Roy PJ et al. (1995) International C. elegans Meeting "Characterization of Semaphorin I and II."

Roy PJ, Zheng H, Culotti JG

[International C. elegans Meeting, 1995]

Semaphorins are a family of proteins implicated in nerve guidance, found in a number of invertebrates and vertebrates, initially described by Kolodkin et al., Cell 75, 1389-1399. Through degenerate PCR, we have identified a C.elegans semaphorin I (Ce-SemaI) cDNA fragment. By splicing a clone we isolated from a cDNA library together with one obtained from the Coulson cDNA sequencing project, we constructed a full length cDNA (2.7kB) which corresponds to the indicated size on Northern blots. Y. Kohara then isolated Ce-SemaI independently and mapped it to YACS, y53C6 and y54E5. We probed overlapping cosmids defined by the two YACS with Ce-SemaI and identified the cosmids (F14B11 and K09A10) which contain the gene encoding Ce-SemaI. The Ce-SemaI gene physically maps within 80 kB to the left of unc-54 on the right arm of chromosome I. Restriction mapping and sequencing of the gene enabled us to construct two minigenes from the Ce-SemaI gene and cDNA, and several reporter constructs utilizing 3.8 kB of predicted promoter sequence, lac-Z and GFP. We are currently using a subclone which contains only the Ce-SemaI gene, and the two minigenes to try to rescue prospective mutants which lie just to the left of unc-54; i.e., let-49, unc-122, let-207 and let-206. Also, Y. Kohara's cDNA sequencing project recently yielded a clone which encodes a second C. elegans semaphorin which differs from Ce-SemaI. In combination with Kohara's clones, we have generated and used a 5'RACE product to construct a full length cDNA (2.4kB), which matches the predicted size indicated by Northern analysis.

Roy PJ et al. (2002) Nature "Chromosomal clustering of muscle-expressed genes in Caenorhabditis elegans."

Roy PJ, Kim SK, Stuart JM, Lund J

[Nature, 2002]

Chromosomes are divided into domains of open chromatin, where genes have the potential to be expressed, and domains of closed chromatin, where genes are not expressed1. Classic examples of open chromatin domains include puffs on polytene chromosomes in Drosophila and extended loops from lampbrush chromosomes2,3. If multiple genes were typically expressed together from a single open chromatin domain, the position of co-expressed genes along the chromosomes would appear clustered. To investigate whether co-expressed genes are clustered, we examined the chromosomal positions of the genes expressed in muscle of Caenorhabditis elegans at the first larval stage. Here we show that co-expressed genes in C. elegans are clustered in groups of 25 along the chromosomes, suggesting that expression from a chromatin domain can extend over several genes. These observations reveal a higher-order organization of the structure of the genome, in which the order of genes along the chromosome is correlated with their expression in specific tissues.

Roy PJ et al. (1995) Worm Breeder's Gazette "A semaphorin (aka fasciclin IV or collapsin) Gene in C. elegans."

Culotti JG, Roy PJ

[Worm Breeder's Gazette, 1995]

A semaphorin (aka fasciclin IV or collapsin) gene in C elegans Peter Roy and Joe Culotti Samuel Lunenfeld Research Institute of Mt. Sinai Hospital Toronto, Ontario MSG 1X5 Canada The Semaphorins (aka Fasciclin IV in Drosophila or Collapsin in chicken) are a group of related proteins that provide guidance cues for migrating axon growth cones. The chicken Semaphorin has been shown to induce growth cone collapse in vitro. We identified a semaphorin gene from C. elegans using degenerate oligonucleotides (based on known Semaphorin sequences, Kolodkin et al., Cell 7;: 1389-99, 1993) for RT-PCR. After trying several combinations of PCR primers and sequencing over 60 different PCR products, we found a single clone that encodes a real Semaphorin. After identifying genomic clones, we read (in the last WBG) that Y. Kohara's group had identified a fasciclin IV-related gene in the large cDNA sequencing project that they are carrying out. We have since obtained his cDNA from them and shown that it is the same semaphorin that we cloned. This cDNA hybridizes to a 2.7 kb mRNA on Northern blots and encodes a protein of at least 600 amino acids. Because of possible uncertainties in our sequence analysis we do not yet know if this semaphorin gene encodes a transmembrane or immunoglobulin domain, as is found in the Semaphorins of other species. The C. elegans semaphorin gene was mapped by Y. Kohara to cosmid F14B11 on LGI just left of unc-54. We have verified this location by sequencing subclones from this cosmid. We are currently attempting to determine the expression pattern of the C elegans semaphorin gene.

