Genome Annotation for Pinkcreek, a C1 Subcluster Mycobacteriophage from New Orleans, Louisiana.

The mycobacteriophage Pinkcreek (C1 subcluster) was extracted from soil collected on the Dr. Norman C. Francis Parkway Bike Trail in New Orleans, Louisiana. It is a member of the family Myoviridae and infects Mycobacterium smegmatis mc2155. The Pinkcreek genome is 153,184 bp and contains 216 predicted protein-coding genes, 29 tRNAs, and 1 transfer-messenger RNA.

Complete Genome Sequence of Finnry, a Subcluster L3 Mycobacteriophage from Charleston, South Carolina.

Subcluster L3 bacteriophage Finnry was isolated from soil collected in Charleston, South Carolina, using Mycobacterium smegmatis mc2155 as a host. The genome of this temperate siphovirus is 75,632 bp long (130 predicted protein-coding genes, 9 tRNAs, and no transfer-messenger RNAs), and BLASTn alignment revealed 99.86% identity with the genome of L3 mycobacteriophage Samty.

Isolation and Annotation of the Genome Sequences of Bacteriophages InvictusManeo (Subcluster K5) and Netyap (Subcluster L2).

The mycobacteriophages InvictusManeo (K5 subcluster) and Netyap (L2 subcluster) were isolated from soils in Cullowhee Creek, Cullowhee, North Carolina. Both exhibit Siphoviridae morphology and infect Mycobacterium smegmatis mc2155. The InvictusManeo genome is 61,147 bp and contains 96 predicted protein-coding genes, whereas the Netyap genome is 76,366 bp with 131 predicted protein-coding genes.

Instructional Models for Course-Based Research Experience (CRE) Teaching.

The course-based research experience (CRE) with its documented educational benefits is increasingly being implemented in science, technology, engineering, and mathematics education. This article reports on a study that was done over a period of 3 years to explicate the instructional processes involved in teaching an undergraduate CRE. One hundred and two instructors from the established and large multi-institutional SEA-PHAGES program were surveyed for their understanding of the aims and practices of CRE teaching. This was followed by large-scale feedback sessions with the cohort of instructors at the annual SEA Faculty Meeting and subsequently with a small focus group of expert CRE instructors. Using a qualitative content analysis approach, the survey data were analyzed for the aims of inquiry instruction and pedagogical practices used to achieve these goals. The results characterize CRE inquiry teaching as involving three instructional models: 1) being a scientist and generating data; 2) teaching procedural knowledge; and 3) fostering project ownership. Each of these models is explicated and visualized in terms of the specific pedagogical practices and their relationships. The models present a complex picture of the ways in which CRE instruction is conducted on a daily basis and can inform instructors and institutions new to CRE teaching.

Complete Genome Sequence of the Cluster B4 Mycobacteriophage Lolalove, Isolated in Charleston, South Carolina.

Lolalove, a B4 subcluster soil bacteriophage of Mycobacterium smegmatis, was isolated in Charleston, SC. It possesses a 71,111-bp linear double-stranded DNA (dsDNA) genome with 99 protein-coding genes and a GC content of 68.9%. genome BLASTn alignments indicate high sequence identity to the related B4 subcluster M. smegmatis phages BrownCNA, Mithril, and Hangman.

Evaluation of Genome Sequences of the Bacteriophages JeTaime and Luna22.

The mycobacteriophages JeTaime (E cluster) and Luna22 (Q cluster) were isolated from soil in Providence, Rhode Island, and Charleston, South Carolina, respectively, using a Mycobacterium smegmatis mc2 155 host. The genome of JeTaime is 75,099 bp (142 predicted genes), and that of Luna22 is 53,730 bp (87 predicted genes). Both phages exhibit Siphoviridae morphology.

An ancient role for nuclear beta-catenin in the evolution of axial polarity and germ layer segregation.

The human oncogene beta-catenin is a bifunctional protein with critical roles in both cell adhesion and transcriptional regulation in the Wnt pathway. Wnt/beta-catenin signalling has been implicated in developmental processes as diverse as elaboration of embryonic polarity, formation of germ layers, neural patterning, spindle orientation and gap junction communication, but the ancestral function of beta-catenin remains unclear. In many animal embryos, activation of beta-catenin signalling occurs in blastomeres that mark the site of gastrulation and endomesoderm formation, raising the possibility that asymmetric activation of beta-catenin signalling specified embryonic polarity and segregated germ layers in the common ancestor of bilaterally symmetrical animals. To test whether nuclear translocation of beta-catenin is involved in axial identity and/or germ layer formation in 'pre-bilaterians', we examined the in vivo distribution, stability and function of beta-catenin protein in embryos of the sea anemone Nematostella vectensis (Cnidaria, Anthozoa). Here we show that N. vectensis beta-catenin is differentially stabilized along the oral-aboral axis, translocated into nuclei in cells at the site of gastrulation and used to specify entoderm, indicating an evolutionarily ancient role for this protein in early pattern formation.

