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[
International Worm Meeting,
2011]
Dosage compensation is a mechanism that equalizes gene expression from the X chromosomes between heterogametic sexes. In Caenorhabditis elegans, the dosage compensation complex (DCC) binds both hermaphrodite X chromosomes to repress transcription by approximately half, to equal the level expression from the single male X. Although C. elegans and C. briggsae diverged 15-30 million years ago, our analysis has shown that dosage compensation complex (DCC) subunits are conserved between species. Each C. elegans DCC component has a homolog in C. briggsae, and the DCC components DPY-27, MIX-1, and SDC-2 have been shown to have similar functions in C. briggsae dosage compensation. However, while DCC components appear conserved, DCC binding sites appear diverged. The C. elegans consensus motif (MEX, motif enriched on X) pivotal for C. elegans DCC recruitment to X is only enriched 0.6-2-fold on C. briggsae X compared to autosomes, in contrast to the 3.8-24-fold enrichment on the C. elegans X chromosome. Furthermore, we characterized the recruitment potential of several C. elegans recruitment sites and their C. briggsae homologous regions in both species. No C. elegans or C. briggsae sequences tested were able to recruit the DCC in C. briggsae to the same degree as in C. elegans. This suggests that the cis-acting DNA recruitment sites in C. briggsae have diverged. Ongoing ChIP-seq experiments to define the C. briggsae DCC binding sites will reveal the degree of divergence. The identification of DNA binding sequences in C. briggsae will set the stage to allow us to investigate the molecular co-evolution of the DNA sequence motif and the DNA-binding domain of the DCC.
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[
International Worm Meeting,
2015]
Dosage compensation (DC) across Caenorhabditis species exemplifies an essential process that has undergone rapid co-evolution of protein-DNA interactions central to its mechanism. In C. elegans, recruitment elements on X (rex sites) recruit a condensin-like DC complex (DCC) to hermaphrodite X chromosomes to balance gene expression between the sexes. Recruitment assays in vivo showed that C. elegans rex sites do not recruit the DCC of C. briggsae, and vice versa. To understand how DC complexes and X chromosomes evolved to use different X targeting sequences, we compared DCC subunits and binding sites in C. elegans to those in three species of the C. briggsae clade (15-30 MYR diverged): C. briggsae, its close relative C. nigoni (C. sp. 9), and C. tropicalis (C. sp. 11). By raising antibodies and introducing endogenous tags with TALENs or CRISPR/Cas9, we showed that homologs of both SDC-2, the pivotal X targeting factor, and DPY-27, a DCC-specific condensin subunit, bind X chromosomes of XX animals. Although the DCC shares key components across these four species, the binding sites differ. First, ChIP-seq studies in C. briggsae and C. nigoni identified DCC binding sites that are homologous across these close relatives but differ from C. elegans sites in sequence and location. Second, C. elegans sites use motifs enriched on X (MEX and MEXII) to drive DCC binding, but these motifs are not in C. briggsae or C. nigoni DCC sites and are not X-enriched. Third, we found an X-enriched motif at DCC binding sites of C. briggsae and C. nigoni that is not X-enriched in C. elegans. An oligo with the C. briggsae motif recruits the DCC in C. briggsae, but a similar oligo lacking the motif fails to recruit, establishing the importance of the motif. Fourth, another motif was found in C. briggsae and C. nigoni that shares a few nucleotides with MEX, but its functional divergence was shown by C. elegans recruitment assays. Fifth, two endogenous C. briggsae X-chromosome regions with strong C. elegans MEX motifs fail to recruit the C. briggsae DCC, as assayed by ChIP-seq and recruitment assays. None of these DCC motifs is enriched on the C. tropicalis draft X sequence, supporting further binding site divergence within the C. briggsae clade. Ongoing ChIP-seq studies in C. tropicalis will help determine how C. elegans and C. briggsae clade motifs are evolutionarily related. Comparison of DCC targeting mechanisms across these four species allows us to characterize a rarely captured event: the recent co-evolution of a protein complex and its rapidly diverged target sequences across an entire X chromosome.