Roy PJ et al. (2000) West Coast Worm Meeting "Global patterns of expression patterns in muscle using mRNA-Tagging"

Roy PJ, Kim SK

[West Coast Worm Meeting, 2000]

Gene networks that control development must endow each unique cell with distinguishing properties. Microarray technology along with the completed C. elegans genome now makes it possible to know these gene networks in a comprehensive manner. Our microarrays contain PCR fragments representing nearly every predicted C. elegans gene. By hybridizing labeled cDNA probes from different samples to the microarray, one can simultaneously determine the expression differences between those two samples for every gene. A limitation of current microarray technology is that RNA is isolated from whole worms, in turn, making tissue specific expression patterns difficult to detect. We have developed a strategy to circumvent this problem of identifying the mRNA profiles in specific tissues or cells by using an approach we call "mRNA-tagging". To isolate cell-specific mRNA, we are using previously characterized promoters to drive the expression of epitope-tagged protein that binds mRNA in a non-biased fashion in a defined set of cells. By immunoprecipitating the tagged mRNA-binding protein from whole animal lysates, cell-specific mRNA is co-immunoprecipitated. The abundance of each mRNA species isolated from these distinct cells is then assayed using microarrays. To identify genes expressed in muscle, we are using the well-characterized promoter myo-3 (Okkema et al., Genetics 135, 385-404) to drive the expression of the epitope-tagged mRNA-binding protein in body wall muscle. To date, we have successfully and reproducibly co-immunoprecipitated muscle-specific RNA, as assayed through both northern and microarray analysis. Control germ line and neuronal RNA are not enriched. We are currently optimizing our mRNA-tagging protocol to identify gene expressed in muscle, and are also extending our studies to identify neuronal and hypodermal genes.

Roy PJ et al. (1997) International C. elegans Meeting "C.elegans Semaphorin II (els-2) Nulls are let, unc, dpy and Deformed."

Culotti JG, Warren CE, Zheng H, Roy PJ

[International C. elegans Meeting, 1997]

Many members of the semaphorin/collapsin family are required for proper neural development and survival; Drosophila semaphorin II is required for viability and can selectively inhibit proper neural synaptic arborization (Matthes et al., 1995, Cell. 81:631-639) . To further investigate the role of semaphorin during development we have cloned C. elegans semaphorin II (els-2) using a cDNA fragment isolated in Y. Kohara's lab. Reporter genes driven by els-2 promoter and enhancer fragments exhibit a complex embryonic expression pattern including alternating stripes of dorsal hypodermal cells. Neuronal expression is seen in many motorneurons, sensory neurons, and various ganglia. Muscle expression is also observed. An els-2 Tc1 insertion allele (ev573) was isolated from our transposon library. Two els-2 deficiency alleles (ev574 and ev575) were generated from ev573. Both ev574 and ev575 delete the first exon, the 5'UTR and possibly promoter and enhancer elements. Brood size for strains homozygous for ev574 and ev575 are very low. Survivors are uncoordinated, dumpy, and severely deformed, especially near the posterior. Phenotypic and genetic analysis is ongoing.

Roy PJ et al. (2005) Dev Cell "Network news: functional modules revealed during early embryogenesis in C. elegans."

Roy PJ, Morris Q

[Dev Cell, 2005]

The functional module is fast becoming the operational unit of the postgenomics era. A new report in Nature by Gunsalus and colleagues describes, using a multiply supported network, functional modules within early C. elegans embryos and identifies several new components of known molecular machines ().

Roy PJ et al. (2000) Development "mab-20 encodes Semaphorin-2a and is required to prevent ectopic cell contacts during epidermal morphogenesis in Caenorhabditis elegans."

Roy PJ, Zheng H, Culotti JG, Warren CE

[Development, 2000]

The Semaphorins are a family of secreted and transmembrane proteins known to elicit growth cone repulsion and collapse. We made and characterized a putative null mutant of the C. elegans gene semaphorin-2a (Ce-sema-2a). This mutant failed to complement mutants of mab-20 (Baird, S. E., Fitch, D. H., Kassem, I. A. A. and Emmons, S. W. (1991) Development 113, 515-526). In addition to low-frequency axon guidance errors, mab-20 mutants have unexpected defects in epidermal morphogenesis. Errant epidermal cell migrations affect epidermal enclosure of the embryo, body shape and sensory rays of the male tail. These phenotypic traits are explained by the formation of inappropriate contacts between cells of similar type and suggest that Ce-Sema-2a may normally prevent formation or stabilization of ectopic adhesive contacts between these cells.

Prathap N et al. (2023) Sci Rep "Prosopis juliflora hydrothermal synthesis of high fluorescent carbon dots and its antibacterial and bioimaging applications."

