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.

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

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

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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.

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Lecture, chapter 6 - Transcription

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

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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

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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.

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Exam 1

Exam stats:

(click on pic for full size image)

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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.

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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.

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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.

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Module 3, Lab 08 (section 2)Genetic transformation of bacteria with the pGLO plasmid

Aequorea victoria, original source of the green fluorescent protein (GFP)

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Today we used the pGLO plasmid to genetically transform Escherichia coli.

pGLO is a plasmid that has been engineered to contain the Green Fluorescent Protein (GFP) gene, originally isolated from the jelly Aequorea victoria. GFP produces a green fluorescence when excited by blue or UV light.

In order to make the GFP gene a functional one it has been engineered so the sugar arabinose triggers the production of the protein. The genes in the arabinose operon (araB, araA, and araD) have been replaced by the GFP gene. Such genes encode proteins that break down arabinose when it is present in the environment, so they are expressed only if this is the case. The regulatory sequence has been left intact, so in the engineered operon the presence of arabinose turns on the GFP gene and, therefore, GFP is produced.

Another feature of the pGLO plasmid is the presence of the beta-lactamase gene, which provides resistance against the antibiotic ampicillin.

The bacteria were transformed through the heat shock technique, and then plated on LB agar plates containing:

• Just LB (lysogeny broth)
• LB and ampicillin
• LB, ampicillin and arabinose

Plates are being incubated for 24 hours at 37ºC.

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Lecture, chapter 5 - DNA replication

We finished the chapter on DNA replication.

We discussed the process in which the DNA polymerase complex actually replicates DNA, including the DNA looping that allows both strands to be synthesized at the same time.

We also covered eukaryotic DNA replication, emphasizing the differences with prokaryotes.

Watch the following video or access this link to understand the main features of the replication process

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Section 1Module 2, Lab 05 - GAPDH nested PCRAmplifying the GAPC gene

Same lab as with section 2. Click here for information on it.

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Lecture, chapter 5 - DNA replicaiton

Today we started chapter 5, on DNA replication, and the third stop in our roadmap, on how genetic information is preserved and inherited.

We started a discussion on how DNA is replicated in prokaryotes, including the concepts of replication fork and replisome. We described the role of several enzymes on the process of DNA replication (DNA gyrase, DNA helicase, single strand binding (SSB) proteins, primase, DNA polymerase), and the fact that there are a leading and a lagging strand during the replication process, the latter being extended in bursts forming Okazaki fragments.

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Section 2Module 2, Lab 05 - GAPDH nested PCRAmplifying the GAPC gene

Thale cress, Arabidopsis thaliana
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Today we started the exercise in which students will learn the basics of nested PCR. We will work with the gene that encodes one of the GAPDH isomers, GAPC, in the thale cress (Arabidopsis thaliana), the model organism of plants. Some people call it "the fruit-fly of plants".

GAPDH is an enzyme in charge of catalyzing one of the reactions in glycolysis. There are several nuclear genes that encode GAPDH isomers (proteins with different amino acid sequences but with the same function), and we are targeting the gene GAPC in the A. thaliana genome. We ran a first round of PCR, with our initial primers, and on Wednesday we will do the second run, with the nested primers.

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Section 2Module 1, lab 4 - PCR of the PV92 Alu insertion locus

Today section 2 completed lab 4 in module 1. For more information click on PCR of the PV92 Alu insertion locus

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Lecture, chapter 4 - Genes, genomes, and DNA

Organization of the human genome
from Allison, L. 2007. Fundamental Molecular Biology. Blackwell Publishing.
(click on pic for a full size image)
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Today we followed our discussion on how DNA is organized in genomes (second stop in our roadmap), including a discussion on satellite DNA (satellites, minisatellites, and microsatellites), palindromic DNA (mirror-like palindromes, inverted repeats, hairpins, stem-and-loops), junk and selfish DNA, and supercoiling.

On Monday we'll discuss how eukaryotic DNA is compacted enough to fit the nucleus of a cell

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Module 1 (section 1)Lab 3 - PCI DNA extraction from human bloodLab 4 - PCR of the PV92 Alu insertion locus

Lab 3 - PCI DNA extraction from human blood

Yesterday students finished the PCI DNA extraction from their own blood. The steps that were left included adding the PCI, doing some pellet washes with cold ethanol and eluting the DNA in TE buffer. Samples were incubated overnight at 55ºC.

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Lab 4 - PCR of the PV92 Alu insertion locus

The goal of this lab was to introduce students to the Polymerase Chain Reaction (PCR), the most popular in vitro technique to make copies of (amplify) target DNA fragments. We extracted DNA from our cheek cells and used it to set up PCRs.

Our target is the PV92 Alu insertion locus, located on chromosome 16.
Alu elements are a family of short interspersed repetitive elements (SINEs) that have mobilized throughout primate genomes for the last 65 My, by retrotransposition.

There are more than 500,000 Alu elements per haploid genome in humans (about 5% of our genome). Depending on the insertion point they may be associated with some genetic diseases (e.g. some cases of hemophilia, familial hypercholesterolemia, severe combined immune deficiency, or neurofibromatosis type 1). But in most cases it has no effect on the individual's health.

Some Alu insertions are very recent and polymorphic. The most recent are human specific (HS) and such is the case of PV92. Because the PV92 insertion locus is HS, polymorphic, neutral (invisible for natural selection), and easy to detect, it has been widely used in human genetic population studies, and it has been one of the markers used to support the out-of-Africa hypothesis.

In this lab we will test the presence of 0, 1, or 2 PV92 Alu insertions in our genomes.

The following picture illustrates the possible outcomes of our PCRs:

The sample on lane 1 belongs to an individual with no PV92 Alu insertions, lane 2 to an individual with insertions in both chromosomes, and lane 3 to an individual with an insertion in one chromosome.

What is your genotype like?

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Module 1, lab 3 (section 1) - PCI DNA extraction from human blood

Today we started the phenol-chloroform isoamyl alcohol (PCI) DNA extraction from most students' blood. Blood samples were obtained throughout Monday and Tuesday.

Students added SSC buffer (pH stabilization), SDS (cell lysis), sodium acetate (NaOAc; protein precipitation), proteinase K (inactivation of endonucleases), PCI (separation of proteins and nucleic acids), and 100% ethanol (DNA precipitation). Samples were frozen to continue the process tomorrow.

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LectureChapter 3 - DNA, RNA, and ProteinsChapter 4 - Genes, genomes, and DNA

Today we finished chapter 3, mentioning the basics of the different functions of RNA and protein structure.

We then started chapter 4, on genes, genomes and DNA, which is our second strop in the class roadmap: How DNA is organized in organisms and how such organization affects its function.

We discussed how little genetic information is necessary for independent life and the importance of non-coding DNA in eukaryotes. We talked about pseudogenes, introns, and repeated sequences (tandem repeats and intersperse elements).

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