Rosetta Brains: A Strategy for Molecularly-Annotated Connectomics

We propose a neural connectomics strategy called Fluorescent In-Situ Sequencing of Barcoded Individual Neuronal Connections (FISSEQ-BOINC), leveraging fluorescent in situ nucleic acid sequencing in fixed tissue (FISSEQ). FISSEQ-BOINC exhibits different properties from BOINC, which relies on bulk nucleic acid sequencing. FISSEQ-BOINC could become a scalable approach for mapping whole-mammalian-brain connectomes with rich molecular annotations

Distribution of label spacings for genome mapping in nanochannels

In genome mapping experiments, long DNA molecules are stretched by confining them to very narrow channels, so that the locations of sequence-specific fluorescent labels along the channel axis provide large-scale genomic information. It is difficult, however, to make the channels narrow enough so that the DNA molecule is fully stretched. In practice its conformations may form hairpins that change the spacings between internal segments of the DNA molecule, and thus the label locations along the channel axis. Here we describe a theory for the distribution of label spacings that explains the heavy tails observed in distributions of label spacings in genome mapping experiments.Comment: 18 pages, 4 figures, 1 tabl

Identifying a New Gene Required for microRNA-mediated Gene Silencing in \u3cem\u3eDrosophila melanogaster\u3c/em\u3e

Mature microRNAs (miRNA) are ~22 nucleotide long single-stranded ribonucleic acids essential for gene silencing. Silencing occurs when miRNAs are processed via endonucleolytic cleavage and subsequently associate with the miRNA-induced silencing complex (miRISC). miRISC binds via complementary base pairing to target mRNAs, and target mRNAs are silenced by either mRNA degradation, translational block, or both. Knowledge of all genes required for silencing is incomplete. We aim to determine the molecular mechanism of silencing by identifying and characterizing genes required for silencing. A forward genetic screen was performed using EMS mutagenesis of Drosophila melanogaster to generate mutant lines with disrupted gene silencing as visualized by a GFP-based fluorescent reporter of silencing. Locations of EMS-induced mutations are being mapped by determination of recombination frequencies between these mutations and molecularly defined P-element insertions. Preliminary recombination mapping reveals that our mutation of interest (I1-5) is found within a discrete region of the genome on chromosome 3R. A new fly line has also been generated to assist with this preliminary recombination mapping. Future deficiency mapping and complementation tests combining the mutation and alleles of candidate genes will reveal the location of our mutation, and lead us to identify a gene required for micro-RNA mediated gene silencing

Tracking Cell Signals in Fluorescent Images

In this paper we present the techniques for tracking cell signal in GFP (Green Fluorescent Protein) images of growing cell colonies. We use such tracking for both data extraction and dynamic modeling of intracellular processes. The techniques are based on optimization of energy functions, which simultaneously determines cell correspondences, while estimating the mapping functions. In addition to spatial mappings such as affine and Thin-Plate Spline mapping, the cell growth and cell division histories must be estimated as well. Different levels of joint optimization are discussed. The most unusual tracking feature addressed in this paper is the possibility of one-to-two correspondences caused by cell division. A novel extended softassign algorithm for solutions of one-to-many correspondences is detailed in this paper. The techniques are demonstrated on three sets of data: growing bacillus Subtillus and e-coli colonies and a developing plant shoot apical meristem. The techniques are currently used by biologists for data extraction and hypothesis formation

Fluorescence in situ hybridization of YAC clones after Alu-PCR amplification

Alu-PCR protocols were optimized for the generation of human DNA probes from yeast strains containing yeast artificial chromosomes (YACs) with human inserts between 100 and 800 kb in size. The resulting DNA probes were used in chromosome in situ suppression (CISS) hybridization experiments. Strong fluorescent signals on both chromatids indicated the localization of specific YAC clones, while two clearly distinguishable signals were observed in ≥90% of diploid nuclei Signal intensities were generally comparable to those observed using chromosome-specific alphoid DNA probes. This approach will facilitate the rapid mapping of YAC clones and their use in chromosome analysis at all stages of the cell cycle

