Prototypical Example for 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

Means of detecting population of marked molecules:

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.