Lecture, chapter 10 - Gene regulation in eukaryotes

Nucleosomes with deacetylated and acetylated histones

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After summarizing the main points of gene regulation in prokaryotes we started discussing mechanisms used by eukaryotic organisms.

We are building upon what we already know about gene regulation in prokaryotes and highlighting the added complexity found in eukaryotes. Some of the reasons for which additional or more complex mechanisms are required in eukaryotes are:

• DNA is found in a nucleus
• DNA is wrapped around histones
• Nucleosomes can be condensed in heterochromatin
• In multicellular eukaryotes different genes are expressed in different cell types or at different stages of development

We started explaining how a cell solves the problem of accessing DNA that is highly condensed (heterochromatin). Acetylation of histones, specifically of lysine residues, holds the key. We described how histone acetyl transferases (HATs), and histone deacetylases (HDs) add and remove acetyl groups from lysine residues in histones.

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Module 3, Lab 11Native and denaturing polyacrylamide gel electrophoreses (PAGEs) of GFP

Image from Bio Rad

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Today we ran a new kind of electrophoresis: Polyacrylamide Gel Electrophoresis (PAGE) is a process that uses the same principle of agarose gel electrophoresis, but it uses a polyacrylamide gel, a thinner, more expensive kind of gel that provides a higher resolution than its agarose counterpart.

We specifically ran protein samples in two ways.

• A native gel, in which proteins, in their native state, migrate at different rates depending on their size (molecular weight), 3D structure, and charge.
• A denaturing gel, in which the proteins are denatured (linearized) in the presence of a detergent such as Sodium Dodecyl Sulfate (SDS) that coats the proteins with a negative charge. The resulting denatured proteins have an overall negative charge and a similar charge to mass ratio. Since denatured proteins act like long rods instead of having a complex 3D shape, the rate at which they migrate in the gel depends only to their size (molecular weight) and not its charge or shape.
  

The goal was to estimate the size of the green fluorescent protein (GFP) by comparing its migration through each gel with the migration of a molecular weight ruler (a "protein ladder") loaded onto the same gel.

PAGE is used for separating proteins ranging in size from 5 to 2,000 kDa due to the uniform pore size provided by the polyacrylamide gel. Agarose gels can also be used to separate proteins, but they do not have a uniform pore size, so they are optimal only for electrophoresis of proteins that are larger than 200 kDa.

We will be able to compare the results in both gels, and if GFP has any activity in either one of them (through pictures taken under UV light).

Gels were stained with Coomasie G-250 and air dried for analysis.

(click on pic for full size image)

(Note: Lab section 2 performed this procedure on Monday)

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Module 3, Lab 10 - Protein quantitationBradford dtermination of GFP

Today we did a protein quantitation using BioRad's Quick Start™ Bradford Protein Assay, a method in which a dye reagent is used (Bradford reagent, based on Brilliant Blue G-250) to bind to proteins (causing the dye reagent to change from a reddish-brownish color to blue) and measure its absorbance. The more concentrated the protein it binds, the darker the blue resultant color, and the greater the absorbance at 595 nm.

Two relative standard proteins are used, bovine serum albumin (BSA) and bovine gamma-globulin (BGG), to generate absorbance vs. protein concentration curves and then interpolate the absorbance of problem samples (mostly with GFP) to estimate their concentration. The problem samples were fractions obtained from the Hydrophobic Interaction Chromatography (HIC) lab.

This method is applied when researchers in proteomics discover a new protein and are trying to gather information about it. In our case, we "discovered" GFP, although we wouldn't have a name yet, had it been a truly newly discovered protein.

(Note: Section 2 performed this lab on Monday evening)

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Lecture, chapter 9 - Gene regulation in prokaryotes

Today we discussed the the stages within transcription at which gene expression can be regulated, whether it is via positive or negative regulation.

We also discussed the role inducers play in causing a change in shape of activators (a.k.a. transcription factors) and repressors, as well as the role of global and specific regulators in gene expression (including the concept of regulon)

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