Composite polymer membranes for laserinduced fluorescence thermometry

We demonstrate a modified version of laser-induced fluorescence thermometry (LIFT) for mapping temperature gradients in the vicinity of small photothermal devices. Our approach is based on temperature sensitive fluorescent membranes fabricated with rhodamine B and polydimethylsiloxane (PDMS). Relevant membrane features for LIFT, such as temperature sensitivity, thermal quenching and photobleaching are presented for a range of 25 °C to 90 °C, and their performance is evaluated upon obtaining the temperature gradients produced in the proximity of optical fiber micro-heaters. Our results show that temperature measurements in regions as small as 750 μm x 650 μm, with a temperature resolution of 1 °C, can be readily obtained

Fluorescent Surface Mapping of Mastodon Tusk

The subject of study is a Mammut americanum (American Mastodon) tusk. The tusk is currently damaged and in need of repair. To better understand the tusk and the preservation materials previously used, fluorescence spectrometry was used to make a surface map of the tusk. This data will be useful to the conservationists as they plan the care and restoration of the tusk.https://scholarworks.uni.edu/mastodon_posters/1007/thumbnail.jp

Fate mapping identifies the origin of SHF/AHF progenitors in the chick primitive streak

Heart development depends on the spatio-temporally regulated contribution of progenitor cells from the primary, secondary and anterior heart fields. Primary heart field (PHF) cells are first recruited to form a linear heart tube; later, they contribute to the inflow myocardium of the four-chambered heart. Subsequently cells from the secondary (SHF) and anterior heart fields (AHF) are added to the heart tube and contribute to both the inflow and outflow myocardium. In amniotes, progenitors of the linear heart tube have been mapped to the anterior-middle region of the early primitive streak. After ingression, these cells are located within bilateral heart fields in the lateral plate mesoderm. On the other hand SHF/AHF field progenitors are situated anterior to the linear heart tube, however, the origin and location of these progenitors prior to the development of the heart tube remains elusive. Thus, an unresolved question in the process of cardiac development is where SHF/AHF progenitors originate from during gastrulation and whether they come from a region in the primitive streak distinct from that which generates the PHF. To determine the origin and location of SHF/AHF progenitors we used vital dye injection and tissue grafting experiments to map the location and ingression site of outflow myocardium progenitors in early primitive streak stage chicken embryos. Cells giving rise to the AHF ingressed from a rostral region of the primitive streak, termed region ‘A’. During development these cells were located in the cranial paraxial mesoderm and in the pharyngeal mesoderm. Furthermore we identified region ‘B’, located posterior to ‘A’, which gave rise to progenitors that contributed to the primary heart tube and the outflow tract. Our studies identify two regions in the early primitive streak, one which generates cells of the AHF and a second from which cardiac progenitors of the PHF and SHF emerge.Esther Camp, Susanne Dietrich, Andrea Münsterber

2: The Mapping of Chromosome 16

Human chromosome 16 is the main focus of the mapping efforts at Los Alamos. The large photomicrograph on these opening pages illustrates the starting point for those mapping efforts, the evaluation of our chromosome-16-specific library of cloned fragments. Among the 23 pairs of human chromosomes, one pair, chromosome 16, is identified by fluorescence in-situ hybridization. Thousands of yellow fluorescent probes derived from the clone library have hybridized to both copies of chromosome 16. The high density and uniform coverage of the fluorescent signals were a strong indication that we could use the library to construct a map of overlapping cloned fragments spanning the entire length of the chromosome

A mutate-and-map protocol for inferring base pairs in structured RNA

Chemical mapping is a widespread technique for structural analysis of nucleic acids in which a molecule's reactivity to different probes is quantified at single-nucleotide resolution and used to constrain structural modeling. This experimental framework has been extensively revisited in the past decade with new strategies for high-throughput read-outs, chemical modification, and rapid data analysis. Recently, we have coupled the technique to high-throughput mutagenesis. Point mutations of a base-paired nucleotide can lead to exposure of not only that nucleotide but also its interaction partner. Carrying out the mutation and mapping for the entire system gives an experimental approximation of the molecules contact map. Here, we give our in-house protocol for this mutate-and-map strategy, based on 96-well capillary electrophoresis, and we provide practical tips on interpreting the data to infer nucleic acid structure.Comment: 22 pages, 5 figure