Archive for the 'Biology' Category

Reading the Cellular Instruction Manual

January 17, 2014

Guest post By David Knox

It is much easier to use a machine if you can read the manual. But being a typical male I can understand the need to ignore the manual and just play with the knobs and buttons to see what they do.

Molecular biology experiments are doing just that. They isolate a single complex or molecule and play with it, changing its concentration or changing the structure of the molecule and measuring the differences that occur in the cell. By systematically ‘playing with’ each molecule’s knobs and buttons, we have explored how those individual molecules changes the behavior of the system.

DNA

Using this knowledge we try to change the behavior of the cells that are not behaving correctly by drugs that change the active concentrations of molecules or inhibit the cell from making the wrong molecules. We try turning the knobs in order to make the cell more normal. But often we are only looking a what is happening at one place in the cell. Every action within this complex system has many effects everywhere in the cell.

The instruction manual for a cell’s behavior is encoded into the DNA. The problem is that we still don’t know how to read it. We know that some of the molecules in the cells can bind with the DNA and change the behavior by producing more (or less) of a protein. The process of converting the DNA into proteins can be broken down into simple steps: DNA is copied into RNA by a process called transcription; the RNA is used as the working blueprint from which many proteins can be built by a process called translation.

DNA is transcribed into RNA which is translated into Proteins.

The statement above is the central doctrine of biology. The statement should be extended to be circular, because the creation of proteins has an effect on which DNA is transcribed. The cell is in a constant state of flux and the system responds to changes in the external environment or within the cell. The system has many feedback loops creating checks and balances to keep the production of proteins within acceptable ranges.

The DNA is very long manual with contingencies for many different functions, some only during development of the organism, some only activated when the cell needs to divide, and some are only activated when the cell needs to die. Within the entire genome (all the DNA), only a small portion represents the blueprints for producing proteins and these segments are called genes. Some of the other DNA is used to directly control the transcription of the genes, but much of the DNA’s purpose is still unknown.

Mountain View

The proteins that bind to the DNA, collectively called DNA binding factors, can actually read the sequence of DNA and bind in specific positions to control the transcription activity around that position. The DNA encodes the signals of where these molecules bind and molecular biologist have discovered many of the signals used by each individual factor. But, many of these signals are overlapping with in the DNA sequence, which forces the individual factors to compete for binding to the DNA. This competition permits the cellular environment to dictate different binding of proteins depending on the local environment’s concentration of proteins. For example, a low level of a protein in the cell does not activate the production of a gene, but once the concentration reaches a certain level that allows it to out-compete other factors and transcription is initiated. There could also be a feedback loop that causes the cell to stop production when the concentration increases to a level that causes the protein to out-compete in a region that blocks or inhibits transcription. Controlling the production of genes is known as Transcriptional Regulation.

We are only beginning to understand the complex balance of different mechanisms used in the cell to regulate the transcription. As we begin to understand the individual mechanisms down their behavior at individual nucleotides of DNA, we begin to see the small effects that changes in concentration of a factor can have on the system as a whole. It is this complex system of small and sometimes subtle shifts in factors bound to the DNA on which my research if focused. I am creating a model of these DNA buttons and knobs based on our current knowledge of each factors behavior in the attempt to understand some of the signal embedded into the DNA. And maybe someday soon we will begin to understand how to read the manual and begin learning what is written in our DNA.

This article is syndicated from the author’s own blog, Visualizing Biological Processes.

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Linda Crnic Institute Holds 2nd Annual Down syndrome Symposium

December 22, 2013

Guest post By Amber Sorenson

On November 8th, the Linda Crnic Institute for Down Syndrome (LCI) held their second annual Down Syndrome Symposium. The conference was attended by researchers throughout Colorado who are interested in furthering Down syndrome research. Down syndrome (DS) is caused by an extra copy of chromosome 21, resulting in developmental defects and mental retardation.

Tom Blumenthal, the executive director of the LCI, began the day with a brief introduction to DS and the mission of the LCI. He emphasized the LCIs goal in making DS a tractable disease. The goal of the institute is not only to find ways to treat the ill effects of DS, but also to learn more about diseases that afflict patients with DS. In particular, DS individuals suffer from cognitive disabilities, including Alzheimer’s disease, and are prone to developing Leukemia. In order to accomplish these goals, the institute funded researchers in Denver and Boulder to study gene expression, autoimmune disease, leukemia, Alzheimer’s, and more in a Down syndrome context. We would get a chance to hear from some of these researchers later in the day.

