Michelle S Teng et al. (2005) International Worm Meeting "Reprogramming olfactory behavioural response in C elegans"

Andy Fraser, John McCafferty, Nigel Carter, Bee Ling Ng, Michelle S Teng, Gert Jansen

[International Worm Meeting, 2005]

Odour detection in animals is achieved by virtue of a large family of Olfactory Receptors (OR) expressed in the olfactory epithelium. In humans, the repertoire of odorant receptor genes consists of 1000 genes, each of which encodes a G Protein Coupled Receptor or GPCR. About half of human ORs are pseudogenes. C elegans has 1000 putative OR genes, of which 30% are pseudogenes. Functional expression of ORs in C elegans provides a valuable and novel research tool to efficiently screen for olfactory ligands as well as to study receptor ligand interaction at mid-throughput using chemotaxis response as a quantitative measure. Rat I7 has been functionally expressed in AWA and AWB neurons, which generate attraction and avoidance responses respectively (Milani et al. 2002), resulting in a response to an altered response to octanal and other ligands. We have replicated and extended the initial published observations using a wider range of ligands. Using MoFlo, we have shown that it is possible to separate live transgenic worms co-expressing elt-2 GFP marker from the non-expressors as well as sort them at various stages of the life cycle based on size prior to using the adult animals for chemotaxis assay. We have also extended AWA/AWB targeted expression to another mammalian OR- hOR17-4, which has previously shown to be expressed in both olfactory epithelium and human spermatozoa where it appears to play a role in chemosensory signalling pathways and sperm chemotaxis (Parmentier et al. 1992; Vanderhaeghen et al. 1993). hOR17-4 has been shown to respond to bourgeonal and lilial in HEK293 cells expressing hOR17-4 and in in vitro sperm chemotaxis assays. We were able to reproduce this observation in a simple chemotaxis assay by expressing hOR17-4 driven by odr-10 promoter that targets expression in the AWA neuron. Our preliminary results show a dose dependent migration towards bourgeonal in these transgenic strains at similar concentrations to that published by Spehr et al. (2004).

Stewart HI et al. (1997) International C. elegans Meeting "Recessive Lethal Mutations and Deficiency Analysis and correlation of the genetic and physical maps, in Caenorhabditis elegans, on LGIII(left) and Balanced by sDp3 ."

Liao Y, Franz NW, Stewart HI, Materi WP, Janke DL, Baillie DL, Ha TT

[International C. elegans Meeting, 1997]

We have isolated 201, EMS induced, recessive lethal mutations on the left arm of LGIII. The free duplication sDp3 (III;f) was used to rescue recessive mutations linked to dpy-17 (recovery rate 1.9%). A subset, of 62 recessive lethals, are also marked with ncl-1 and will facilitate the mosaic analysis of these lethal mutations. So far, we have identified 78 new essential genes let-700 to let-792. Of these new essential genes, 24 have multiple alleles. Utilizing mutations in sDf125, to estimate satauration, (see abstract Nigel ONeil et al ,this meeting) we estimate that we have within this set, 123 of the essential genes in this area . As there are about 4.8 Mb of DNA, in the interval between unc-93 and dpy-19, we estimate that there will be approximately 150 essential genes, in this region. Both gamma rays and ultraviolet light were used to generate a set of overlapping deficiencies. We have shown that duplications can be used to rescue large deletions such as sDf121, sDf125, sDf127 and sDf130 , as well as smaller deficiencies. This indicates that there are no holes or lethal mutations along the length of sDp3 in the areas uncovered by these large deficiencies. We have shown that duplications, such as sDp3, are valid and useful as balancer systems and have utilized deficiency mapping to resolve the genetic mapping of essential genes. Selected visible mutations have also been complementation tested with our deficiencies. PCR analysis was utilized, with these deficiencies, to correlate the genetic and physical maps and to further define the end points of these deficiencies At this time, 11 of the essential genes have been rescued, (See abstracts; GregVatcher et al, Nigel ONeil et al ,this meeting), utilizing transgenic strains constructed in this laboratory (see abstract; Diana Janke et al, this meeting). This work has been funded by a grant from NIH (National institute of Health), U.S.A. to Ann. Rose; D. Riddle and D.L. Baillie and MRC of Canada to D.L. Baillie.

