Detecting Mutations the Easy Way

Recent Brandeis Ph.D graduate, Tracey Seier (Molecular and Cell Biology Program), Professor Sue Lovett, Research Assistant Vincent Sutera, together with former Brandeis undergraduates Noor Toha, Dana Padgett and Gal Zilberberg have developed a set of bacterial strains that can be used as “mutational reporters”.  Students in the Fall 2009 BIOL155a, Project Laboratory in Genetics and Genomics, course also assisted in the development of this resource. This work has recently been published in the journal Genetics.

These Escherichia coli strains carry mutations in the lacZ (β-galactosidase) gene that regain the ability to metabolize lactose by one, and only one, specific type of mutation. This set allows environmental compounds to be screened for effects on a broad set of potential mutations, establishing mutagen status and the mutational specificity in one easy step.

This strain set is improved over previous ones in the inclusion of reporters that are specific for certain types of mutations associated with mutational hotspots in gene. Mutations at these sites occur much more frequently than average and involve DNA strand misalignments at repeated DNA sequences rather than DNA polymerase errors. Such mutations are associated with human diseases, including cancer progression, and have been under-investigated because of the lack of specific assays. Using this strain set, Seier et al. also identified a mutagen, hydroxyurea, used in the treatment of leukemia and sickle cell disease, which affects only the “hotspot” class of mutations. This strain set, which will be deposited in the E. coli Genetic Stock Center,  will facilitate the screening of potential mutagens, environmental conditions or genetic loci for effects on a wide spectrum of mutational events.



Left: E. coli colonies showing lacZ mutant revertants (blue pimples) arising on a white colony on growth medium containing the beta-galactosidase indicator dye,  X-gal


yet more papers in the wild

More papers appearing recently:

Current Brandeis authors noted in boldface, past Brandeis trainees shown in italics

Older Adults are Better at Spotting Fake Smiles

Studies of aging and the ability to recognize others’ emotional states tend to show that older adults are worse than younger adults at recognizing facial expressions of emotion, a pattern that parallels findings on non-social types of perception. Most of the previous research focused on the recognition of negative emotions such as anger and fear. In a study “Recognition of Posed and Spontaneous Dynamic Smiles in Young and Older Adults” recently published in Psychology and Aging, Derek Isaacowitz’s Emotion Laboratory set out to investigate possible aging effects in recognizing positive emotions; specifically, the ability to discriminate between posed or “fake” smiles and genuine smiles. They video-recorded different types of smiles (posed and genuine) from younger adults (mean age = 22) and older adults (mean age = 70). Then we showed those smiles to participants who judged whether the smiles were posed or genuine.

Across two studies, older adults were actually better at discriminating between posed and genuine smiles compared to younger adults. This is one of the only findings in the social perception literature suggesting an age difference favoring older individuals. One plausible reason why older adults may be better at distinguishing posed and spontaneous smiles is due to their greater experience in making these nuanced social judgments across the life span; this may then be a case where life experience can offset the effects of negative age-related change in cognition and perception.

This was the first known study to present younger and older adult videotaped smiles to both younger and older adult participants; using dynamic stimuli provides a more ecologically valid method of assessing social perception than using static pictures of faces. The findings are exciting because they suggest that while older adults may lose some ability to recognize the negative emotions of others, their ability to discriminate posed and genuine positive emotions may remain intact, or even improve.

The Emotion Laboratory is located in the Volen Center at Brandeis. First author Dr. Nora Murphy (now Assistant Professor of Psychology at Loyola Marymount University) conducted the research as a postdoctoral research fellow, under the supervision of Dr. Isaacowitz, and second author Jonathan Lehrfeld (Brandeis class of 2008) completed his Psychology senior honors thesis as part of the project. The research was funded by the National Institute of Aging.

Lights, Camera, Splice!

In their paper “Ordered and Dynamic Assembly of Single Spliceosomes” appearing in Science this week, Brandeis postdoc Aaron Hoskins and co-workers use a combination of yeast genetic engineering, chemical biology, and multiwavelength fluorescence microscopy to work out the kinetic mechanism by which the spliceosome assembles on a model pre-messenger RNA prior to splice out an intron in the RNA. The work is a collaboration between Jefl Gelles’s lab in Biochemistry,  Melissa Moore’s lab at UMass Medical School, and  Virginia Cornish’s lab at Columbia.

Hoskins et al. use a single-molecule fluorescence approach that dubbed “CoSMoS” (Co-localization Single Molecule Spectroscopy), originally developed in the Gelles lab by Larry Friedman and Johnson Chung, that is a powerful method to study the assembly and function of the complex macromolecular machines that perform a wide variety of biological functions. In this movie, shown 150x faster than real time, the comings and goings of many U1 spliceosome components on a surface-tethered pre-mRNA are shown as the appearance and disappearances of white spots.  The white spots orginate from the fluorescence emission of specifically labeled U1 components upon excitation with a 532nm laser.