The first speaker of the day was our keynote speaker, Jeanne Lawrence from the University of Massachusetts. This past year Jeanne published remarkable work in which she was able to silence the extra copy of chromosome 21 in DS with the XIST gene that is known the silence the extra X chromosome in women. Not only was Jeanne a fantastic speaker, she was also humble and sweet. She told a great story about how her interest in translational epigenetics and basic biology lead her to serendipitously address a clinical problem. Jeanne began her career interested in chromosome and nuclear structure. Her lab demonstrated that the XIST gene on the X chromosome transcribes a stable RNA that paints the inactive X chromosome. They also noticed that when the X chromosome was translocated onto an autosome, the autosome was partially silenced. This led them to ask if they could silence an autosome with XIST. However, how could they silence an autosome and maintain the cell viability? This led them to DS fiberblasts. They reprogrammed fiberblasts from DS patients to create stem cells. They then used zinc-finger editing to target XIST to chromosome 21. Chromosome 21 silencing in these cells is apparent after 5 days and fully silenced after 20 days of induction. The silencing remains stable, even after the XIST RNA expression is turned off. After silencing of the extra copy of chromosome 21 proliferation was increased in the cells to wildtype levels and the cultures were able to form neural progenitors earlier than the trisomy 21 cells. Jeanne was clear to point out that her discovery does not represent a cure of DS, but is a great research tool. And she hopes that further research may yield therapies for patients with DS.

The remainder of the day we heard from researchers who won the first round of grants from the LCI. Because this was the first year of awards from the LCI, the researchers have only had 6 months of time to get their projects off the ground. Much of the work by these researchers has been stunted by unexpected problems, such as culturing the DS iPS cells. Some of the highlights are summarized below:

• Matt Kennedy spoke about beta amyloid plaques in the brains of DS patients and the beta amyloid induced loss of dendritic spines.

• Rui Yi spoke about using quantitative miRNA-seq, mRNA seq, ribosomal profiling, and HITS-CLIP on trisomy 21, and wildtype iPS cells to better understand the mis-regulation in DS cells.

• James DeGregori hypothesized that the reduced fitness of trisomy 21 hematopoietic stem cells selects for adaptive mutations that lead to a higher risk of leukemia.

• Karl Pfenninger showed that DS neurons in culture exhibit distinct phenotypes in culture including increase in growth cone area, filopodia, axon length and adhesion. He would like to better understand brain development in DS patients, in particular the effect of increased amyloid progenitor protein (APP) on neuronal wiring.

• Rich Spitz reported that 50-80% of DS individuals have some form of auto-immune disease. His lab is profiling UBASH3A, associated with auto-immune disease, on chromosome 21 in DS individuals to determine if they have a higher rate of disease causing alleles.

For more information about the Linda Crnic Institute for Down Syndrome visit their website.

Butcher Symposium 2013

November 22, 2013
Guest post by David Knox

The Butcher Symposium was initiated in 2002 to bring together scientists from across the CU system to promote new collaborations. The Symposium also shares the progress of research previously funded by Butcher Seed Grants. The symposium is held every other year and this year it was held on November 1, 2013, in Westminster, Colorado. Traditionally the Symposium has been restricted to only CU-affiliated faculty and all attendees are required to present a poster at the symposium to be eligible to apply for the Seed grant. This year, for the first time, graduate students were also invited to participate. In parallel with the faculty program, students were required to participate in the poster session to apply for the BioFrontiers Science Alliance (BFSA) Seed Grant program.

The format for the Symposium featured recipients of the 2012 grants giving short talks outlining their progress (scientific, publications and continuing funding) of their proposal. The morning session consisted of 10 talks with a strong medical flavor featuring speakers from National Jewish Health, Anschutz Medical campus, and CU Boulder. Lunch is served at the very dynamic poster session. This year there were nearly 150 faculty and another 40 student posters. The poster session is a perfect venue for finding collaborations. People from five different campuses are able to interact. It is among the most energetic poster session I have attended. Unfortunately, there are too many people and interesting posters to visit in the two hours dedicated to the session. I had a list of 12 posters I wanted to visit and only made it to 6 of them, as I needed to be at my poster for half the time.

The afternoon session continued with longer talks from Dr. Robin Dowell – MCDB Boulder, Dr. David Schwartz – Chair of Medicine at Anschutz Medical Campus, and Dr. Lee Niswander – Children’s Hospital and Developmental Biology at Anschutz. The keynote talk was from Nobel laureate Dr. Jack Szostak about his research into the mechanisms and chemistry that could have been used in the earliest primitive environments and creation of self-replicating membranes in a primordial environment.

The newly created seed grant program for postdoctoral researchers and graduate students, BioFrontiers Science Alliance Seed Grant Award Program, is modeled on the successful Butcher Program that awards seed grants to fund collaborative and potentially transformative bioscience research. BFSA aims to build on existing institutional resources to encourage scientific creativity by supporting the independent research projects designed and proposed by graduate students and postdocs.

The primary goals of this award are to provide graduate students and postdocs with opportunities to:

• Tap into their own creativity and independently design and follow through on their own research ideas (not those of their faculty advisor)
• Pursue interdisciplinary collaborative projects that represent a significant new research direction
• Collaborate with diverse colleagues at CU Boulder and at other Colorado research organizations
• Participate in and expand the membership of the BioFrontiers Science Alliance
• Gain practical experience in the grant review and award processes.

For more information about BFSA and these events, please visit us at the BioFrontiers Science Alliance website.