Karuppiah P, Prathap N, Venkatesan S, Shivakumar MS, Balla P, Ramasamy K, Periyasami G

[Sci Rep, 2023]

Carbon dots have stimulated the curiosity of biomedical researchers due to their unique properties, such as less toxicity and high biocompatibility. The synthesis of carbon dots for biomedical application is a core area in research. In the current research, an eco-friendly hydrothermal technique was employed to synthesize high fluorescent, plant-derived carbon dots from Prosopis juliflora leaves extract (PJ-CDs). The synthesized PJ-CDs were investigated by physicochemical evaluation instruments such as fluorescence spectroscopy, SEM, HR-TEM, EDX, XRD, FTIR, and UV-Vis. The UV-Vis absorption peaks obtained at 270 nm due to carbonyl functional groups shifts of n→π*. In addition, a quantum yield of 7.88 % is achieved. The synthesized PJ-CDs showing the presence of carious functional groups O-H, C-H, C=O, O-H, C-N and the obtained particles in spherical shape with an average size of 8 nm. The fluorescence PJ-CDs showed stability against various environmental factors such as a broad range of ionic strength and pH gradient. The antimicrobial activity of PJ-CDs was tested against a Staphylococcus aureus, and a Escherichia coli. The results suggest that the PJ-CDs could substantially inhibit the growth of Staphylococcus aureus. The findings also indicate that PJ-CDs are effective materials for bio-imaging in Caenorhabditis elegans and they can be also used for pharmaceutical applications.

Ginzburg VE et al. (2000) East Coast Worm Meeting "Semaphorin 1a and 1b mutants exhibit male tail morphological defects."

Ginzburg VE, Roy PJ, Culotti JG

[East Coast Worm Meeting, 2000]

The semaphorin protein family has been implicated in axon guidance and morphogenesis in vertebrates and invertebrates, as described by Kolodkin et al., Cell 75, 1389-1399, 1993. Analysis of the C.elegans genome has revealed three genes that encode semaphorins designated Sema-1a, Sema-1b and Sema-2a according to their predicted protein structure. Recently, P. Roy has isolated and characterized mutants of the secreted semaphorin Sema-2a and found it has mild axon guidance defects and epidermal cell migration defects (P. Roy et., al 2000). To understand what role transmembrane semaphorins play in the development of C.elegans we decided to utilize reverse genetics to isolate Sema-1a and Sema-1b mutants. With mutants of all three semaphorin genes, we can study their functions using classical genetics, and moreover we can look for downstream components by designing various enhancer and suppressor screens. First, we have constructed a deletion library of approximately 1.7 million haploid genome, using a standard mutagenesis protocol. We have designed several sets of nested PCR primers for Sema-1a and Sema-1b genes. Screening of the deletion library found several candidates for deletions in the genes and we isolated two alleles of Sema-1a (ev715, ev708) and one allele of Sema-1b (ev709). The alleles were sequenced and revealed that ev715 is a deletion of ~300bp that puts the next three exons out-of-frame, ev708 is a first intron deletion of ~800bp and ev709 is a deletion of two exons. We found that backcrossed versions of the mutants exhibit several abnormalities, which we are further characterizing. Ectopic male tail ray1 (R1) and swollen head are two common phenotypes for both genes. The penetrance of an ectopic R1 in ev715 is 41% (n=201), and in ev709 is 17% (n=170). The penetrance of this unique phenotype is enhanced in a Sema-1a, Sema-1b double mutant (ev715 ev709) to 91% (n=202). Currently, we are attempting to rescue these phenotypes with the cloned genes. We are also further characterizing single, double and triple semaphorin mutants. P>P>

Dixon SJ et al. (2005) Development "Muscle arm development in Caenorhabditis elegans."

Roy PJ, Dixon SJ

[Development, 2005]

In several types of animals, muscle cells use membrane extensions to contact motor axons during development. To better understand the process of membrane extension in muscle cells, we investigated the development of Caenorhabditis elegans muscle arms, which extend to motor axons and form the postsynaptic element of the neuromuscular junction. We found that muscle arm development is a highly regulated process: the number of muscle arms extended by each muscle, the shape of the muscle arms and the path taken by the muscle arms to reach the motor axons are largely stereotypical. We also investigated the role of several cytoskeletal components and regulators during arm development, and found that tropomyosin (LEV-11), the actin depolymerizing activity of ADF/cofilin (UNC-60B) and, surprisingly, myosin heavy chain B (UNC-54) are each required for muscle arm extension. This is the first evidence that UNC-54, which is found in thick filaments of sarcomeres, can also play a role in membrane extension. The muscle arm phenotypes produced when these genes are mutated support a ''two-phase'' model that distinguishes passive muscle arm development in embryogenesis from active muscle arm extension during larval development.