CHEMISTRY
Dynamic, artificial cells and vesicles
Scott Long et al. have constructed synthetic cells comprised of lipid bilayer membranes surrounding a two-phase, aqueous polymer solution that can be reversibly converted to a single phase. An understanding of compartmentalization in cell function has been hampered by the lack of an experimental model system. Using a previously developed method, the authors constructed artificial cells by encapsulating a poly-(ethylene glycol) (PEG)/dextran aqueous two-phase system (ATPS) within a giant lipid vesicle. The researchers tagged each macromolecule, PEG, dextran, and lipid, with an individual fluorescent marker to monitor transitions by confocal microscopy. The two encapsulated phases were found to contain different polymer concentrations, and the authors were able to create microcompartments within the synthetic cells by introducing molecules with varying affinities for PEG or dextran. Fluorescently tagged streptavidin accumulated preferentially in the PEG-rich phase of a vesicle containing biotinylated PEG, whereas SBA, a carbohydrate-binding lectin, partitioned into the dextran-rich phase of the vesicles. Through osmotically driven dehydration, Long et al. were able to partition single-stranded DNA oligonucleotides into the dextran-rich phase of an ATPS-containing vesicle.
Figure 1.
Synthetic cell vesicle.
“Dynamic microcompartmentation in synthetic cells” by M. Scott Long, Clinton D. Jones, Marcus R. Helfrich, Lauren K. Mangeney-Slavin, and Christine D. Keating (see pages 5920-5925)
BIOCHEMISTRY
An efficient, mutant RNA polymerase
Jean Guillerez et al. have engineered a bacteriophage T7 RNA polymerase (RNAP) that reduces abortive cycling over a variety of initial sequences. Previous research has shown that the initial transcription complex formed by this monomeric RNAP, a popular tool for producing RNA in vitro, is unstable; it often dissociates before it can undergo structural rearrangement to yield the elongation complex, releasing small abortive transcripts. Guillerez et al. mutagenized an inefficient RNAP, the I810S mutant, and screened for a recovery in transcriptional efficiency. Substitution of proline for leucine at codon 266 (P266L) reduced synthesis of abortive transcripts and favored runoff transcription. The authors observed this effect in either the single P266L or double P266L/I810S mutant. Decreased synthesis of abortive transcripts by the P266L mutant was caused by stabilization of the initial transcription complex, according to the researchers. The authors constructed additional mutants at position 266 and found that, regardless of the inserted amino acid (alanine, serine, or tyrosine), the removal of the conserved proline was responsible for this stabilization. In a preparative RNA synthesis experiment with [α-32P]ATP-labeled transcripts, the P266L mutant was superior to wild-type enzyme with respect to synthesis rate, product yield, and low contamination with abortive products.
Figure 2.
P266 on the structure of T7 RNAP.
“A mutation in T7 RNA polymerase that facilitates promoter clearance” by Jean Guillerez, Pascal J. Lopez, Florence Proux, Hélène Launay, and Marc Dreyfus (see pages 5958-5963)
MEDICAL SCIENCES
Characterizing melanomas by microarray
Christopher Haqq et al. report the identification of gene expression patterns for each stage of melanoma progression. The research team found that melanomas lost the expression of cell adhesion and extracellular matrix molecules, such as cadherin 3 (CDH3) and matrix metalloproteinase 10 (MMP10), in the transition from radial to vertical tumor growth. Immunohistochemical analysis confirmed that CDH3 and MMP10 expression was stronger in the radial tumor growth phase. The authors used cDNA expression array profiling to characterize the patterns of transcript modulation in normal skin, nevi, primary melanomas, and two types of metastatic melanoma. By two-class significance analysis of microarrays (SAM), Haqq et al. pinpointed genes whose expression distinguished benign nevi from primary melanomas. The up-regulation of genes such as osteopontin, known to play a role in melanoma progression, and down-regulation of genes such as Wnt inhibitory factor 1, known to have potential tumor suppressor activity, were observed. By multiclass SAM comparison, the researchers detected >2,000 genes that distinguished skin, nevi, and primary and metastatic melanoma.
Figure 3.
Stages of melanoma growth.
“The gene expression signatures of melanoma progression” by Christopher Haqq, Mehdi Nosrati, Daniel Sudilovsky, Julia Crothers, Daniel Khodabakhsh, Brian L. Pulliam, Scot Federman, James R. Miller III, Robert E. Allen, Mark I. Singer, Stanley P. L. Leong, Britt-Marie Ljung, Richard W. Sagebiel, and Mohammed Kashani-Sabet (see pages 6092-6097)
NEUROSCIENCE
Neural cliques record experiences and generate brain codes
By simultaneously recording the activity of hundreds of neurons in live, behaving mice, Longnian Lin et al. report a method that may provide a clearer picture of how memories are formed and stored. Although brain activity is typically measured one or a few neurons at a time, complex behaviors like learning and memory depend on large sets of neurons acting together. To determine how such multineuron groups work together to allow memory formation, Lin et al. developed a 96-electrode array that can simultaneously record activity patterns in up to 260 individual neurons of the mouse hippocampus, which mediates memory. Using different startle-inducing stimuli and differing environments of stimulus occurrence, the researchers identified basic coding units in the mouse hippocampus, termed neural cliques. These neural cliques responded uniquely to each stimulus and, according to the researchers, provide a plausible, real-time neural basis of memory formation. In addition, the authors demonstrated that the activation patterns of neural cliques can generate a set of neural activation codes that, like the genetic code, seem to be universal across individuals and species.
Figure 4.
Neural firing patterns from startle stimuli.
“Identification of network-level coding units for real-time representation of episodic experiences in the hippocampus” by Longnian Lin, Remus Osan, Shy Shoham, Wenjun Jin, Wenqi Zuo, and Joe Z. Tsien (see pages 6125-6130)
ANTHROPOLOGY
Humans, not climate change, likely cause of elephant extinctions
One million years ago, proboscideans lived in most of Africa, Europe, Asia, and the Americas. Today, wild elephants are found only in portions of sub-Saharan Africa and South Asia. Whether Pleistocene extinctions in the order Proboscidea could be attributed to climate change or human hunting has been a source of debate. Todd Surovell et al. examined 41 kill/scavenge sites, with ages ranging from 1.8 million to 10,000 years, that showed evidence of human-elephant spatiotemporal contemporaneity. The authors found that the archaeological record of these sites is preferentially located on the edges of the human geographic range over the entire time span. The finding is commensurate with global overkill, the authors say, because extinction due to climate change would likely result in these sites being found not only along the frontier of dispersal but also well behind it. Expansion of the prehistoric human range likely resulted in localized extinction events, and proboscideans through time have survived in refugia, such as tropical forest biomes, that are largely in-accessible to human populations.
Figure 5.
Global elephant kill/scavenge sites.
“Global archaeological evidence for proboscidean overkill” by Todd Surovell, Nicole Waguespack, and P. Jeffrey Brantingham (see pages 6231-6236)