Gemini,a Bifunctional Enzymatic and Fluorescent Reporter of Gene Expression

Background

The development of collections of quantitatively characterized standard biological parts should facilitate the engineering of increasingly complex and novel biological systems. The existing enzymatic and fluorescent reporters that are used to characterize biological part functions exhibit strengths and limitations. Combining both enzymatic and fluorescence activities within a single reporter protein would provide a useful tool for biological part characterization.

Methodology/Principal Findings

Here, we describe the construction and quantitative characterization of Gemini, a fusion between the β-galactosidase (β-gal) α-fragment and the N-terminus of full-length green fluorescent protein (GFP). We show that Gemini exhibits functional β-gal activity, which we assay with plates and fluorometry, and functional GFP activity, which we assay with fluorometry and microscopy. We show that the protein fusion increases the sensitivity of β-gal activity and decreases the sensitivity of GFP.

Conclusions/Significance

Gemini is therefore a bifunctional reporter with a wider dynamic range than the β-gal α-fragment or GFP alone. Gemini enables the characterization of gene expression, screening assays via enzymatic activity, and quantitative single-cell microscopy or FACS via fluorescence activity. The analytical flexibility afforded by Gemini will likely increase the efficiency of research, particularly for screening and characterization of libraries of standard biological parts.     

Combined next‐generation sequencing and morphology reveal fine‐scale speciation in Crocodile Skinks (Squamata: Scincidae: Tribolonotus)

Next‐generation sequencing has vast potential to revolutionize the fields of phylogenetics and population genetics through its ability to collect genomic scale data sets of thousands of orthologous loci. Despite this potential, other types of data (e.g. morphology, ecology) remain important, particularly for studies endeavouring to delimit species. Here, we integrate next‐generation sequencing with morphology to examine divergence between populations of Tribolonotus pseudoponceleti on the islands of Buka and Bougainville in the Solomon Archipelago. We used the Ion Torrent PGM to collect over 648 Mbp of sequence data for 12 samples, representing 1526 loci recovered from all samples, and 3342 were recovered from at least six samples. Genetic structure analyses strongly support the distinctiveness of these two populations, and Bayes factor delimitations decisively select speciation between Buka and Bougainville. Principal components and discriminant function analyses reveal concordant morphological divergence. Finally, demographic analyses via diffusion approximation and approximate Bayesian computation prefer a complex model of mid‐Pleistocene divergence with migration, and a later decrease or cessation of migration and population size shift, suggesting a scenario in which migration was enabled by Pleistocene merging of these two islands, and limited when isolated by higher sea levels. Further analysis of four Sanger sequenced loci in IMa2 had limited power to distinguish among models including and excluding migration, but resulted in similar population size and divergence time estimates, although with much broader confidence intervals. This study represents a framework for how next‐generation sequencing and morphological data can be combined and leveraged towards validating putative species and testing demographic scenarios for speciation.     

Fate of bacteria ingested by larvae of the freshwater mayfly,Ephemera danica

The fate of bacteria in the food of a common freshwater invertebrate has been studied both in controlled laboratory experiments and in a stream sediment. The animal chosen was the larva of the burrowing mayfly,Ephemera danica. It ingested all available bacteria nonselectively. More bacteria were found associated with the hindgut than with the mesenteron despite continuous plug flow of food through the alimentary canal. Species of bacteria were affected in different ways.Aeromonas hydrophila andCitrobacter freundii were both digested, the former selectively.Flavobacterium sp. and other unidentified species appeared to attach to the hindgut wall. Digestion of bacteria was not due to a sudden change in pH.     

Unique thylakoid membrane architecture of a unicellular N2-fixing cyanobacterium revealed by electron tomography

Cyanobacteria, descendants of the endosymbiont that gave rise to modern-day chloroplasts, are vital contributors to global biological energy conversion processes. A thorough understanding of the physiology of cyanobacteria requires detailed knowledge of these organisms at the level of cellular architecture and organization. In these prokaryotes, the large membrane protein complexes of the photosynthetic and respiratory electron transport chains function in the intracellular thylakoid membranes. Like plants, the architecture of the thylakoid membranes in cyanobacteria has direct impact on cellular bioenergetics, protein transport, and molecular trafficking. However, whole-cell thylakoid organization in cyanobacteria is not well understood. Here we present, by using electron tomography, an in-depth analysis of the architecture of the thylakoid membranes in a unicellular cyanobacterium, Cyanothece sp. ATCC 51142. Based on the results of three-dimensional tomographic reconstructions of near-entire cells, we determined that the thylakoids in Cyanothece 51142 form a dense and complex network that extends throughout the entire cell. This thylakoid membrane network is formed from the branching and splitting of membranes and encloses a single lumenal space. The entire thylakoid network spirals as a peripheral ring of membranes around the cell, an organization that has not previously been described in a cyanobacterium. Within the thylakoid membrane network are areas of quasi-helical arrangement with similarities to the thylakoid membrane system in chloroplasts. This cyanobacterial thylakoid arrangement is an efficient means of packing a large volume of membranes in the cell while optimizing intracellular transport and trafficking.     

Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture

Mouse embryonic stem (ES) cells are competent for production of all fetal and adult cell types. However, the utility of ES cells as a developmental model or as a source of defined cell populations for pharmaceutical screening or transplantation is compromised because their differentiation in vitro is poorly controlled. Specification of primary lineages is not understood and consequently differentiation protocols are empirical, yielding variable and heterogeneous outcomes. Here we report that neither multicellular aggregation nor coculture is necessary for ES cells to commit efficiently to a neural fate. In adherent monoculture, elimination of inductive signals for alternative fates is sufficient for ES cells to develop into neural precursors. This process is not a simple default pathway, however, but requires autocrine fibroblast growth factor (FGF). Using flow cytometry quantitation and recording of individual colonies, we establish that the bulk of ES cells undergo neural conversion. The neural precursors can be purified to homogeneity by fluorescence activated cell sorting (FACS) or drug selection. This system provides a platform for defining the molecular machinery of neural commitment and optimizing the efficiency of neuronal and glial cell production from pluripotent mammalian stem cells.     

Characterization of cyclic AMP-binding proteins in rat Sertoli cells using a photoaffinity ligand

Summary Protein-bound cyclic AMP (cAMP) levels in cultured rat Sertoli cells have been determined after exposure to follicle-stimulating hormone (FSH) and agents which elevate intracellular cAMP or mimic cAMP action. Changes in the content of protein-bound cAMP were correlated with changes in receptor availability determined by measuring [³H] cAMP binding. Using the photoaffinity analog of cAMP, 8-N₃ [³²P] cAMP, two major cAMP-binding proteins in Sertoli cell cytosol, with molecular weights of 47 000 and 53 000 daltons, were identified as regulatory subunits of type I and type II cAMP-dependent protein kinases, respectively. Densitometric analysis of autoradiograms demonstrated differential activation of the two isozymes in response to treatment with FSH and other agents. Results of this study demonstrate the value of measuring changes in protein-bound cAMP and the utility of the photoaffinity labeling technique in correlating hormone-dependent processes in which activation of cAMP-dependent protein kinase occurs.     

Genome-wide SNP typing reveals signatures of population history

Single-nucleotide polymorphism (SNP) arrays have become a popular technology for disease-association studies, but they also have potential for studying the genetic differentiation of human populations. Application of the Affymetrix GeneChip Human Mapping 500K Array Set to a population of 102 individuals representing the major ethnic groups in the United States (African, Asian, European, and Hispanic) revealed patterns of gene diversity and genetic distance that reflected population history. We analyzed allelic frequencies at 388,654 autosomal SNP sites that showed some variation in our study population and 10% or fewer missing values. Despite the small size (23-31 individuals) of each subpopulation, there were no fixed differences at any site between any two subpopulations. As expected from the African origin of modern humans, greater gene diversity was seen in Africans than in either Asians or Europeans, and the genetic distance between the Asian and the European populations was significantly lower than that between either of these two populations and Africans. Principal components analysis applied to a correlation matrix among individuals was able to separate completely the major continental groups of humans (Africans, Asians, and Europeans), while Hispanics overlapped all three of these groups. Genes containing two or more markers with extraordinarily high genetic distance between subpopulations were identified as candidate genes for health differences between subpopulations. The results show that, even with modest sample sizes, genome-wide SNP genotyping technologies have great promise for capturing signatures of gene frequency difference between human subpopulations, with applications in areas as diverse as forensics and the study of ethnic health disparities.     

Surprising dependence on postsegregational killing of host cells for maintenance of the large virulence plasmid of Shigella flexneri

Low-copy-number plasmids all encode multiple systems to ensure their propagation, including replication, partition (active segregation), and postsegregational killing (PSK) systems. PSK systems kill those rare cells that lose the plasmid due to replication or segregation errors. PSK systems should not be used as the principle means of maintaining the plasmid. The metabolic cost of killing the many cured cells that would arise from random plasmid segregation is far too high. Here we describe an interesting exception to this rule. Maintenance of the large virulence plasmid of Shigella flexneri is highly dependent on one of its PSK systems, mvp, at 37 degrees C, the temperature experienced during pathogenesis. At 37 degrees C, the plasmid is very unstable and mvp efficiently kills the resulting cured bacterial cells. This imposes a major growth disadvantage on the virulent bacterial population. The systems that normally ensure accurate plasmid replication and segregation are attenuated or overridden at 37 degrees C. At 30 degrees C, a temperature encountered by Shigella in the outside environment, the maintenance systems function normally and the plasmid is no longer dependent on mvp. We discuss why the virulent pathogen tolerates this self-destructive method of propagation at the temperature of infection.