O'Connor SL et al. (1995) International C. elegans Meeting "smg-1, A GENE INVOLVED IN NONSENSE-MEDIATED mRNA DECAY."

O'Connor SL, Pulak RA, Anders KR, Anderson P

[International C. elegans Meeting, 1995]

The informational suppressor genes smg-1 through smg-7 play a role in identifying and degrading mRNAs that contain nonsense codons. In smg(+) genetic backgrounds, unc-54 nonsense and frameshift mutation-containing mRNAs are unstable. Mutations in any of the seven smg genes cause these mutant mRNAs to accumulate to near wild-type levels (see Pulak and Anderson. 1993. Genes and Dev. 7: 1885; Cali and Anderson, Abstract this meeting). We used three factor mapping and a microinjection assay to identify two overlapping cosmids, F56F2 and C14A12, that contain smg-1 (O'Connor, 1993 Meeting Abstract). Subsequent subcloning and microinjection experiments localized the smg-1 rescuing activity to a 10 kb region. Southern analysis of ten smg-1 alleles isolated in a mut-2(r459) background identifies insertions in three alleles (r904, r910, and r913). All three insertions map to the 10 kb rescuing region. A 4 kb subclone from the middle of the rescuing region identifies a single transcript of 7.8 kb on an N2 northern blot. The size of this transcript is altered in smg-1(r904) and smg-1(r913) mutants. Thus, it seems likely that this 7.8 kb transcript is smg-1. The large size of the putative smg-1 mRNA is consistent with the large size of the smg-1 mutational target. Partial cDNAs of this transcript have been identified and the isolation of full length clones is underway. Sequence analysis of genomic and cDNA clones predicts that SMG-1 protein contains a kinase domain. The C-terminal region of SMG-1 has approximately 50% identity over 400 AA to the kinase domain of several phosphatidylinositole 3-kinases. This homology appears to be limited to the kinase domain; no significant homologies to other regions of SMG-1 have been identified. Sequence analysis is continuing.

O'Connor SL et al. (1993) International C. elegans Meeting "Transformation rescue of smg-1, a gene involved in degrading aberrant mRNAs."

O'Connor SL, Anderson P, Anders KR, Pulak RA

[International C. elegans Meeting, 1993]

O'Connor SL et al. (1997) International C. elegans Meeting "smg-1, A C. ELEGANS GENE INVOLVED IN NONSENSE-MEDIATED mRNA DECAY."

O'Connor SL, Anderson P

[International C. elegans Meeting, 1997]

The seven C. elegans smg genes are necessary for identifying and degrading mRNAs that contain premature nonsense codons. In smg(+) genetic backgrounds, nonsense mutation-containing mRNAs are less stable than their WT counterparts. Mutations in any of the seven smg genes eliminate the system of nonsense-mediated mRNA decay (NMD), which cause nonsense-mutant mRNAs to accumulate to near WT levels (Pulak and Anderson. 1993. Genes and Dev. 7: 1885). NMD is a non-essential system and smg(-) animals have a near WT phenotype. We are working on the cloning and characterization of the smg genes to gain a better understanding of this system. We have taken advantage of the well characterized genetic and physical maps of C. elegans in the cloning of smg-1. After three-factor mapping, a transformation rescue assay was used to identify smg-1-containing clones. We have localized smg-1 to a 13 kb region on the cosmid F56F2. Southern blot analysis of ten mutator-induced smg-1 alleles identifies insertions in three alleles (r904, r910, and r913). All three insertions map to the 13 kb rescuing region. Clones from this region identify a single transcript of 7.8 kb in N2. This transcript is larger in smg-1(r913) and absent in smg-1(r904) mutants. Thus, this 7.8 kb transcript is likely smg-1, and smg-1(r904) is demonstrated to be a null allele. The phenotype of smg-1(r904) is indistinguishable from other smg alleles, proving that smg-1 is a non-essential gene. The large size of the smg-1 mRNA is consistent with the fact that smg-1 alleles are the most commonly isolated alleles in general screens for smg(-) mutations. Sequence analysis demonstrates that the C-terminal region of SMG-1 is approximately 30% identical over 300 AA to the kinase domain of several phosphatidylinositol-3-kinases, including the human gene mutated in ataxia telangiectasia (ATM). This homology appears to be limited to the kinase domain; no significant homologies to other regions of SMG-1 have been identified. Since some PI-3 kinase-like proteins are inhibited by the immunosuppressant drugs rapamycin and FK506, we tested whether these drugs could inhibit the smg system. Even at concentrations 1000 fold higher than those used in cell culture, we were unable to observe any loss of nonsense-mediated mRNA decay. To determine where this decay process occurs in cells, we are generating anti-SMG-1 antibodies. Antibodies to SMG-1 show strong nuclear staining in the germline and in embryos. This staining is reduced but not absent in smg-1(r904) animals. We are presently making additional antibodies and further purifying the existing ones in order to confirm the possible nuclear localization of SMG-1.

