-
[
MicroPubl Biol,
2021]
For El Mouridi, S; AlHarbi, S; Frkjr-Jensen, C (2021). A histamine-gated channel is an efficient negative selection marker for C. elegans transgenesis. microPublication Biology. 10.17912/micropub.biology.000349.
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[
Annu Rev Microbiol,
1992]
Oxygenases that incorporate one or two atoms of dioxygen into substrates are found in many metabolic pathways. In this article, representative oxygenases, principally those found in bacterial pathways for the degradation of hydrocarbons, are reviewed. Monooxygenases, discussed in this chapter, incorporate one hydroxyl group into substrates. In this reaction, two atoms of dioxygen are reduced to one hydroxyl group and one H2O molecule by the concomitant oxidation of NAD(P)H. Dioxygenases catalyze the incorporation of two atoms of dioxygen into substrates. Two types of dioxygenases, aromatic-ring dioxygenases and aromatic-ring-cleavage dioxygenases, are discussed. The aromatic-ring dioxygenases incorporate two hydroxyl groups into aromatic substrates, and cis-diols are formed. This reaction also requires NAD(P)H as an electron donor. Aromatic-ring-cleavage dioxygenases incorporate two atoms of dioxygen into aromatic substrates, and the aromatic ring is cleaved. This reaction does not require an external reductant. All the oxygenases possess a cofactor, a transition metal, flavin or pteridine, that interacts with dioxygen. The concerted reactions between dioxygen and carbon in organic compounds are spin forbidden. The cofactor is used to overcome this restriction. For the oxygenases that require the NAD(P)H cofactor, the enzyme reaction is separated into two steps, the oxidation of NAD(P)H to generate two reducing equivalents, and the hydroxylation of substrates. Flavoprotein hydroxylases that catalyze the monohydroxylation of the aromatic ring carry out these two reactions on a single polypeptide chain. In other oxygenases, the NAD(P)H oxidation and a hydroxylation reaction are catalyzed by two separate polypeptides that are linked by a short electron-transport chain. Two reducing equivalents generated by the oxidation of NAD(P)H are transferred through the electron-transport chain to the cofactor on a hydroxylase component that they reduce. Dioxygen couples with the reduced cofactor and subsequently hydroxylates substrates. The electron-transport chains associated with oxygenases contain at least two redox centers. The first redox center is usually a flavin, while the second is an iron-sulfur cluster. The electron transport is initiated by a single two-electron transfer from NAD(P)H to a flavin, followed by two single-electron transfers from the flavin to an iron-sulfur cluster. The primary sequences of many oxygenases have been determined, and according to their sequence similarities, the oxygenases can be grouped into several protein families. Among proteins of the same family, the sequences in regions involved in cofactor binding are strongly conserved. Local sequence similarities are also observed among oxygenases from different families, primarily in regions involved in cofactor binding.
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[
Neuron,
2012]
The adult mammalian central nervous system exhibits restricted regenerative potential. Chen etal. (2011) and El Bejjani and Hammarlund (2012) used Caenorhabditis elegans to uncover intrinsic factors that inhibit regeneration of axotomized mature neurons, opening avenues for potential therapeutics.
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[
Oncogene,
1997]
The
p53 protein is known to trans-activate a number of genes by specific binding to a consensus sequence containing two decamers of the type: PuPuPuCA/TT/AGPyPyPy. In order to identify new
p53 trans-activated genes, we defined a set of criteria for computer search of
p53-responsive elements. Based on experimental data, we proposed an extended consensus sequence composed of the two decamers of the El-Deiry consensus sequence flanked by two additional ones. A maximum of 3 bp substitutions was accepted for the two decamers of the El-Deiry consensus sequence, as well as for each additional decamer, except when the two decamers of the El-Deiry consensus sequence are contiguous. In this case, each additional decamer is allowed to bear one base insertion or deletion between the median C and G. This set of criteria was validated by identifying within the promoter region of the IGF-BP3 gene the existence of a novel
p53-responsive element whose functional significance was verified. By limiting our computer search to Vertebrate genes involved in cell cycle regulation, cellular adhesion or metastatic processes and to gene families most often found in HOVERGEN database, 7785 gene sequences were first analysed. Among the oncogenes, kinases, proteases and structural proteins, 55 new genes were selected; six of them were retrieved in more than one species.
