Subcluster L3 bacteriophage Finnry was isolated from soil collected in Charleston, South Carolina, using Mycobacterium smegmatis mc(2)155 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.

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 mc(2) 155. The Pinkcreek genome is 153,184 bp and contains 216 predicted protein-coding genes, 29 tRNAs, and 1 transfer-messenger RNA.

Subcluster F1 bacteriophage KingMidas was isolated from soil collected in Providence, Rhode Island, using Mycobacterium smegmatis mc(2)155 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.

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for protein synthesis by translating codons in mRNA into amino acid sequences. tRNAs were long considered as house-keeping molecules that lacked regulatory functions. However, a growing body of evidence indicates that cellular tRNA levels fluctuate in correspondence to varying conditions such as cell type, environment, and stress. The fluctuation of tRNA expression directly influences gene translation, favoring or repressing the expression of particular proteins. Ultimately comprehending the dynamic of protein synthesis requires the development of methods able to deliver high-quality tRNA profiles. The method that we present here is named SPOt, which stands for Streamlined Platform for Observing tRNA. SPOt consists of three steps starting with metabolic labeling of cell cultures with radioactive orthophosphate, followed by guanidinium thiocyanate-phenol-chloroform extraction of radioactive total RNAs and finally hybridization on in-house printed macroarrays. tRNA levels are estimated by quantifying the radioactivity intensities at each probe spot. In the protocol presented here we profile tRNAs in Mycobacterium smegmatis mc(2)155, a nonpathogenic bacterium often used as a model organism to study tuberculosis.

Snapping shrimps (Alpheus spp.) exhibit extreme asymmetry of the chelae, presenting a large snapper and a small pincer, which are used for different behaviors. Like most crustaceans, snapping shrimps are able to autotomize, or drop, limbs when threatened. Although some limbs can be regenerated following autotomy, when the snapper is autotomized, the shrimp undergoes claw reversal, growing a new pincer at the site of the autotomized snapper and transforming the previous pincer into a new snapper over a series of molts. Although autotomy is costly in terms of loss of function and limb re-growth, failing to autotomize a limb could be lethal. We document the breakage plane of Alpheus angulosus (McClure, 2002) and examined variation in "drop latency," the latency to autotomize the snapper claw in response to a standardized physical threat. We measured drop latency of mature snapper claws (i.e. with morphological features typical of adult snappers not recently autotomized) in a large cohort of the shrimp (N = 93). We found autotomy to be dependent on threat intensity rather than threat duration among all shrimp. Specifically, autotomy occurrences were distributed in clusters around threat intensity changes, rather than normally distributed throughout threat time. We also found significantly longer drop latency in males than females, suggesting a greater cost of mature snapper autotomy in males (P = 0.004). After initial snapper autotomy, we also measured latency to autotomize the transforming claw based on molt stage. Transforming claws exhibited significantly shorter average drop latency than autotomy of mature snappers (P = 0.008). Our assessment of the behaviors of the autotomy of mature and transforming claws provides insight into the costs of autotomy relative to claw value, as well as the evolutionary costs of autotomy and claw transformation in snapping shrimps.

The snapping shrimp, Alpheus angulosus McClure, 2002, is a small crustacean with bilaterally asymmetric claws that serve distinct behavioural and sensory functions. If the large claw is lost, the organism switches handedness, transforming its small pincer claw into a large snapping claw while simultaneously developing a small claw on the contralateral side. To better understand the mechanisms required to adapt to this radical change in body composition, we examined developmental plasticity by tracing changes in sensory setae distribution on the claws throughout transformation. We observed only two broad types of setae, simple and plumose. Quantitative analysis across molt stages revealed significant alterations in setae composition and numbers that occurred primarily on the edge of the propodus, where the most drastic morphological changes also occur. These results suggest that previous developmental mechanisms are re-engaged to support the proliferation and differentiation of new setae during transformation.