O'Connor SL et al. (1997) International C. elegans Meeting "NEW CLEAVAGE AND POLYADENYLATION SIGNALS IN THE 3'UTR OF THE unc-54(r293) TRANSCRIPT RESTORE MESSAGE STABILITY."

Shang H, O'Connor SL, Anderson P

[International C. elegans Meeting, 1997]

Functions of the C. elegans smg genes are required for nonsense-mediated mRNA decay (NMD), a surveillance system that degrades transcripts which contain premature stop codons. smg mutants were isolated in our lab as phenotypic suppressors of unc-54(r293). The 3' end of the 6.5 kb wild-type unc-54 transcript is formed using a cleavage and polyadenylation signal (CPS) located 270 nt downstream of the termination codon. The unc-54(r293) 3'UTR contains a 256 nt deletion that removes the normal CPS. This mutant transcript is 8.3 kb and accumulates to approximately 8% of wild-type unc-54 levels. A CPS located approximately 1.8 kb downstream of the normal CPS is presumably used in unc-54(r293). The abnormal 3'UTR of unc-54(r293) mRNA causes the normal termination codon to be recognized as a premature stop codon by the NMD system either because stabilizing elements have been removed by the deletion or because smg-specific destabilizing elements have been introduced as part of the unc-54(r293) 3'UTR. smg mutations phenotypically suppress the muscle defects of unc-54(r293) because they eliminate NMD. Besides alleles of the seven smg genes, a number of intragenic revertants were isolated during unc-54(r293) suppressor screens. Unlike the smg alleles, the intragenic alleles are dominant and inseparable from the unc-54 locus. These screens used either chemical mutagens or a mut-5 genetic background. We have characterize five EMS, one ENU, and three mut-5-induced alleles using Southern, Northern, and sequence analysis. Northern analysis shows that all nine alleles accumulate transcripts that are nearly wild-type in size and abundance. To determine the molecular basis of these revertants we sequenced PCR products generated from genomic DNA. These analyses demonstrate that all six chemically-induced mutants contain a single G>A transition located 189 bp downstream of the unc-54(r293) termination codon. This single base change creates the sequence AATGAA, which we believe serves as a new cleavage and polyadenylation signal. Each of the three mut-5-induced alleles contain Tc1 insertions located either 60 bp or 70 bp downstream of the unc-54(r293) termination codon. We are currently working to demonstrate that the predicted new cleavage and polyadenylation signals are actually utilized in the revertants and to characterize any destabilizing elements in the longer unc-54(r293) 3'UTR.

O'Connor SL et al. (1996) Midwest Worm Meeting "smg-1, A C. ELEGANS GENE INVOLVED IN NONSENSE-MEDIATED mRNA DECAY."

Anderson P, O'Connor SL

[Midwest Worm Meeting, 1996]