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[
International Worm Meeting,
2005]
We have developed a systematic approach for inferring cis-regulatory logic from whole-genome microarray expression data.[1] This approach identifies local DNA sequence elements and the combinatorial and positional constraints that determine their context-dependent role in transcriptional regulation. We use a Bayesian probabilistic framework that relates general DNA sequence features to mRNA expression patterns. By breaking the expression data into training and test sets of genes, we are able to evaluate the predictive accuracy of our inferred Bayesian network. Applied to S. cerevisiae, our inferred combinatorial regulatory rules correctly predict expression patterns for most of the genes. Applied to microarray data from C. elegans[2], we identify novel regulatory elements and combinatorial rules that control the phased temporal expression of transcription factors, histones, and germline specific genes during embryonic and larval development. While many of the DNA elements we find in S. cerevisiae are known transcription factor binding sites, the vast majority of the DNA elements we find in C. elegans and the inferred regulatory rules are novel, and provide focused mechanistic hypotheses for experimental validation. Successful DNA element detection is a limiting factor in our ability to infer predictive combinatorial rules, and the larger regulatory regions in C. elegans make this more challenging than in yeast. Here we extend our previous algorithm to explicitly use conservation of regulatory regions in C. briggsae to focus the search for DNA elements. In addition, we expand the range of regulatory programs we identify by applying to more diverse microarray datasets.[3] 1. Beer MA and Tavazoie S. Cell 117, 185-198 (2004). 2. Baugh LR, Hill AA, Slonim DK, Brown EL, and Hunter, CP. Development 130, 889-900 (2003); Hill AA, Hunter CP, Tsung BT, Tucker-Kellogg G, and Brown EL. Science 290, 809812 (2000). 3. Baugh LR, Hill AA, Claggett JM, Hill-Harfe K, Wen JC, Slonim DK, Brown EL, and Hunter, CP. Development 132, 1843-1854 (2005); Murphy CT, McCarroll SA, Bargmann CI, Fraser A, Kamath RS, Ahringer J, Li H, and Kenyon C. Nature 424 277-283 (2003); Reinke V, Smith HE, Nance J, Wang J, Van Doren C, Begley R, Jones SJ, Davis EB, Scherer S, Ward S, and Kim SK. Mol Cell 6 605-616 (2000).
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[
West Coast Worm Meeting,
2004]
Cell fusion is essential for fertilization and formation of syncytia during organogenesis. About one-third of the somatic cells in C. elegans fuse to form an invariant pattern of syncytia during embryonic and postembryonic development. Most cell fusion in the embryo occurs in the epidermis during body elongation. Nearly all cell fusions require the integral membrane protein EFF-1: virtually all epidermal cells fail to fuse in
eff-1 mutants (Mohler, el al ., Dev. Cell 2: 355 (2002)). The proper spatiotemporal regulation of
eff-1 activity appears to ensure that cell fusion occurs between the appropriate cells during C. elegans development. We previously reported a zygotic embryonic lethal mutant,
fus-1 , in which the embryonic epidermal cells undergo hyperfusion (Kontani, el al ., IWM (2003)). In
fus-1 mutant embryos, formation of the epidermal syncytia is nearly normal during early embryogenesis; however, most or all epidermal cells, except the lateral seam cells, abnormally fuse into a single large syncytium in late stage embryos. This hyperfusion phenotype is suppressed by an
eff-1 mutation, indicating that
fus-1 represses
eff-1 -mediated cell fusion. We positionally cloned
fus-1 and found that it encodes the e subunit of the vacuolar H + -ATPase (V-ATPase). Immunoreactive FUS-1 is first detected in a punctate pattern in gut cells of comma-stage embryos. It is first detected in the epidermis of ~2-fold stage embryos and immunoreactivity increases in all epidermal cells except the lateral seam cells during late embryogenesis. Staining is particularly prominent around the surfaces of these epithelial cells. Together with the previous observation that hyperfusion is detected only in late stage
fus-1 mutant embryos, we speculate that FUS-1 acts to suppress
eff-1 -mediated cell fusion late in embryogenesis. V-ATPases are multi-subunit proton pumps that play important roles in various membrane transport, protein sorting, and degradation processes. The role of the e subunit of the V-ATPase is unknown; however, the yeast homolog of FUS-1 has been implicated in assembly of V-ATPase subunits. We performed RNAi to test the function of other V-ATPase subunits in proper cell fusion and found that interference of these genes induced hyperfusion of epidermal cells, similar to the phenotype seen in
fus-1 embryos. These results suggest that the V-ATPase may be required for proper localization of EFF-1 in epidermal cells to prevent inappropriate cell fusion. Alternatively, given the cell surface localization of the protein that we have observed, and implication of the V-ATPase in intracellular membrane fusion events (Bayer, el al ., J Cell Biol. 162: 211 (2003)), it may act more directly to regulate the membrane fusion-promoting activity of EFF-1.