Fruit flies of the genus Drosophila have been an attractive and effective genetic model organism since Thomas Hunt Morgan and colleagues made seminal discoveries with them a century ago. Work with Drosophila has enabled dramatic advances in cell and developmental biology, neurobiology and behavior, molecular biology, evolutionary and population genetics, and other fields. With more tissue types and observable behaviors than in other short-generation model organisms, and with vast genome data available for many species within the genus, the fly’s tractable complexity will continue to enable exciting opportunities to explore mechanisms of complex developmental programs, behaviors, and broader evolutionary questions. This primer describes the organism’s natural history, the features of sequenced genomes within the genus, the wide range of available genetic tools and online resources, the types of biological questions Drosophila can help address, and historical milestones.

Fruit flies of the genus Drosophila have been an attractive and effective genetic model organism since Thomas Hunt Morgan and colleagues made seminal discoveries with them a century ago. Work with Drosophila has enabled dramatic advances in cell and developmental biology, neurobiology and behavior, molecular biology, evolutionary and population genetics, and other fields. With more tissue types and observable behaviors than in other short-generation model organisms, and with vast genome data available for many species within the genus, the fly’s tractable complexity will continue to enable exciting opportunities to explore mechanisms of complex developmental programs, behaviors, and broader evolutionary questions. This primer describes the organism’s natural history, the features of sequenced genomes within the genus, the wide range of available genetic tools and online resources, the types of biological questions Drosophila can help address, and historical milestones.

Wolfram syndrome (WFS) is a progressive neurodegenerative disease characterized by diabetes insipidus, diabetes mellitus, optic atrophy, and deafness. WFS1 and WFS2 are caused by recessive mutations in the genes Wolfram Syndrome 1 (WFS1) and CDGSH iron sulfur domain 2 (CISD2), respectively. To explore the function of CISD2, we performed genetic studies in flies with altered expression of its Drosophila orthologue, cisd2. Surprisingly, flies with strong ubiquitous RNAi-mediated knockdown of cisd2 had no obvious signs of altered life span, stress resistance, locomotor behavior or several other phenotypes. We subsequently found in a targeted genetic screen, however, that altered function of cisd2 modified the effects of overexpressing the fly orthologues of two lysosomal storage disease genes, palmitoyl-protein thioesterase 1 (PPT1 in humans, Ppt1 in flies) and ceroid-lipofuscinosis, neuronal 3 (CLN3 in humans, cln3 in flies), on eye morphology in flies. We also found that cln3 modified the effects of overexpressing Ppt1 in the eye and that overexpression of cln3 interacted with a loss of function mutation in cisd2 to disrupt locomotor ability in flies. Follow-up multi-species bioinformatic analyses suggested that a gene network centered on CISD2, PPT1 and CLN3 might impact disease through altered carbohydrate metabolism, protein folding and endopeptidase activity. Human genetic studies indicated that copy number variants (duplications and deletions) including CLN3, and possibly another gene in the CISD2/PPT1/CLN3 network, are over-represented in individuals with developmental delay. Our studies indicate that cisd2, Ppt1 and cln3 function in concert in flies, suggesting that CISD2, PPT1 and CLN3 might also function coordinately in humans. Further, our studies raise the possibility that WFS2 and some lysosomal storage disorders might be influenced by common mechanisms and that the underlying genes might have previously unappreciated effects on developmental delay.

The ability to regenerate lost tissues, organs or whole body parts is widespread across animal taxa; in some animals, regeneration includes transforming a remaining structure to replace the one that was lost. The transformation of one limb into another involves considerable plasticity in morphology, physiology and behavior, and snapping shrimp offer excellent opportunities for studying this process. We examined the changes required for the transformation of the small pincer to a mature snapping claw in Alpheus angulosus. First molt claws differ from mature claws in overall shape as well as in morphology related to snapping function; nonetheless, shrimp with first molt claws do produce snaps. While most shape variables of second molt claws do not differ significantly from mature claws, the plunger (structure required for snap production) does not reach mature size until the third molt for females, or later for males. Thus, the pincer claw can be transformed into a functional snapping claw in one molt, although both the underlying morphology and superficial shape are not fully regenerated at this stage. The rapid production of a functional snapping claw that we observe in this study suggests that this particular function is of significant importance to snapping shrimp behavior and survival.