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[
Biochemistry,
2012]
Decapping scavenger (DcpS) enzymes catalyze the cleavage of a residual cap structure following 3' 5' mRNA decay. Some previous studies suggested that both m(7)GpppG and m(7)GDP were substrates for DcpS hydrolysis. Herein, we show that mononucleoside diphosphates, m(7)GDP (7-methylguanosine diphosphate) and m(3)(2,2,7)GDP (2,2,7-trimethylguanosine diphosphate), resulting from mRNA decapping by the Dcp1/2 complex in the 5' 3' mRNA decay, are not degraded by recombinant DcpS proteins (human, nematode, and yeast). Furthermore, whereas mononucleoside diphosphates (m(7)GDP and m(3)(2,2,7)GDP) are not hydrolyzed by DcpS, mononucleoside triphosphates (m(7)GTP and m(3)(2,2,7)GTP) are, demonstrating the importance of a triphosphate chain for DcpS hydrolytic activity. m(7)GTP and m(3)(2,2,7)GTP are cleaved at a slower rate than their corresponding dinucleotides (m(7)GpppG and m(3)(2,2,7)GpppG, respectively), indicating an involvement of the second nucleoside for efficient DcpS-mediated digestion. Although DcpS enzymes cannot hydrolyze m(7)GDP, they have a high binding affinity for m(7)GDP and m(7)GDP potently inhibits DcpS hydrolysis of m(7)GpppG, suggesting that m(7)GDP may function as an efficient DcpS inhibitor. Our data have important implications for the regulatory role of m(7)GDP in mRNA metabolic pathways due to its possible interactions with different cap-binding proteins, such as DcpS or eIF4E.
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[
J Infect Dis,
2015]
BACKGROUND: Elimination of onchocerciasis and lymphatic filariasis is targeted for 2020. Given the coincident Loa loa infections in Central Africa and the potential for drug resistance development, the need for new microfilaricides and macrofilaricides has never been greater. With the genomes of L. loa, Onchocerca volvulus, Wuchereria bancrofti, and Brugia malayi available, new drug targets have been identified. METHODS: The effects of the tyrosine kinase inhibitors imatinib, nilotinib, and dasatinib on B. malayi adult males, adult females, L3 larvae, and microfilariae were assessed using a wide dose range (0-100 M) in vitro. RESULTS: For microfilariae, median inhibitory concentrations (IC50 values) on day 6 were 6.06 M for imatinib, 3.72 M for dasatinib, and 81.35 M for nilotinib; for L3 larvae, 11.27 M, 13.64 M, and 70.98 M, respectively; for adult males, 41.6 M, 3.87 M, and 68.22 M, respectively; and for adult females, 42.89 M, 9.8 M, and >100 M, respectively. Three-dimensional modeling suggests how these tyrosine kinase inhibitors bind and inhibit filarial protein activity. CONCLUSIONS: Given the safety of imatinib in humans, plans are underway for pilot clinical trials to assess its efficacy in patients with filarial infections.
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Meyer, Barbara J., Thomas, Cristel G., Korf, Ian, Ralston, Edward J., Schartner, Caitlin M., Haag, Eric S., Schwarz, Erich M., Yin, Da
[
Evolutionary Biology of Caenorhabditis and Other Nematodes,
2014]
Sexual mode evolves rapidly in some eukaryotic lineages. This is expected to have pronounced consequences for population genetics, sexual differentiation and the nature and intensity of sexual selection, all of which may be reflected in the genome. C. elegans is a self-fertile species, derived recently from an obligately outcrossing male-female ancestor.This trait has evolved in at least two other species of the Elegans sub-genus, C. briggsae and C. sp. 11 (Kiontke et al. 2011). Previous studies indicate that selfing species have smaller genomes and several thousand fewer protein-coding genes than their outcrossing ancestors (Thomas et al. 2012). This reproducibility may be stimulated by an interaction between partial selfing and segregation distortion affecting large indels in male meiosis (Wang et al. 2010). However, the size, location, and gene content of specific deletions remain unknown for any natural system. To characterize the process of genome shrinkage, we have produced a genome assembly from the closest known outcrossing relative of C. briggsae, C. nigoni (formerly C.sp. 9; Felix et al. 2014). The C. nigoni genome is roughly 20 Mb (20%) larger than that of C. briggsae. By comparing C. nigoni contigs with the chromosome-level assembly for C. briggsae, we created an approximation of the C. nigoni physical map. Genome-wide sequence alignment showed the majority of the size reduction is located on the two arms of the five autosomes. Using C. sp.5 as an outgroup, we are able to identify gene family reductions, as well as specific genes recently lost in the C. briggsae lineage. Finally, we present detailed characterization of a family of rapidly evolving proteins that were independently lost in C. elegans and C. briggsae, the MSS (male-specific secreted) family, We have characterized their temporal and spatial expression, and find they are likely to be transferred to the female reproductive tract. We are now developing assays to reveal potential physiological responses to MSS proteins in females, including using calcium imaging to localize putative responder cells in female reproductive tract. References Felix, M.-A., C. Braendle, et al. (2014). "A streamlined system for species diagnosis in Caenorhabditis (Nematoda: Rhabditidae) with name designations for 15 distinct biological species." PLoS One 9:
e94723.Kiontke, K., M.-A. Felix, et al. (2011). "A phylogeny and molecular barcodes for Caenorhabditis, with numerous new species from rotting fruits." BMC Evol Biol 11: 339.Thomas, C. G., R. Li, et al. (2012). "Simplification and desexualization of gene expression in self-fertile nematodes." Curr Biol 22: 2167-2172.Wang, J., P. J. Chen, et al. (2010). "Chromosome size differences may affect meiosis and genome size." Science 329: 293.
