Author: Prof. Andresen

Today I spent the morning brushing up on some reading, looking at a review article done by Bai, et al. This article described research very similar to my own, because they are also using spectroscopy to look at the ion atmosphere around Nucleic Acids. They did not use precipitated DNA though, rather they use a buffer equilibrium technique to preserve the atmosphere around the DNA. They found that the total ions around the DNA almost always was within error of exactly compensating for the DNAs negative charge. They also studied the competition between different molecules with similar charges, finding that bulkier polyions were often less competitive than the alkali and alkaline earth ions. I then spent the afternoon brushing up on my OES skills, preparing and running a test series and calibrations set.

As promised, learning the experimental ropes began today. It began with a crash course in stoichiometry, led by John, the student veteran of the Andresen lab. Once we had recalled the number crunching of high school chemistry, we were sent to the pipettes to create solutions of varying concentrations of Mg, P, and Co. We created these samples so we could be trained on the main machine of the lab, the Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES). To quickly summaries what this machine does, it pumps solutions through a high temperature plasma, which blasts the molecules in solution apart and detects the resulting ions by analyzing the discrete wave lengths detected at different regions throughout the machine.
Using the ICP, we can send DNA on NCP molecules that have been bathed in different ionic solutions, we can detect the ions that attach themselves to molecules. It’s important to note that the main error in the experiments comes from pipetting, so the Foreign Factor will be getting lots of practice on that, competitions galore.

Kurt was not around today so John was in charge. We had a session on lab safety early on, to start the day. Jeremy Kumar gave us essential information on how to handle situations gone wrong in the lab and he gave us helpful information about appropriate lab behavior.

Next, John showed us how to prepare solutions of Cobalt, Magnesium and Phosphorus. We learned how to use the pipettes and brushed up our old school chemistry skills in stoichiometry as we calculated concentrations of solutions.

The most exciting part of the day was learning how to actually run the Spectrometer as John lead the way!!

We rounded up reading the articles we had to cover today. We read the “Physics of Chromatin” article by Helmut Schiessel. This article was like an all round summary of all the other articles we read during the week. First , it talked about the structure of the Nucleosomes, then proceeded to say that DNA is packed in a way that the information would be easy to access even though it is packed in a complex way.

Next, we read about how an increase in ionic strength makes the DNA thicker and hydrogen bonding being responsible for the linking between the DNA- backbones and the histones surrounded by the DNA.

The paper also confirmed once more that counter ion release was responsible for overcharging. The paper ended by using a sphere ad chain model to explain the DNA and histone interaction in three different cases.
1) weakly charged
2) strongly charged and
3) physiological condition

With today being the last day of reading and next week the beginning of learning experimentation procedures, it seems appropriate to discus a few of the experiments we have read about during these past few reading days.

With certain aspects of these nucleosome structures fairly ambiguous, such as how they are packed along the genome, investigation into these uncertain aspects of nucleosome function is currently being looked into. The main work we have looked at is the paper, Structure and Phase Diagram of Nucleosome Core Particles Aggregated by Multivalent Cations, by Aurélie Bertin, Stéphanie Mangenot, Madalena Renouard, Dominique Durand, and Françoise Livolant, where they examined the behavior of NCP in various polyvalent ion solutions. The general trend was that when a solution of NCP had polyvalent ions added, the NCP rapidly fell out of solution. However, as more solution was added the NCPs went back into solution until it reached its original concentration, an unusual trend that that could suggest certain processes and structures of chromatin and NCP.

Ben has basically said all we did today at the lab and has given a little lecture on the Necleosome core particles(NCP),but i will add a little to that. I will explain a little about the experiment that was done on the NCP he talked about. As he said already, the NCP has a net negative charge and so the experiment involved adding two different cations ,namely a magnesium ion and a spermidine ion to a solution containing the NCP.These ions were added separately in two different experiments. The behavior of the NCP was observed as they precipitated when the ions were added but also came out in re-dissolution when the number of ions added reached a certain threshold. They used different concentrations of the NCP to see the different behaviors it will have. We learned that, the lower the concentration of the NCP was, the greater the interaction it had with the ions. We will keep you posted, but for now the second half of the international factor is out till tomorrow.