Watts JL et al. (1991) International C. elegans Meeting "ANALYSIS OF ZYG-11 PROTEIN IN EARLY EMBRYOS OF C. ELEGANS"

Kemphues KJ, Watts JL

[International C. elegans Meeting, 1991]

zyg-11 is a maternally acting gene which is required for embryogenesis in C. elegans (Kemphues et al., Dev. Biol. 113, 449-460, 1986). The zyg-l l gene product is necessary for the proper completion of meiosis as well as for certain cytoplasmic and nuclear events of the first cell cycle. The sequence of zyg-11 does not reveal any significant homologies to other EMBL-GenBank sequences (Carter et al., Mol Gen Genet (1990) 221:72-80). Even though zyg-l 1 mutations show a strict maternal effect, zyg-l 1 RNA is not germline specific, as it is expressed in bn2 worms which lack a germ line. We have raised polyclonal antibodies against a trpE- zyg -11 fusion protein. Our antibody recognizes the fusion protein as well as a prominent band of approximately 90kD in N2 worm and embryo preps, corresponding to the predicted size of the zyg-11 protein. Affinity purified antibody stains the nuclear envelope of early embryos in a cell cycle dependent fashion and also stains muscle in larvae and adult worms. We are currently analyzing the localization and abundance of zyg-l l protein in zyg-ll mutants and bn2 worms.

Balasubramaniyan M et al. (2015) Int J Biol Macromol "Immunopotentiating nano-chitosan as potent vaccine carter for efficacious prophylaxis of filarial antigens."

Santhanam M, Perumal K, Devasena T, Balasubramaniyan M

[Int J Biol Macromol, 2015]

Lymphatic filariasis (LF), a morbid vector-borne parasitic infection affects millions in tropical areas. Complete eradication can only be achieved by the development of a potent vaccine. Among the various filarial antigens that have been characterized, antigens Brugia malayi thioredoxin (TRX) and abundant larval transcript (ALT) have produced recognizable level of protection in Jirds, thereby evidenced to be good vaccine candidates. In this study an attempt was made to enhance their immunoprophylactic activity by encapsulating them in natural polysaccharide chitosan forming nanospheres (CN). High encapsulation efficiency for TRX (93%) in CN (TCN) and ALT-2 (90%) in CN (ACN) was achieved. Morphological studies confirmed the spherical and uniform distribution of nanospheres to be 220 nm. The electrostatic interaction between chitosan and the antigens were confirmed using differential scanning calorimetry and FT-IR. The study revealed the immunostimulatory property of chitosan providing enhanced level of proliferation for encapsulated antigens in peripheral blood mononuclear cells from endemic normal personals, at low concentration (TCN mean stimulation index (SI)=4.23+/-0.15 and ACN (SI)=4.05+/-0.33) compared to stimulation obtained by antigens alone. Hence, our study demonstrated that natural macromolecule derived CN can be used as efficacious immunostimulatory vaccine carter for LF thereby diminishing pathological sequel.

Tsai MC et al. (2007) J Cell Biol "Microtubules are involved in anterior-posterior axis formation in C. elegans embryos."

Tsai MC, Ahringer J

[J Cell Biol, 2007]

Microtubules deliver positional signals and are required for establishing polarity in many different organisms and cell types. In Caenorhabditis elegans embryos, posterior polarity is induced by an unknown centrosome-dependent signal. Whether microtubules are involved in this signaling process has been the subject of controversy. Although early studies supported such an involvement (O''Connell, K.F., K.N. Maxwell, and J.G. White. 2000. Dev. Biol. 222:55-70; Wallenfang, M.R., and G. Seydoux. 2000. Nature. 408:89-92; Hamill, D.R., A.F. Severson, J.C. Carter, and B. Bowerman. 2002. Dev. Cell. 3:673-684), recent work involving RNA interference knockdown of tubulin led to the conclusion that centrosomes induce polarity independently of microtubules (Cowan, C.R., and A.A. Hyman. 2004. Nature. 431:92-96; Sonneville, R., and P. Gonczy. 2004. Development. 131: 3527-3543). In this study, we investigate the consequences of tubulin knockdown on polarity signaling. We find that tubulin depletion delays polarity induction relative to wild type and that polarity only occurs when a small, late-growing microtubule aster is visible at the centrosome. We also show that the process of a normal meiosis produces a microtubule-dependent polarity signal and that the relative levels of anterior and posterior PAR (partitioning defective) polarity proteins influence the response to polarity signaling. Our results support a role for microtubules in the induction of embryonic polarity in C. elegans.

Stewart HI et al. (1996) West Coast Worm Meeting "Rescue of New Essential Genes on LGIII(left), in the Nematode Caenorhabditis elegans."