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Schwarz, Erich M., Ralston, Edward J., Yin, Da, Haag, Eric S., Schartner, Caitlin M., Meyer, Barbara J.
[
International Worm Meeting,
2013]
Sexual mode evolves rapidly in some eukaryotic lineages. This is expected to have pronounced consequences for population genetics, sexual differentiation and the nature and intensity of sexual selection, all of which may be reflected in the genome. C. elegans is a self-fertile species, derived recently from an obligately outcrossing male-female ancestor. This trait has evolved in at least two other species of the Elegans sub-genus, C. briggsae and C. sp. 11. Previous studies indicate that selfing species have smaller genomes and several thousand fewer protein-coding genes than their outcrossing ancestors. This reproducibility may be stimulated by an interaction between partial selfing and segregation distortion affecting large indels in male meiosis. However, the size, location, and gene content of specific deletions remain unknown for any natural system. To characterize the process of genome shrinkage, we have produced a genome assembly from the closest known outcrossing relative of C. briggsae, C. sp.9. The C. sp. 9 genome is roughly 20 Mb (20%) larger than that of C. briggsae. By comparing C. sp.9 contigs with the chromosome-level assembly for C. briggsae, we created an approximation of the C. sp.9 physical map. This allowed us to examine the size and location of C. sp.9-specific sequences with respect to chromosome, and to relate them to known domains of gene density and recombination within a chromosome. Using an outgroup, we are also able to infer the identity of specific genes recently lost in the C. briggsae lineage. In this poster we present details of these analyses, along with some of their implications.
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[
Worm Breeder's Gazette,
1976]
We have studied maternal effects in 23 zyg ts mutants to estimate the times of expression of genes whose products are required in embryogenesis. We have used the following three tests, called arbitrarily A, B, and C. A test: Heterozygous (m/+) L4's are shifted to 25 C and allowed to self-fertilize. If 100% of their eggs yield larvae (25% of which express the mutant phenotype as adults), then the mutant is scored as maternal (M). If 25% of the F1 eggs fail to hatch, then the mutant is scored as non-maternal (N). An M result indicates that expression of the + allele in the parent allows m/m zygotes to hatch and grow to adulthood. A result of N indicates the opposite: that the + allele must be expressed in the zygote for hatching to occur. Out of 23 zyg mutants tested, 3 were scored N and 20 were scored M in the A test. Therefore, for most of the genes defined by these mutants, expression in the parent is sufficient for zygote survival, even if the gene is not expressed in the zygote. B test: Homozygous (m/m) hermaphrodites reared at 25 C are mated with N2 (+/+) males. If eggs fail to hatch at 25 C, but mated hermaphrodites shifted to 16 C produce cross progeny to give proof of mating, then the mutant is scored M. If cross progeny appear in the 25 C mating, then the mutant is scored N. An M result indicates that expression of the + allele in the zygote is not sufficient to allow m/+ progeny of an m/m hermaphrodite to survive. Conversely an N result indicates either that zygotic expression of the + allele is sufficient for survival, or that a sperm function or factor needed for early embryogenesis can be supplied paternally (see C test below). Out of the 23 zyg mutants tested, 11 were scored M and 12 were scored N. The combined results of A and B tests and their simplest interpretation are as follows. Ten mutants are M,M; the genes defined by these mutants must be expressed in the hermaphrodite parent for the zygote to survive. Ten mutants are M,N; these genes can be expressed either in the parent or in the zygote. Two mutants are N,N; these genes must be expressed in the zygote. One mutant is N,M; this gene must be expressed both in the maternal parent and in the zygote. C test: Homozygous (m/m) hermaphrodites reared at 25 C are mated with heterozygous (m/+) males. If rescue by a +/+ male in the B test depends on the + allele, then only half the cross progeny zygotes of a C test mating (m/+ male x m/m hermaphrodite) should survive. However, if rescue depends on a function or cytoplasmic component from the male sperm, then all the cross progeny zygotes in a C test should survive. Of the 10 M,N mutants, 6 have been C tested; one exhibited paternal rescue independent of the + allele. The A and B tests also were carried out on 16 mutants that arrest before the L3 molt (acc mutants). In the A test on 2 of these mutants, all m/m progeny of m/+ parents grew to adulthood at 25 C. Therefore, parental contributions are sufficient to overcome a progeny mutational block as late as the L2 stage. All 16 acc mutants scored N in the B test.