Infantile-onset neuronal ceroid lipofuscinosis (INCL) is a severe pediatric neurodegenerative disorder produced by mutations in the gene encoding palmitoyl-protein thioesterase 1 (Ppt1). This enzyme is responsible for the removal of a palmitate group from its substrate proteins, which may include presynaptic proteins like SNAP-25, cysteine string protein (CSP), dynamin, and synaptotagmin. The fruit fly, Drosophila melanogaster, has been a powerful model system for studying the functions of these proteins and the molecular basis of neurological disorders like the NCLs. Genetic modifier screens and tracer uptake studies in Ppt1 mutant larval garland cells have suggested that Ppt1 plays a role in endocytic trafficking. We have extended this analysis to examine the involvement of Ppt1 in synaptic function at the Drosophila larval neuromuscular junction (NMJ). Mutations in Ppt1 genetically interact with temperature sensitive mutations in the Drosophila dynamin gene shibire, accelerating the paralytic behavior of shibire mutants at 27 degrees C. Electrophysiological work in NMJs of Ppt1-deficient larvae has revealed an increase in miniature excitatory junctional potentials (EJPs) and a significant depression of evoked EJPs in response to repetitive (10 hz) stimulation. Endocytosis was further examined in Ppt1-mutant larvae using FM1-43 uptake assays, demonstrating a significant decrease in FM1-43 uptake at the mutant NMJs. Finally, Ppt1-deficient and Ppt1 point mutant larvae display defects in locomotion that are consistent with alterations in synaptic function. Taken together, our genetic, cellular, and electrophysiological analyses suggest a direct role for Ppt1 in synaptic vesicle exo- and endocytosis at motor nerve terminals of the Drosophila NMJ.

Alpheus angulosus McClure, 2002 is one of several species of snapping shrimp that live along the east coast of the United States and belong to the edwardsii group of Alpheus. The genus Alpheus presents with bilateral asymmetry in their chelipeds, specifically a large snapper and a smaller pincer. This is an extreme example of the asymmetry found in many other crustaceans. A significant amount of work has been done on the adult behavior, physiology, and transformation/regeneration of the two claws, but less is known about the early development of the nervous system that underlies this asymmetry. The work reported here begins to establish an atlas of embryonic development in this species staged by using both eye index and percent development connected to yolk depletion during embryogenesis. This represents the first step toward a more comprehensive understanding of embryonic development that can be used to address future neuro-developmental questions regarding limb asymmetry and plasticity.

Although powerful bioinformatics tools are available for free on the web and are used by neuroscience professionals on a daily basis, neuroscience students are largely ignorant of them. This Neuroinformatics module weaves together several bioinformatics tools to make a comprehensive unit. This unit encompasses quantifying a phenotype through a Quantitative Trait Locus (QTL) analysis, which links phenotype to loci on chromosomes that likely had an impact on the phenotype. Students then are able to sift through a list of genes in the region(s) of the chromosome identified by the QTL analysis and find a candidate gene that has relatively high expression in the brain region of interest. Once such a candidate gene is identified, students can find out more information about the gene, including the cells/layers in which it is expressed, the sequence of the gene, and an article about the gene. All of the resources employed are available at no cost via the internet. Didactic elements of this instructional module include genetics, neuroanatomy, Quantitative Trait Locus analysis, molecular techniques in neuroscience, and statistics-including multiple regression, ANOVA, and a bootstrap technique. This module was presented at the Faculty for Undergraduate Neuroscience (FUN) 2011 Workshop at Pomona College and can be accessed at http://mdcune.psych.ucla.edu/modules/bioinformatics.

