Dilute-’N’-Go sequencing

Prof. Larry Wangh and his lab are interested in detecting changes in mitochondrial genomic sequences that result from aging, disease, or drugs.  To do this, they use LATE-PCR, an advanced form of asymmetric PCR, to detect mutations in the mitochondria by using multiplexes to study many mitochondrial genes at the same time.  LATE-PCR generates single DNA strands that are easily diluted for sequencing.  In the past. they have only been able to sequence one DNA strand from these multiplex reactions.

In a recent publication in Nucleic Acid Research, staff members Yanwei Jia and John Rice, along with Molecular and Cell Biology grad student Adam Osborne, describe the development of a blocking reagent that allows them to sequence both strands of the product DNA, thus allowing for the easy verification of mutations.

The figure at right shows that without a blocker (BLK), one is not able to obtain the excess (XP) strand sequence from a multiplex reaction.  Using a blocker one is able to get not only the limiting (LP) strand, but also the excess strand from the same multiplex

Post-translational modifications of Ras Oncogenes

Ras oncogenes were first discovered almost 40 years ago, when scientists experimenting with what would later be known as retroviruses found that virus taken from a leukemic rat could induce sarcomas in other rodents.  Today we know that activating mutations of human RAS are present in ~30% of all human cancers, including both solid tumors of nearly every tissue type as well as hematological malignancies.  But despite decades of study and a wealth of knowledge collected about RAS family proteins as well as their upstream activators and downstream signaling effectors, targeting oncogenic RAS has remained elusive.

Recently, much effort had been focused on developing RAS farnesyltransferase inhibitors, which target an essential post-translational modifying enzyme which adds a farnesyl-lipid necessary for membrane anchoring to RAS, which is normally tethered to the inner face of the plasma membrane.  However, these inhibitors have not been effective as hoped, as a geranylgeranyl-lipid can be alternatively added  by another enzyme when farnesyltransferase is inhibited.  Targeting both enzymes subsequently proved to be too toxic to normal cells.

A recent paper in Blood by Ben Cuiffo, a Molecular and Cell Biology graduate student, and Prof. Ruibao Ren, sets the sights on another post-translational modification: palmitoylation.  Palmitoylation serves as a second membrane anchor for some RAS isoforms, allowing them entry to the secretory pathway to traffic from the Golgi to the plasma membrane.  The necessity for palmitoylation for oncogenic transformation was previously unclear as RAS signaling scenarios from the Golgi have been characterized in a variety of cell types in culture.  Cuiffo and Ren made use of a mouse leukemia model driven by oncogenic N-RAS to drive leukemogenesis in vivo. They found that a point mutation that prevents palmitoylation but does not affect RAS activation was unable to drive leukemogenesis in this model.  The finding that palmitoylation is essential for N-RAS leukemogenesis in vivo exposes a potentially powerful new cancer target, not only for N-RAS driven leukemias, but for a variety of malignancies that rely on N-RAS to transduce oncogenic signals.

Naegleria Genome

Chan Fulton, Professor Emeritus of Biology at Brandeis, has been for many years a pioneer in using Naegleria gruberi, a single-cell eukaryote, as a research organism. This eukaryote can differentiate from ameobae to swimming flagellates in response to environmental cues.

In a paper published this week in Cell by Chan and his co-workers, the genome sequence of this organism is revealed and analyzed. The sequence information provides new insights into metabolic diversity (aerobic-anaerobic switching) in eukaryote evolution and in the early branching in the development of signalling pathways.

Brandeis users can read the full text on the web.

Research quickies

Some of our recent publications (descriptions are mine, not the authors’)

Lau: Finding new insect viruses by sequencing small RNAs (siRNA and piRNA)

Katz Lab: Taste affects smell

Sengupta Lab: Stress early in life causes epigenetic changes in worms

HPC cluster hits milestone

Our high-performance computing cluster passed the 1000 core mark this month, thanks to computer purchases for the computational biophysics and computational neuroscience groups and infrastructure support from Library and Technology Services. I’m looking forward to another great year working with you all.

Back online

We’ve been off-line for a while, and now we’ve moved into the new version of WordPress supported by the campus IT folks. It should now be relatively easy for labs to post themselves to this blogs.

What have we missed? Well, for one, Michael Rosbash and Jeff Hall are getting the Gruber Neuroscience Prize, together with Michael Young (Rockefeller U), for their work on genetics of circadian rhythms.

I’ll post some more of the “backdated” news when I get a chance. Feel free to ask for an account so you can do it yourself…

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