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Fennell, Francesca, Yang, Yuqi, Bowerman, Bruce, Jud, Molly, Miller, Alex, Lowry, Josh, Bao, Zhirong, Tran, Nhah, Padilla, Thalia, Shao, Hong
[
International Worm Meeting,
2019]
Morphogenesis involves coordinated cell migrations and cell shape changes that generate tissues and organs. Cell adhesion and the cytoskeleton are important for morphogenesis, but the signaling pathways involved remain poorly understood. As genes required for morphogenesis often have earlier roles in development, temperature-sensitive embryonic lethal (TS-EL) alleles are useful tools for investigating morphogenesis. From a collection of ~1,000 TS-EL mutants, we have classified the terminal phenotypes of 191 with normal early embryogenesis, after upshifts from the permissive (15 deg C) to the restrictive temperature (26 deg C) at the L4 stage, to identify 79 with highly penetrant elongation defects. We identify causal mutations using whole genome sequencing (WGS) and genetic complementation tests. So far, we have identified 30 alleles representing 21 genes. Many of these genes likely regulate gene expression globally with roles in cell fate specification (e.g.
glp-1, which encodes a Notch receptor involved in blastomere specification) or differentiation (e.g.
chaf-1, which encodes a chromatin assembly factor subunit), while others appear to be weak alleles of cell division genes (e.g.
zwl-1, which encodes a kinetochore protein). To identify mutants that are specifically defective in morphogenesis, we upshifted mutant embryos just prior to the bean stage, after most cell division, cell fate specification and differentiation has occurred, and identified 12 with penetrant elongation defects. These include two with causal mutations in
rib-1 and
rib-2, which encode heparin sulfate synthesis proteins that regulate morphogenesis in other organisms, although how post-translational sugar modifications influence morphogenesis is still unclear. We are now using WGS to identify genes for 6 more of these 12 late-upshift mutants. We also are characterizing morphogenetic defects in fixed mutant embryos, using antibodies against epidermal cell membrane proteins to assess cell shape dynamics. Our long term goal is to advance our understanding of morphogenesis by identifying more genes that influence this process.
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[
C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany,
2010]
Left-right (LR) patterning is an intriguing but poorly understood process of bilaterian embryogenesis. We report a novel mechanism to break LR symmetry, whereby the embryo uncouples its midline from the anteroposterior (AP) axis. Specifically, the eight-cell C. elegans embryo establishes a midline that tilts rightward from the AP axis and positions more cells on the left, allowing subsequent differential LR fate inductions. To establish the tilted midline, cells exhibit LR asymmetric protrusions and a handed collective movement. This process, termed chiral morphogenesis, is based on differential regulation of cortical contractility between a pair of sister cells that are bilateral counterparts fate-wise, and is activated by non-canonical Wnt signaling. Chiral morphogenesis is timed by the division furrow of a neighbor of the sister pair, suggesting a nov el developmental clock and a novel signaling mechanism from the contractile ring to adjacent cells.