Talks From the 2013 MCDB Retreat

November 8, 2013

Guest post by Timothy Read

The 2013 MCDB retreat was held in Vail, Colorado the weekend of October 11th and as filled with 3 days of excellent science as presented during talks and at posters. I will summarize a couple of highlights from the scientific talks in this post.

The talks began Friday evening with an interesting take on the role of ploidy in evolution, presented by Dr. Robin Dowell. Robin highlighted results from her collaboration with the Pellman lab at the Dana Farber. By competing two alternately fluorescently labeled yet isogenic strains of S.cerevisiae and observing mutations in DNA sequence that accumulated over 240 generations, researchers were able to pinpoint mutations in pathways important for adaptation to raffinose containing media. Not surprisingly, mutations in hexose transporter genes were shown to be important for survival. However, more surprisingly, researchers showed that having an increased ploidy leads to more rapid adaptation. Results such as this are very important from an evolutionary standpoint and have broad applications for cancer research, as cancer cells are known to be wrought with aneuploidy.

Another cancer related theme was presented by Dr. Joaquin Espinosa. Dr. Espinosa showed compelling data on the transcription factor, p53, a tumor suppressor that is mutated in over 50% of cancers. A major take home point from this talk was that p53 acts solely as a transcriptional activator in the immediate response to activation. The discrepancy between this new finding and previous reports can be explained by the use of a new high throughput technology called GRO-seq, or global run on followed by sequencing. This technology is able to directly measure transcription, rather than the steady state transcript as measured by RNA-seq and expression microarray. Dr. Espinosa’s talk also examined a newly emerging class of RNA molecules termed eRNAs, or enhancer RNAs. After defining the complete list of transcriptional targets of p53, they found that eRNAs were expressed near the target genes and directly overlapping known p53 binding sites, suggesting that p53 binding may potentially cause the expression of these RNAs, or alternatively that these RNAs somehow influence binding of p53 to sequences in the DNA.

Another interesting talk was presented by Katie Heiser, a graduate student in the lab of Dr. Bob Garcea. Katie described ‘virus factories’ as domains within the nucleus of cells infected with mouse polyomavirus (MPyV) that are implicated in viral DNA replication. These factories form tube-like structures in infected nuclei imaged by EM. She described how infection of cells can lead to a drastic re-localization of host proteins, including a shift of DNA damage proteins toward these virus factories. Katie also showed that these factories are sites of viral DNA replication. Katie’s results could help elucidate more general mechanisms used by viruses to aid in infection and replication.

Overall, the 2013 MCDB retreat was a great success and helped stimulate discussion about projects going on in every lab. The MCDB retreat takes place every other year. On alternate years students put together a symposium in which several researchers from other Universities are invited to give talks at the University of Colorado. Planning for the 2014 MCDB graduate student symposium is underway and will be held in the fall. Keep an eye out for more information about the symposium!

Review of the 2013 MCDB Retreat

October 28, 2013

Guest post by Jessica Vera

This year’s MCDB retreat (my third as a grad student in the department) was held in Vail, CO. Before getting into the science of it all, I just need to say that we were blessed with some spectacular weather and beautiful fall mountain scenery. Everything went off without a hitch and a thanks must be given to Robin Dowell and Gia Voeltz as well as Eric Hedl (IT) and Kathy Lozier(Admin) for their efforts in organizing the retreat. This being my third retreat, and quite possibly my last, I found myself getting nostalgic and thinking of previous retreat experiences. I was a first year when I first attended my first MCDB retreat, and as such little was required of me other than to attend the talks and check out some posters. My first retreat was also Robin’s first retreat and it was there that I distinctly remember meeting her for the first time. Little did I know that some six months later I would be joining her lab.

Each lab in the department is allotted a 20 minute talk during the retreat. I would like to elaborate on two such talks. First, Eric Davis of the Shen lab gave a talk entitled ‘Genome-wide analysis of gene-trap insertions and essential genes in human cells’. This was a continuation of work presented in his MMB talk just one week prior. What I like about his project is that he has utilized very new techniques to address lingering questions in the field of GPI-anchored protein trafficking. He has been mutagenizing a human haploid lymphoblast cell line via gene-trap insertion and then putting these cells under various selective conditions. He then performs next-gen sequencing to locate gene-trap insertion sites and identify genes involved in mediating the selected phenotype. At the retreat he specifically focused on his analytical methods for finding statistically significant hits in his experiments. I know firsthand how challenging certain aspects of his project can be and I am impressed with the quality of his work and with how he presented it to the department at large.

Second, I was happy to have caught James Orth’s talk ‘Follow the SINE: selective inhibitors of nuclear export as anti-cancer agents’. Being that James is a new assistant research professor in the department, I confess having known little about his research until the retreat. He is making use of the cell cycle biosensor system named FUCCI which allows for single cell assignment and monitoring of cell cycle progression via fluorescence microscopy. This system provides a good basis for exploring the effects of anti-cancer drugs. I predict this system may also prove useful to many other MCDB research labs!