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[
Worm Breeder's Gazette,
1994]
cej-1 Encodes a Novel Protein with Poly-Threonine Motif M. L. A. Khanl, M. Tabish, T. Fukushigel1 S. Tsukita2, M. Itoh , Sh. Tsukita , and S. S. Siddiqui. (1): Lab. of Molecular Biology, Dept of Ecological Engg. Toyohashi Univ. Technology, Toyohashi 441, and (2). National Institute for Physiological Sciences, Okazaki 444, Japan.
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[
Mech Ageing Dev,
2009]
Energy production via oxidative phosphorylation generates a mitochondrial membrane potential (DeltaPsi(m)) across the inner membrane. In this work, we show that a lower DeltaPsi(m) is associated with increased lifespan in Caenorhabditis elegans. The long-lived mutants
daf-2(
e1370),
age-1(
hx546),
clk-1(
qm30),
isp-1(
qm150) and
eat-2(
ad465) all have a lower DeltaPsi(m) than wild type animals. The lower DeltaPsi(m) of
daf-2(
e1370) is
daf-16 dependent, indicating that the insulin-like signaling pathway not only regulates lifespan but also mitochondrial energetics. RNA interference (RNAi) against 17 genes shown to extend lifespan also decrease DeltaPsi(m). Furthermore, lifespan can be significantly extended with the uncoupler carbonylcyanide-3-chlorophenylhydrazone (CCCP), which dissipates DeltaPsi(m). We conclude that longevity pathways converge on the mitochondria and lead to a decreased DeltaPsi(m). Our results are consistent with the 'uncoupling to survive' hypothesis, which states that dissipation of the DeltaPsi(m) will extend lifespan.
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[
Arch Environ Contam Toxicol,
2005]
Fungi (Cunninghamella elegans ATCC 9245, Mucor ramannianus R-56, Aspergillus niger VKMF-1119, and Phanerochaete chrysosporium BKMF-1767) were tested to elucidate the biologic fate of the topical insect repellent N,N-diethyl-m-toluamide (DEET). The elution profile obtained from analysis by high-pressure liquid chromatography equipped with a reverse-phase C-18 column, showed that three peaks occurred after incubation of C. elegans, with which 1 mM DEET was combined as a final concentration. The peaks were not detected in the control experiments with either DEET alone or tested fungus alone. The metabolites produced by C. elegans exhibited a molecular mass of 207 with a fragment ion (m/z) at 135, a molecular mass of 179 with an m/z at 135, and a molecular mass of 163 with an m/z at 119, all of which correspond to N,N-diethyl-m-toluamide-N-oxide, N-ethyl-m-toluamide-N-oxide, and N-ethyl-m-toluamide, respectively. M. ramannianus R-56 also produced N, N-diethyl-m-toluamide-N-oxide and N-ethyl-m-toluamide but did not produce N-ethyl-m-toluamide-N-oxide. For the biologic toxicity test with DEET and its metabolites, the freshwater zooplankton Daphnia magna was used. The biologic sensitivity in decreasing order was DEET > N-ethyl-m-toluamide > N,N-diethyl-m-toluamide-N-oxide. Although DEET and its fungal metabolites showed relatively low mortality compared with other insecticides, the toxicity was increased at longer exposure periods. These are the first reports of the metabolism of DEET by fungi and of the biologic toxicity of DEET and its fungal metabolites to the freshwater zooplankton D. magna.