Collaborative and international scientific efforts continue to be of increasing importance in the development of successful educational and research programs. The goal of our study abroad program, Neuroscience Seminar in Germany, is to bring this fact to light for undergraduates and make them aware of the global opportunities that exist in the neurosciences and related biological sciences. Here we discuss our experience of conducting a four-week summer study abroad course in collaboration with two universities associated with the German Graduate Schools of Neuroscience: Munich Center for Neurosciences - Ludwig-Maximilians-Universität (MCN-LMU) and Charité - Universitätsmedizin, Berlin (a joint institution of the Freie Universität and the Humboldt-Universität). This course combined the historical foundations of neuroscience in Germany with current research programs at these two prominent German research universities. Two weeks were spent at each location and faculty members from the participating universities provided seminars, laboratory exercises, demonstrations and tours. Students were presented with background reading and lecture material prior to the seminars and activities. Additionally, they were responsible for leading seminar-style class discussions through brief presentations and submitting written critical analyses of primary research papers associated with the laboratory exercises. These assignments provided a means to assess learning outcomes, coupled with course evaluations. Overall, this experience may serve as a template for those interested in study abroad course development and research opportunities in the neurosciences.

Infantile-onset Neuronal Ceroid Lipofuscinosis (INCL) is a severe pediatric neurodegenerative disorder produced by mutations in the gene encoding palmitoyl-protein thioesterase 1 (Ppt1). This enzyme is responsible for the removal of a palmitate post-translational modification from an unknown set of substrate proteins. To better understand the function of Ppt1 in neurons, we performed an unbiased dominant loss-of-function genetic modifier screen in Drosophila using a previously characterized Ppt1 gain-of-function system. The enhancers and suppressors identified in our screen make novel connections between Ppt1 and genes involved in cellular trafficking and the modulation of synaptic growth. We further support the relevance of our screen by demonstrating that Garland cells from Ppt1 loss-of-function mutants have defects in endocytic trafficking. Endocytic tracer uptake and ultrastructural analysis of these non-neuronal cells points to Ppt1 playing a role in modulating the early stages of vesicle formation. This work lays the groundwork for further experimental exploration of these processes to better understand their contributions to the INCL disease process.

The Internet is enhancing and challenging traditional approaches to teaching undergraduate neuroscience. In addition to the new FUN-supported development of a Society for Neuroscience Portal for higher education, there is a wealth of available teaching resources currently housed on the web. This article discusses the current state of digital libraries and introduces a series of exemplary web-based classroom resources.

Palmitoylation is the post-translational addition of a palmitate moiety to a cysteine residue through a covalent thioester bond. The addition and removal of this modification is controlled by both palmitoyl acyl-transferases and thioesterases. Using bioinformatic analysis, we identified 22 DHHC family palmitoyl acyl-transferase homologs in the Drosophila genome. We used in situ hybridization, RT-PCR, and published FlyAtlas microarray data to characterize the expression patterns of all 22 fly homologs. Our results indicate that all are expressed genes, but several, including CG1407, CG4676, CG5620, CG6017/dHIP14, CG6618, CG6627, and CG17257 appear to be enriched in neural tissues suggesting that they are important for neural function. Furthermore, we have found that several may be expressed in a sex-specific manner with adult male-specific expression of CG4483 and CG17195. Using tagged versions of the DHHC genes, we demonstrate that fly DHHC proteins are primarily located in either the Golgi Apparatus or Endoplasmic Reticulum in S2 cells, except for CG1407, which was found on the plasma membrane. We also characterized the subcellular localization and expression of the three known thioesterases: Palmitoyl-protein Thioesterase 1 (Ppt1), Palmitoyl-protein Thioesterase 2 (Ppt2), and Acyl-protein Thioesterase 1 (APT1). Our results indicate that Ppt1 and Ppt2 are the major lysosomal thioesterases while APT1 is the likely cytoplasmic thioesterase. Finally, in vivo rescue experiments show that Ppt2 expression cannot rescue the neural inclusion phenotypes associated with loss of Ppt1, further supporting distinct functions and substrates for these two thioesterases. These results will serve as the basis for a more complete understanding of the protein palmitoylome’s normal cellular functions in the fly and will lead to further insights into the molecular etiology of diseases associated with the mis-regulation of palmitoylation.

(2007). We Hold These Truths to be Self-evident, that All Flies and Men Are Created Equal: Recent Progress on Human Disease Models. Fly: Vol. 1, No. 2, pp. 118-122.