Complete Genome Sequence of the Cluster F1 Mycobacteriophage KingMidas.

Subcluster F1 bacteriophage KingMidas was isolated from soil collected in Providence, Rhode Island, using Mycobacterium smegmatis mc2155 as the host. The genome is 57,386 bp and contains 105 predicted protein-coding genes but no transfer-messenger RNAs or tRNAs. This siphovirus has an icosahedral head, with a genome 99.1% identical to that of F1 mycobacteriophage Scottish.

Genome Sequences of the Mycobacteriophages Awesomesauce and LastJedi.

Bacteriophages Awesomesauce and LastJedi infect Mycobacterium smegmatis mc2155. While the Awesomesauce genome is 57,054 bp with 94 protein-coding genes, the LastJedi genome is 55,149 bp with 94 protein-coding genes. Nucleotide sequence comparison in Phamerator detected synteny between Awesomesauce gp49 to gp61 and singleton LilSpotty. Whole-genome BLASTn alignments revealed that LastJedi strongly resembles Clifton (99.41% identity).

Restricted expression of karyopherin alpha mRNA in the sea urchin suggests a role in neurogenesis.

Karyopherin alpha (KAP-α) proteins are critical for the transport of many molecules into the nucleus. In this study, we identified three members of the KAP-α family in the sea urchin Lytechinus variegatus and described the developmental expression of these proteins. Although many importins are assumed to have ubiquitous expression, we found that all three genes were differentially expressed. Both LvKPNA1/5/6 and LvKPNA3/4 accumulated at high levels during cleavage, exhibiting cyclic expression as cells divided. By the blastula and gastrula stages expression decreased, remaining highest in the vegetal plate and archenteron, and by the prism/pluteus stages expression was restricted to the oral surface and gut. Expression of a third KAP-α gene, LvKPNA2/7, was examined in embryos from the mesenchyme blastula to pluteus stages. LvKPNA2/7 mRNA is present in vegetal cells of the mesenchyme blastula and, during gastrulation, it is localized to the archenteron and appears in additional groups of ectodermal cells. Prism/pluteus stage embryos expressed LvKPNA2/7 in the gut and scattered distribution of transcripts in the ciliary band resembled expression patterns of neural cells. We hypothesize that LvKPNA2/7 maintains pluripotency in the neural precursors prior to activation of neural differentiation and believe that this study is an important first step in an effort to better understand the roles of importins during embryogenesis.

Nuclearization of ß-catenin in ectodermal precursors confers organizer-like ability to induce endomesoderm and pattern a pluteus larva.

BACKGROUND: In many bilaterians, asymmetric activation of canonical Wnt (cWnt) signaling at the posterior pole is critical for anterior-posterior (AP) body axis formation. In 16-cell stage sea urchins, nuclearization of ß-catenin in micromeres activates a gene regulatory network that defines body axes and induces endomesoderm. Transplanting micromeres to the animal pole of a host embryo induces ectopic endomesoderm in the mesomeres (ectoderm precursors) whereas inhibiting cWnt signaling blocks their endomesoderm-inducing activity and the micromeres become ectoderm-like. We have tested whether ectopic activation of cWnt signaling in mesomeres is sufficient to impart the cells with organizer-like abilities, allowing them to pattern normal embryonic body axes when recombined with a field of mesomeres. RESULTS: Fertilized eggs were microinjected with constitutively active Xenopus ß-catenin (actß-cat) mRNA and allowed to develop until the 16-cell stage. Two mesomeres from injected embryos were then recombined with isolated animal halves (AH) from uninjected 16-cell stage embryos. Control chimeras produced animalized phenotypes (hollow balls of ectoderm) and rarely formed skeletogenic mesoderm (SM)-derived spicules, endoderm or pigment cells, a type of non-skeletogenic mesoderm (NSM). In contrast, over half of the 0.5 pg/pL actß-cat mesomere/AH chimeras formed a partial or complete gut (exhibiting AP polarity), contained mesenchyme-like cells similar to SM, and produced pigment cells. At three days, chimeras formed plutei with normal embryonic body axes. When fates of the actß-cat mRNA-injected mesomeres were tracked, we found that injected mesomeres formed mesenchyme-like and pigment cells, but endoderm was induced. Higher concentrations of actß-cat mRNA were less likely to induce endoderm or pigment cells, but had similar mesenchyme-like cell production to 0.5 pg/pL actß-cat mesomere/AH chimeras. CONCLUSIONS: Our results show that nuclear ß-catenin is sufficient to endow naïve cells with the ability to act as an organizing center and that ß-catenin has both cell-autonomous and non-autonomous effects on cell fate specification in a concentration-dependent manner. These results are consistent with the hypothesis that a shift in the site of early cWnt signaling in cleaving embryos could have modified polarity of the main body axes during metazoan evolution.