Stewart HI, Franz NW, Barbazuk BW, Baillie DL, Ha TT

[West Coast Worm Meeting, 1996]

We utilized trangenic strains containing cosmids, sequenced by the sequencing consortium, to rescue the lethal phenotype of several essential genes (See presentation by Diana Janke et al, this session). The rescued genes are positioned on LGIII(left) balanced by the duplication sDp3(see poster; Stewart et al, this session). A set of overlapping deficiencies was utilized to define areas within which to focus our rescue attempts). PCR analysis was utilized to further define the end points of these deficiencies, facilitating our analysis ( see poster ; Norm Franz et al, this session). We concentrated our analysis on areas to the right of dpy-17, in the interval between dpy-17 to dpy-19. Lethals mapping to sDf125 that were not rescued by sDp8 are presented in a poster by Nigel O'Niel et al, this session. We have rescued several essential genes. Our rescues will facilitate the functional analysis of these new genes. These rescues have also helped us to align and correlate the physical and genetic maps. Our estimates are that there will be more than two essential genes per cosmid. A total of 202 recessive lethal mutations have been generated, using EMS as a mutagen. We intend to make molecular assignments of all of these elements, through our cosmid rescue experiments. As we have estimated that there is only a 28% saturation of the area for essential genes, we will be generating more recessive lethal genes, to facilitate this analysis. This work has been funded by grants from the National Institute of Health (N.I.H.), U. S.A. to Ann Rose; D. Riddle and David Baillie and Canadian Genome and Advanced Technologies (C.G.A.T.), Canada to Ann Rose and David Baillie.

Jillian L. Youds et al. (2006) European Worm Meeting "The DOG-1 Helicase, Genomic Instability and Fanconi Anemia"

Ann M. Rose, Nigel J. O'Neil, Simon J. Boulton, Louise J. Barber, Jillian L. Youds

[European Worm Meeting, 2006]

Jillian L. Youds1, Nigel J. O''Neil1, Louise J. Barber2, Simon J. Boulton2 & Ann M. Rose1. In Caenorhabditis elegans, DOG-1 is required for the maintenance of polyG/polyC-tracts (G-tracts). In the absence of DOG-1, it is thought that G-tracts form secondary structures that block replication, leading to deletions that initiate in the G-tracts. Using our assay for deletions forming in the absence of DOG-1, we have assayed the in vivo contribution of various repair genes to the maintenance of these tracts. We show that DOG-1 and the BLM ortholog, HIM-6, act synergistically during replication; simultaneous loss of function of both genes results in replicative stress and an increase in the formation of small deletions that initiate in G-tracts. Similarly, we show that genes implicated in homologous recombinational repair and trans-lesion synthesis are required to prevent G-tract deletions in the dog-1 background. However, genes essential to the non-homologous end-joining and nucleotide excision repair pathways do not appear to be involved in deletion prevention or formation. By investigating the mechanisms that maintain genomic stability in dog-1 mutants, we might better understand the genomic instability associated with Fanconi anemia, as the human gene most similar to dog-1, BRIP1/FANCJ, was recently shown to be mutated in a subset of patients with Fanconi anemia, implicating it in interstrand cross-link (ICL) repair. In collaboration with the Boulton lab, we are currently investigating the possibility that DOG-1 and BRIP1 have functionally conserved roles in DNA repair. Our preliminary data indicate that dog-1 mutants are sensitive ICL-inducing agents and we are assessing the relationship to fcd-2 and other genes involved in ICL repair. . This research is funded by the Natural Sciences and Engineering Council and the Michael Smith Foundation for Health Research.

Pilgrim DB et al. (2000) West Coast Worm Meeting "Maternal UNC-45 protein co-localizes with NMY-2, a non-muscle myosin at the cleavage furrow of early embryos"

Pilgrim DB, Ao W

[West Coast Worm Meeting, 2000]