Pre-mRNAs are spliced in a complex cycle wherein the spliceosome assembles, is activated for catalysis, performs two transesterification reactions, and disassembles on every turnover.  Steps between the isolatable intermediates depicted in this cycle involve the coordinated association and dissociation of many spliceosome components.  A key finding by Hoskins et al. is that spliceosome assembly is reversible, and this is represented by the dashed arrows between the pre-mRNA, A, and B complexes.

The multi-wavelength, total internal reflection fluorescence (TIRF) microscope built by Larry Friedman and Johnson Chung in the Gelles laboratory uses lasers of different wavelengths to excite spectrally distinguishable fluorophores on various spliceosome components. Photo by Diane Katherine Hunt.

According to Hoskins, who will leave Brandeis to take up a faculty position in the Biochemistry Department at the University of Wisconsin, Madison

By far, the most challenging aspect of the project was determining two completely orthogonal methods for attaching fluorophores to endogenous spliceosomes in whole cell extract.  Since these experiments are quantitative, we needed to find methods that give a very high degree of fluorophore incoporation and specificity (in other words, 10% labeling would not cut it!).

The novel part, for me, is that for decades spliceosome kinetics have been “off-limits” to enzymologists due to the complexity of the system.  However, by developing the correct analytical tools, the spliceosome can be studied in detail usually reserved for enzymes orders of magnitude smaller.

Hoskins plans to continue these single molecule studies of the spliceosome in his new lab in Wisconsin and will be focusing on splice site selection and  coupling of nuclear RNA processing events.  He also aims to develop new methodologies for fluorescent labeling of ribonucleoproteins in vitro and in vivo.

Separating proteins and manipulating live cells using magnetic nanoparticles

Brandeis grad students Yue Pan (Chemistry) and Marcus Long (Biochemistry), together with Professors Lizbeth Hedstrom and Bing Xu, have synthesized novel 6 nm diameter magnetic nanobeads (comparable in size to a globular protein) and used them to separate specific proteins from a cell lysate and manipulate live cells. This work has just appeared online in the journal Chemical Science.

Selectively binding glutathione-S-transferase fusion proteins using
glutathione-decorated iron oxide nanoparticles and down-stream applications

These small, magnetic beads have numerous advantages over larger traditional glutathione-modified beads, including rapid purification, and ultra low non-specific binding. Importantly, both the purified GST and the protein of interest (POI) preserve their innate properties. They also demonstrate that functionalized iron oxide nanoparticles can be used to manipulate live cells. This work  establishes design principles for decorating magnetic nanoparticles that will ultimately should lead to a general and comprehensive platform for studying biological interactions and biological systems using a magnetic force.

Barry and Dogic receive 2010 Cozzarelli Prize

Physics graduate student Edward Barry and Professor Zvonimir Dogic have been selected to receive the 2010 Cozzarelli Prize in Engineering and Applied Sciences from the Proceedings of the National Academy of Sciences (PNAS) for their work entitled “Entropy driven self-assembly of non-amphiphilic colloidal membranes.”

The work of Barry and Dogic was selected for exploring a novel pathway for the self-assembly of 2D fluid-like surfaces or monolayer membranes from non-amphiphilic molecules. Amphiphilic molecules consist of immiscible components, such as a hydrophobic tail and a hydrophilic head, which are irreversibly linked to each other, thus frustrating their bulk separation. When added to water, these molecules self-assemble into a variety of structures in order to satisfy competing affinities for the solvent. One particular structure, a bilayer membrane, which is a thin flexible sheet with remarkable mechanical and chemical properties, plays an essential role in biology, physics, and material science. Over the past decade the paramount example of conventional amphiphilic self-assembly has inspired the synthesis of numerous amphiphilic-type building blocks for studies of membrane self-assembly including various block-copolymers, heterogeneous nanorods, and hybrid protein-polymer complexes. Underlying all of these studies is the belief that amphiphilic molecules are an essential requirement for membrane assembly.

Barry and Dogic, using a combination of theory and experiments, describe for the first time a set of design principles required for the assembly of non-amphiphilic membranes in which the constituent rod-like molecules are chemically homogeneous.  Using a simple mixture of filamentous bacteriophages and non-adsorbing polymer, they were able to assemble macroscopic membranes roughly 4-5 orders of magnitude larger than the constituent molecules themselves. Due to unique properties of their system, Barry and Dogic were able to characterize the physical behavior of the resulting non-amphiphilic membranes at all relevant length scales and provide an entropic mechanism that explains their stability. The importance of these results lies in their potential to establish a fundamentally different route toward solution based self-assembly of 2D materials.

Papers selected for the Cozzarelli Prize were chosen from more than 3,700 research articles published by PNAS in 2010 and represent the six broadly defined classes under which the National Academy of Sciences is organized. The award was established in 2005 and named the Cozzarelli Prize in 2007 to honor late PNAS Editor-in-Chief Nicholas R. Cozzarelli. The annual award acknowledges recently published papers that reflect scientific excellence and originality. The 2010 awards will be presented at the PNAS Editorial Board Meeting, and awardees are recognized at the awards ceremony, during the National Academy of Sciences Annual Meeting on May 1, 2011, in National Harbor, Maryland.

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