A gathering of minds: expanding understanding of the origins of biological diversity and the evolution of developmental mechanisms.

This paper is a short report on the 2012 Society of Integrative and Comparative Biology Annual Meeting. Charleston, South Carolina, USA. 3-7 January 2012 (abstracts freely available at http://www.sicb.org/meetings/2012/).

Characterization of circadian behavior in the starlet sea anemone, Nematostella vectensis.

BACKGROUND: Although much is known about how circadian systems control daily cycles in the physiology and behavior of Drosophila and several vertebrate models, marine invertebrates have often been overlooked in circadian rhythms research. This study focuses on the starlet sea anemone, Nematostella vectensis, a species that has received increasing attention within the scientific community for its potential as a model research organism. The recently sequenced genome of N. vectensis makes it an especially attractive model for exploring the molecular evolution of circadian behavior. Critical behavioral data needed to correlate gene expression patterns to specific behaviors are currently lacking in N. vectensis. METHODOLOGY/PRINCIPAL FINDINGS: To detect the presence of behavioral oscillations in N. vectensis, locomotor activity was evaluated using an automated system in an environmentally controlled chamber. Animals exposed to a 24 hr photoperiod (12 hr light: 12 hr dark) exhibited locomotor behavior that was both rhythmic and predominantly nocturnal. The activity peak occurred in the early half of the night with a 2-fold increase in locomotion. Upon transfer to constant lighting conditions (constant light or constant dark), an approximately 24 hr rhythm persisted in most animals, suggesting that the rhythm is controlled by an endogenous circadian mechanism. Fourier analysis revealed the presence of multiple peaks in some animals suggesting additional rhythmic components could be present. In particular, an approximately 12 hr oscillation was often observed. The nocturnal increase in generalized locomotion corresponded to a 24 hr oscillation in animal elongation. CONCLUSIONS/SIGNIFICANCE: These data confirm the presence of a light-entrainable circadian clock in Nematostella vectensis. Additional components observed in some individuals indicate that an endogenous clock of approximately 12 hr frequency may also be present. By describing rhythmic locomotor behavior in N. vectensis, we have made important progress in developing the sea anemone as a model organism for circadian rhythm research.

Blocking Dishevelled signaling in the noncanonical Wnt pathway in sea urchins disrupts endoderm formation and spiculogenesis, but not secondary mesoderm formation.

Dishevelled (Dsh) is a phosphoprotein key to beta-catenin dependent (canonical) and beta-catenin independent (noncanonical) Wnt signaling. Whereas canonical Wnt signaling has been intensively studied in sea urchin development, little is known about other Wnt pathways. To examine roles of these beta-catenin independent pathways in embryogenesis, we used Dsh-DEP, a deletion construct blocking planar cell polarity (PCP) and Wnt/Ca(2+) signaling. Embryos overexpressing Dsh-DEP failed to gastrulate or undergo skeletogenesis, but produced pigment cells. Although early mesodermal gene expression was largely unperturbed, embryos exhibited reduced expression of genes regulating endoderm specification and differentiation. Overexpressing activated beta-catenin failed to rescue Dsh-DEP embryos, indicating that Dsh-DEP blocks endoderm formation downstream of initial canonical Wnt signaling. Because Dsh-DEP-like constructs block PCP signaling in other metazoans, and disrupting RhoA or Fz 5/8 in echinoids blocks subsets of the Dsh-DEP phenotypes, our data suggest that noncanonical Wnt signaling is crucial for sea urchin endoderm formation and skeletogenesis.

Differential stability of beta-catenin along the animal-vegetal axis of the sea urchin embryo mediated by dishevelled.

beta-Catenin has a central role in the early axial patterning of metazoan embryos. In the sea urchin, beta-catenin accumulates in the nuclei of vegetal blastomeres and controls endomesoderm specification. Here, we use in-vivo measurements of the half-life of fluorescently tagged beta-catenin in specific blastomeres to demonstrate a gradient in beta-catenin stability along the animal-vegetal axis during early cleavage. This gradient is dependent on GSK3beta-mediated phosphorylation of beta-catenin. Calculations show that the difference in beta-catenin half-life at the animal and vegetal poles of the early embryo is sufficient to produce a difference of more than 100-fold in levels of the protein in less than 2 hours. We show that dishevelled (Dsh), a key signaling protein, is required for the stabilization of beta-catenin in vegetal cells and provide evidence that Dsh undergoes a local activation in the vegetal region of the embryo. Finally, we report that GFP-tagged Dsh is targeted specifically to the vegetal cortex of the fertilized egg. During cleavage, Dsh-GFP is partitioned predominantly into vegetal blastomeres. An extensive mutational analysis of Dsh identifies several regions of the protein that are required for vegetal cortical targeting, including a phospholipid-binding motif near the N-terminus.