LectureChapter 10 - Gene regulation in eukaryotesChapter 11 - Regulation at the RNA level

Today we talked about the important role that the histone remodeling complex plays in allowing proteins access to DNA in nucleosomes. We related the action of this enzyme, as well as HATs and HDs in the gene expression process as a whole.

We then discussed the role of methylation patterns in gene silencing and provided an example in genetic imprinting.

We also started a discussion on chapter 11, on how gene expression is regulated between transcription and translation (control at the mRNA level). We mentioned the several ways in which the translation of a mRNA molecule can be controlled, and then we focused in one of them: Controlling the stability and the rate of degradation of the mRNA (usually via ribonucleases a.k.a. RNases).

----------------------

Lecture, chapter 10 - Gene regulation in eukaryotes

Nucleosomes with deacetylated and acetylated histones

____________________________________________________________

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.

----------------------

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

Image from Bio Rad

_______________________________________________________________

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)

----------------------

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)

-----------------------

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)

----------------------

Lecture Chapter 7 - Protein structure and functionChapter 9 - Gene regulation in prokaryotes(And... The PCR Song)

We finished chapter 7, and the third stop in our road map, on how DNA organization influences protein function. We discussed the most common structural motifs of DNA binding proteins and agents that may denature or even degrade proteins.

We also started chapter 9, on regulation of gene expression in prokaryotes. We discussed the basics of positive and negative regulation and the differences in frequency of gene regulation events at transcription, translation and in between.

__________________________________________________

The PCR song

The highlight of the day was, with no doubt, the performance of The PCR Song by students in this class. You can see the video of the original song by clicking here, or accessing the previous entry in this blog.

The exercise, meant to reinforce the reagents and steps of PCR, ended up being delightful and enjoyable one, even leading to the suggestion to use more songs that summarize processes (a molecular biology greatest hits CD?).

I am open tu suggestions and new ideas!

(the mp3 file of the Winter 10-11 Biol 217 Ensemble is available upon request!)

----------------------

PCR song

After exam 1, which showed that several people in the class are confused on what is required to perform a PCR and on the steps of the thermal cycle, I decided to reinforce the basics of the procedure that were explained in labs 4 and 5.

Students were allowed to vote on having a quiz on Friday or learning by heart and singing the PCR song. A nearly unanimous decision favoring the latter was reached. The PCR song highlights:

• Who invented PCR
• The main reagents of PCR
• The steps of the thermal cycle
• Some important applications of PCR

Tomorrow (Friday) students will sing the song in class for the opportunity to 1) properly learn the basics of one of the most popular molecular techniques and 2) earn a few points.

The song: Students in previous quarters have found this song useful and some have even used it as a ringtone... (Warning: Cheesy!)

----------------------

Module 2Lab 6 - Gel electrophoresis of the pJet1.2+GAPC gene R.E.D.Lab 7 - Sequencing reactions of the GAPC gene

Lab 6

Today we ran the gel electrophoresis of yesterday's R.E.D. to confirm the successful ligation of the GAPC gene from Arabidopsis, which we amplified through nested PCR, with the pJet1.2 plasmid.

The results were used to determine which plasmid DNA purified samples were to be used in lab 7, when setting the sequencing reactions for the GAPC gene.

Lab 7

We added forward and reverse sequencing primers (pJET SEQ F and pJET SEQ R) to the plasmid DNA purified samples that had the GAPC gene insert and put them in a 96-well plate. The plate will be shipped to the DOE Joint Genome Institute (JGI) to be sequenced as part of their Sequencing Training Program (STR). The results should be in in 2-5 weeks, ready to be used in the bioinformatics labs

We will discuss the DNA sequencing technique most commonly used: Dye-terminator sequencing, a modification of Sanger's chain termination sequencing protocol, which allowed the automation of the DNA sequencing process.

(Note: Section 2 followed these protocols on Monday)

----------------------

Module 2, lab 6 - GAPC gene cloningPlasmid DNA purification ('miniprep')

Today we used the E. coli cultures Salesha and Jess inoculated yesterday to do a small-scale plasmid DNA extraction ('miniprep') using Promega's Wizard® Plus SV Minipreps DNA Purification System. Then restriction enzyme digestions with Bgl II were set to confirm that the insert (GAPC gene) is present in the plasmid (pJet1.2).

(Section 2 did the procedure on Monday [MLK day]. Thanks to Stacy for inoculating the media the day before!)

Here's a recap of what we have done so far in the last couple of exercises:

Lab 5 - Nested PCR of the GAPC gene from Arabidopsis

Lab 6 - Cloning he GAPC gene from Arabidopsis

• Ligation of PCR amplified GAPC gene onto the pJet1.2 plasmid
• Genetic transformation of E. coli with the pJet1.2 plasmid
• Cloning of genetically transformed E. coli
• Minipreps (purification of pJet1.2 plasmid)

Next, running an agarose gel to confirm the results of the R.E.D. (lab 6) and setting DNA sequencing reactions (lab 7)

-----------------------

Lecture, chapter 7 - Protein structure and function

Today we talked about the quaternary level of structure in proteins, and the nomenclature of proteins that are composed by several subunits.