Today the foreign factor was churning through papers again, this time looking at prior experimentation done with Nucleosome Core Particles (NCLs). These publications are along the lines of the work us internationals will be doing around the lab, so as we know what its like to be in a strange land, for those of you who are foreign to NCLs, we’ll turn this post into a lesson on NCLs and how they act in certain environments.

If you can imagine wrapping a thick string around a cylinder, that is a basic picture of an NCL, with DNA strands being the string and a histone octamer, or protein being the cylinder. As seen in the image to the right, the ‘nucleosome’ refers to this whole string and ball structure of a 146-147bp (base pair) DNA strand wrapped around the histone. This tiny structure (10nm) has a lesser negative charge than isolated DNA, due to the positive core, with an overall charge of -150. The nucleosomes can be thought of as the building blocks of chromosomes, as multiple nucleosomes can be wound together to form chromatin fibers, which be further arranged to form chromosomes. The reason we are interested in these nucleosomes is that due to the small, dense and complex structures, not much is known about the processes that compacts nucleosomes into chromatin fibers.

We’ll let that information be absorbed and talk about experimentation tomorrow. Foreign factor gone.

We moved on to different article that also tries to explain why the liked charged macroions/polyelectrolytes (DNA in particular) will want to stay attached together,in a condensed form ,when counterions are added to a solution in which they are in. We also learned ,from kurt, about the role that enthalpy and entropy play in this procedure. We learned about why the system as a whole chooses to preserve its entropy at the expense of its enthalpy by using its highest mutli-valent ion in the bonding process. I have learned a lot in just two days. It is going to be a great summer!!

Today, we basically had to catch up with the readings for research. We have a lot of fun stuff to read and get acquainted with, because the research involves a decent measure of biology. So I decided to get going with it right away and discover why two molecules(DNA) of the same charge will want to sit beside one another holding hands(be attracted to one another). They should be enemies according to the physics we all know. So to get to the bottom of this, Ben and I read our first article and then bombarded Kurt with all our uncertainties(there most always be uncertainties in labs right?)and he made them all clear. Tomorrow,we will be on to the next one.

p.s make sure you laugh at the uncertainty joke. Thanks

With a summer theme of Bio-Physics, it would seem important for Fash and Benjamin (the Foreign factor of the lab), the young Nigerian and New Zealand aspiring physicist to brush up on the prefix (Bio). Currently, although we are well versed in physical ideas, such as electrostatic repulsion, Fash’s and my biological knowledge doesn’t extend further then a semester of bio 112; leaving us about as lost as we felt line dancing at the local rodeo last weekend (although Fash did suit his pink rodeo hat).

Today — and most likely the remainder of the week — will be dedicated to becoming acclimated with the biological and chemical content of the Andressen lab, and also getting a good grasp on prior work that has been conducted on the unusual properties of DNA.

From the literature we covered today (DNA-Inspired Electrostatics – W. Gilbert, Electrostatics of Strongly Charged Biological Polymers: Ion-Mediated Interactions and Self-Organization in Nucleic Acids and Proteins – G. Wong, L. Pollack) the interesting behavior of DNA came to the forefront of Fash and my bilingual discussions. Switching effortlessly between New Zealandish, English and American, we conversed about the theme of electrostatics. We agreed that one of the most striking aspects of DNA was its charge to length ratio. The long thin DNA coil contians one unit of negative fundamental charge every 0.17nm of length. Even more remarkable than this fact, are the experimental observations that despite the repulsive effects one might expect from such dense, like charge, the DNA attracts itself under a range of solution conditions. This was a recurring theme in the literature studied, with numerous theories including Poisson-Boltzmann Mean-Field Theory, presented in an attempt to explain this unintuitive phenomena.

Also applicable to the work we will be doing was the descriptions of how DNA acts in varying ionic solutions. When placed in “physiological conditions,” (1MolL-1 NaCl) the DNA follows the shape of a coil, however, when placed in a highly dilute solution, the DNA forms into a torus (donut) shape with an average radius of 50nm. This resembles the experiments the foreign factor will most likely be looking into. However, instead of dealing with isolated DNA, we will be looking at how DNA, with nucleosomes still present within the structure, will act in varying solutions.

That seems like enough of an introduction, apologies for the accents, and until tomorrow, the Foreign half of team Andressen is out.