unc-45 is an essential gene for normal body wall muscle thick filament development and mutants show a partial maternal effect. The unc-45 protein product (UNC-45) contains tetratricopeptide (TPR) repeats and similarity to fungal proteins, but its biochemical function is still unknown. We have previously shown that UNC-45 is a component of muscle thick filaments and co-localizes with myosin heavy chain B but not myosin heavy chain A in the body wall muscles of adult worms[1]. Previous genetic evidence also suggests that UNC-45 may interact with myosin heavy chain isoforms in the muscle cells [2]. We show here that UNC-45 is also contributed maternally to the embryos and present in all cells of the early embryo. Zygotic UNC-45 is only detected in the developing muscle cells of the embryo. Moreover, our yeast two-hybrid screens show that UNC-45 interacts specifically with NMY-2, a non-muscle myosin. These two proteins are also co-localized at the cleavage furrow of the early embryos. The localization of UNC-45 at the cleavage furrow is dependent on the presence of NMY-2. NMY-2 has been previously shown to be required for embryonic polarity and cytokinesis [3,4]. Our results suggest that the maternal UNC-45 may have a function in the early embryo which is independent of muscle function. References 1. Ao, W., and Pilgrim, D. (2000). J. Cell Biol. 148, 375-384. 2. Venolia, L., and R.H. Waterston. (1990). Genetics. 126, 345-354. 3. Guo, S., and Kemphues, K. J. (1996). Nature, 382, 455-458. 4. Shelton, C. A., Carter, J. C., Ellis, G. C., and Bowerman, B. (1999). J. Cell Biol. 146, 439-451.

Schein JE et al. (1996) West Coast Worm Meeting "CONSTRUCTION AND UTILIZATION OF THE CHROMOSOME IV COSMID TRANSGENIC LIBRARY"

Schein JE, Rose AM, Baillie DL, Franz NW, Janke DL

[West Coast Worm Meeting, 1996]

Construction of a chromosome IV cosmid transgenic library of C. elegans strains carrying sequenced cosmids in extrachromosomal arrays has begun as part of our labs' collaboration with the Genome Sequencing Consortium (see abstract by D. Janke et al.). There are 20 chromosome IV cosmids available in transgenic strains at the time of this writing, and we estimate a total of 40-50 will be available to the community by the end of July. In order to enhance the utility of this library, we are also using these strains to localize approximately 50 essential gene mutations, previously identified in the Baillie lab, to cosmids by way of transgenic rescue experiments (for results of similar efforts on chromosomes III and V, see abstracts by Nigel O'Neil et al., Helen Stewart et al., and Brad Barbazuk et al.). Correlation of the genetic and physical maps in this manner will facilitate the selection of candidate cosmids for use in further rescue experiments, and thereby increase the speed with which the connection between genetic mutations and DNA sequence is made. Currently these rescue experiments are focused in the sDf2 region, in particular in the lin-3 - let-60 interval, which contains 13 essential gene mutations and 21 overlapping cosmids. The two genes bracketing this interval, lin-3 and let-60(ras), were previously placed on the physical map by others. To date, two additional essential genes have been positioned to cosmids on the physical map; let-70(ubc2) (in collaboration with Mei Zhen and Peter Candido) and let-64. It is anticipated that by the time of the meeting several additional mutations will have been positioned to cosmids. This work is being funded by a grant from the Canadian Genome and Associated Technologies Program to Ann Rose and David Baillie.

Brister JR et al. (1991) International C. elegans Meeting "MOLECULAR CHARACTERIZATION OF THE FER-15 REGION."

Brister JR, L'Hernault SW

[International C. elegans Meeting, 1991]

fer-15 interferes with the transition of sessile spermatids to active crawling spermatozoa both in vivo and in vitro. This gene has been three factor and deficiency mapped to a narrow interval of chromosome 11 near the emb-27 gene. (S. L'Hernault and S. Ward, unpublished observations). This region is in a large contig of cosmids and the placement of zyg-11, which is to the left of fer15, on the physcial map (Carter, P. and K. Kemphues Mol. Gen. Genet. 1990 227: 72) greatly aided our search. We have prepared heterozygous DNA from a number of deficiency and one putative lethal point allele that affect this region and probed Southerns of these DNA's with various cosmids ( courtesy of J. Sulston and A, Coulson) that span this interval. A number of deficiency breakpoints have been discovered, and DNA from at least part of the fer-15 gene has been identified by these Southern blot analyses. The cosmid C30D12 detected several deletion breakpoints that define the genetic location of fer-15. More precise localization of the deletion breakpoints around fer-15 occurred when we probed deficiency DNA's with various restriction fragments derived from C30D12. A 2.5 kb Pstl fragment hybridizes to deficiency breakpoint from mnDf98 (which breaks just outside fer-15) and mnDf101 (which breaks within fer-15). This fragment has been used to probe male vs female Northerns [male= RNA from Mog fem-3(q23gf); female= RNA from fem-1(hc171f)] and it detects a 2.6 kb RNA that is not sex specific. Either fer-15 encodes a rare message or its product is not sex limited in expression. Transgenic animals, constructed by microinjecting the cosmid C30D12 into fer-15 (hc89ts) animals, resulted in transient rescue of the sterile phenotype associated with this mutation.