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[
Worm Breeder's Gazette,
2001]
RNAi is being used routinely to determine loss-of-function phenotypes and recently large-scale RNAi analyses have been reported (1,2,3). Although there is no question about the value of this approach in functional genomics, there has been little opportunity to evaluate reproducibility of these results. We are engaged in RNAi analysis of a set of 762 genes that are differentially expressed in the germline as compared to the soma (4 -- "Germline"), and have reached a point in our analysis that allows us to look at the issue of reproducibility. We have compared the RNAi results of genes in our set that were also analyzed by either Fraser et al. (1 -- Chromosome 1 set "C1") or Gonczy et al. (2 -- Chromosome 3 set "C3"). In making the comparison we have taken into account the different operational definition of "embryonic lethal" used by the three groups. In the C3 study, lethal was scored only if there were fewer than 10 surviving larva on the test plate, or roughly 90% lethal. In our screen and the C1 screen the percent survival was determined for each test. To minimize the contribution of false positives from our set, in our comparison with the C1 set we defined our genes as "embryonic lethal" if at least 30% of the embryos did not hatch, but included all lethals defined by Fraser et al. (> 10%). For our comparison with the C3 set, we used a more restrictive definition of "embryonic lethal" that required that 90% of the embryos did not hatch. (This means that in Table 1, five genes from our screen that gave lethality between 30-90% were included in the not lethal category; one of these was scored as lethal by Gonczy et al.). We have analyzed 149 genes from the germline set that overlap with the C1 set and 132 genes that overlap with the C3 set. The table below shows the number of genes scored as embryonic lethal (EL) or not embryonic lethal (NL) in each study. (Note that these comparisons do not include data from our published collection of ovary-expressed cDNAs.) Table 1. Comparing RNAi analysis of the same genes in different studies. Germline Chromosome 1 Germline Chromosome 3 NL (117) EL (32) NL (97) EL (35) NL (104) 100 4 NL (89) 87 2 EL (45) 17 28 EL (43) 10 33 Overall, the degree of reproducibility is high. The concordance between our results and the published results was 86% with C1 (128/149 genes) and 90% with C3 (120/132). However, we scored a larger number of genes as giving rise to embryonic lethal phenotypes than the other studies did. What does this mean? One possibility is that we are generating a large number of false positives (God forbid!). The other interpretation is that there is a fairly high frequency of false negatives in each screen (4-8% in our screen (2/45; 4/49); 22% in the C3 screen (10/45); and 35% (17/49) in the C1 screen). It is no surprise that the different methods used by the three groups resulted in slightly different outcomes and we can only speculate on which methodological variation contributed most. In comparing our methods to those used in the C3 study we note that our two groups used different primer pairs for each gene; that we tested genes individually while they tested genes in pairs; and that the operational definition of "embryonic lethal" differed. Considering the latter two differences, we speculate that even with pools of two, the competition noted by Gonczy et al. in dsRNA pools could reduce levels of lethality below the 90% cutoff. The major difference between our approach and the C1 approach is feeding vs. injection, raising the possibility that for some genes feeding may be a less effective means of administering dsRNA. Whatever the basis for the difference, these comparisons indicate that genes scored as "non-lethal" in any single study may show an embryonic lethal RNAi phenotype when reanalyzed. It therefore seems useful to have more than one pass at analyzing C. elegans genes via RNAi. We are indebted to P. Gonczy for very useful comments. Fraser, A. G., Kamath, R. S., Zipperlen, P., Martinez-Campos, M., Sohrmann, M. and Ahringer, J. (2000). Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature 408 , 325-330. Gonczy, P., Echeverri, G., Oegema, K., Coulson, A., Jones, S. J., Copley, R. R., Duperon, J., Oegema, J., Brehm, M., Cassin, E. et al. (2000). Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III. Nature 408 , 331-336. Piano, F., Schetter, A. J., Mangone, M., Stein, L. and Kemphues, K. J. (2000). RNAi analysis of genes expressed in the ovary of Caenorhabditis elegans. Curr Biol 10 , 1619-1622. Reinke, V., Smith, H. E., Nance, J., Wang, J., Van Doren, C., Begley, R., Jones, S. J., Davis, E. B., Scherer, S., Ward, S. et al. (2000). A global profile of germline gene expression in C. elegans. Mol Cell 6 , 605-616.
-
[
International Worm Meeting,
2013]
Chromosome mutations are considered changes in the chromosome number or structure. They are due to gametogenesis inborn error (meiosis) or during the zygote first cellular divisions. All these alterations might be observed during metaphase from the cellular cycle, where DNA loses are seen (clastogenic processes) due to DNA repair processes deficiency o total absence, among others. 4617 chromosomal studies were performed at Hospital Para El Nino Poblano (Pediatric Hospital) in Mexico. During 19 years period of time (from 1992 to 2011) were 34.6% (1596 patients) showed different chromosomal alterations. Among the studies population, male and female pediatric patients with different genetic diseases were chosen. These chromosome changes are classified as numeric or structural alterations, respectively. Another group of genetic alterations are known as mutations and can be inherited among generations. A wide variety of pediatric patients with genetic diseases due to chromosome aberrations are described in this study analyzing their clinical characteristics, medical or surgical treatments and their medical evolution according to their genetic change. An early patient diagnosis is important for a better quality of life.