We discussed a classification of proteins from a molecular biology perspective and also how DNA binding proteins can read information contained in DNA sequences without undoing the double helix.

----------------------

Lecture, chapter 7 - Protein structure and function

Today we started the final chapter on the third stop in our road map, specifically on how genetic information affects protein function.

We discussed the structure of amino acids and how they are linked into polypeptides thanks to peptide bonds (a special case of covalent bonds).

We also discussed the basics of the four levels of organization in proteins, but focused specially on the secondary and tertiary levels of structure. We discussed the importance of hydrogen bonds in maintaining alpha-helices and beta-sheets, an how these are folded into 3-D structures that are crucial for protein function.

----------------------

Module 2, lab 6 (section 1) GAPC gene cloning

Click here for info on the same procedure, performed by students in section 2.

-----------------------

Module 3, lab 9 (section 1) Hydrophobic Interaction Chromatography (HIC) of GFP

Click here for info on the procedure performed by students in section 2.

-----------------------

Lecture, chapter 6 - Transcription

DNA looping and interaction of general and specific transcription
factors with RNA polymerase in eukaryotic cells during transcription
___________________________________________________

Today we finished chapter 6, on transcription.

We discussed how the RNA polymerase recognizes specific DNA sequences in the promoter of prokaryotic DNA, and how it forms a transcription bubble in order to read the template strand on the DNA and synthesize the RNA molecule. We commented how the terminator causes the formation of a hairpin in the RNA transcript that causes the RNA ploymerase to "fall off" the DNA.

We highlighted the differences in transcription between prokaryotes and eukaryotes:

• the use of three kinds of RNA polymerase (I, II, and III) for transcribing different kinds of genes
• the use of general and specific transcription factors
• the role of enhancer regions in aiding the transcription process
• the need for DNA looping and the use of a mediator complex for all the transcription factors to interact with the transcription apparatus

CLICK HERE to watch a good Youtube video on transcription, or HERE for a video from the Dolan DNA Learning Center!

----------------------

Module 3, lab 9 (section 2) Hydrophobic Interaction Chromatography (HIC) of GFP

Yesterday we performed a Hydrophobic Interaction Chromatography (HIC) to separate the green fluorescent protein (GFP), produced in our bacterial cultures, from other proteins commonly found in bacteria.

A sample of bacterial culture was concentrated and then resuspended in a solution in which they were lysed. The high salt solution, containing all the proteins found in the bacteria, was then passed through a hydrophobic interaction column where molecules of GFP bound to the hydrophobic beads. The high salt solution increased the hydrophobicity of GFP by further exposing its hydrophobic amino acid residues.

A series of washes with buffers of decreasing salinity allows proteins with various levels of hydrophobicity to gradually unbind from the beads and be collected in a test tube. By switching collection tubes each time a buffer is added, different proteins can be collected. One of them was GFP and the tube in which it was collected glowed with a green color (of course).

Diagram of Hydrophobic Interaction Chromatography (HIC)

GFP molecules are represented by black triangles

-----------------------

Module 2, lab 6 (section 2)GAPC gene cloning

Yesterday we started the process of cloning the GAPC gene from Arabidopsis, which we amplified via nested PCR in lab 5. The lab was divided in three main steps

• Ligation (of GAPC gene on to the pJet1.2 plasmid)
• Preparation of competent cells
• Genetic transformation of E. coli

We spent most of the lab manipulating bacteria to make them competent (i.e. get them ready to uptake extracellular naked DNA). Once this was achieved, the GAPC gene from Arabidopsis, obtained via nested PCR, was ligated to the pJet1.2 plasmid.
The plasmid was then used to genetically transform E. coli, which were spread on LB agar/Amp/IPTG plates and incubated.

----------------------

Exam 1

Exam stats:

(click on pic for full size image)

----------------------

Module 3, lab 08 (section 1)Genetic transformation of E. coli with the pGLO plasmid

We followed the same process we followed with section 2.

For more information click here.

-----------------------

Module 2, lab 5 (section 1)GAPC gene nested PCRGel electrophoresis (PCR round 2) and PCR purification

Today we ran the agarose gel electrophoresis for the second round of the nested PCR of the Arabiodopsis GAPC gene (which we will clone, and eventually sequence), and also purified the reaction products, in order to eliminate leftover reagents and keep only the DNA.

----------------------

Lecture, chapter 6 - Transcription

Today we started chapter 6, on transcription, covering the basics of the process.

We discussed how to decide which are the template (sense) and coding (anti-sense) strands in DNA, the reasons for which some genes are expressed all the time (housekeeping genes) and some others are expressed only some times, and included some transcription-specific terminology.

----------------------