The seven C. elegans smg genes are necessary for identifying and degrading mRNAs that contain premature nonsense codons. In smg(+) genetic backgrounds, nonsense mutation-containing mRNAs are less stable than their WT counterparts. Mutations in any of the seven smg genes eliminate the system of nonsense-mediated mRNA decay, which cause nonsense-mutant mRNAs to accumulate to near WT levels (Pulak and Anderson. 1993. Genes and Dev. 7: 1885). Nonsense-mediated mRNA decay is a non-essential system and smg(-) animals have a near WT phenotype. We are working on the cloning of several smg genes to gain a better understanding of this system. We have taken advantage of the well characterized genetic and physical maps of C. elegans in the cloning of smg-1. After three-factor mapping, a transformation rescue assay was used to identify smg-1-containing clones. The smg-1 rescuing region is contained within a 13 kb genomic clone. Southern blot analysis of ten smg-1 alleles isolated in a transposon-active background identifies insertions in three alleles (r904, r910, and r913). All three insertions map to the 13 kb rescuing region. Clones from this region identify a single transcript of 7.8 kb in N2. This transcript is larger in smg-1(r913) and absent in smg-1(r904) mutants. Thus, this 7.8 kb transcript is likely smg-1 and smg-1(r904) is a null. The phenotype of smg-1(r904) is indistinguishable from other viable smg alleles. Like smg-2 (Carr, B. Meeting Abstracts, 1993), smg-1 is a non-essential gene, confirming the smg-1 null phenotype suggested by genetic analysis. The large size of the putative smg-1 mRNA is consistent with the fact that smg-1 alleles are the most commonly isolated alleles in general screens for smg(-) mutations. Partial cDNAs from oligo-d(T)- and randomly primed libraries have been identified and assembled into a contig comprising most of the smg-1 transcript. Sequence analysis of genomic and cDNA clones demonstrates that the C-terminal region of SMG-1 is approximately 30% identical over 300 AA to the kinase domain of several phosphatidylinositol-3-kinases, including the recently cloned human gene ATM. This homology appears to be limited to the kinase domain; no significant homologies to other regions of SMG-1 have been identified. We are continuing to sequence smg-1 and are preparing polyclonal antibodies to investigate the SMG-1 protein and its subcellular localization.

O'Connor G et al. (1998) East Coast Worm Meeting "The C. elegans Amyloid Precursor Related Protein, APL-1, May Signal Through Goa"

Li C, O'Connor G

[East Coast Worm Meeting, 1998]

Mutations in the human Amyloid Precursor Protein (APP) gene have been linked to 2-3% of the familial Alzheimer disease (FAD) cases. These mutations are correlated with an increase in the release of b-amyloid peptide from the full-length APP protein. Human APP is a 695 amino acid, glycosylated single transmembrane domain protein with two conserved extracellular domains, one which is cysteine rich and one which is highly acidic. The cytoplasmic domain of the protein includes a predicted Goa binding site. Mutations in two genes encoding the presenilins PS1 and PS2 have been linked to 60% of FAD cases. In vitro studies have shown that APP interacts with both PS1/2 and Goa. C. elegans APL-1 is related to APP. APL-1 is a 680 amino acid protein that shares many structural similarities with APP, but does not contain the b-amyloid peptide. We have shown that APL-1 is glycosylated. Although APP and related proteins from other species are proteolytically cleaved, we have not been able to detect any cleavage of APL-1 thus far. To study the specific interaction of APL-1 with SEL-12, the C. elegans homologue to human presenilins, and the neuronally expressed C. elegans G-protein, GOA-1, we are determining whether in vitro translated SEL-12 and GOA-1 co-precipitate with endogenous APL-1. We have shown biochemically that GOA-1 interacts with APL-1, and we are further characterizing this binding. To identify genes that act downstream of apl-1, transgenic animals that over-express apl-1 under an inducible (hsp 16-2) or neuronal-specific (VAMP) promoter were generated. We have confirmed that the transgenic lines over-express apl-1 by Western blot analysis. Heat shock-induced and constitutive neural over-expression of APL-1 both cause a decreased mobility in transgenic animals; however, transgenic animals are wildtype in assays for chemotaxis, osmolarity avoidance, and brood size. When GOA-1 function is disrupted, animals exhibit a hyperactive phenotype1. Since this phenotype is opposite that of the APL-1 overexpressing strains and APL-1 contains a possible consensus sequence for Goa binding, epistasis experiments were performed. A double mutant containing a loss-of-function mutation in goa-1 and a gain-of-function apl-1 transgene were produced. The phenotypes exhibited by double mutant animals were the same as the goa-1 single mutation animals. These biochemical and genetic data suggest that GOA-1 signals downstream of apl-1.

Bhagwati P Gupta et al. (2002) West Coast Worm Meeting "Genetic analysis of the vulval mutants in C. briggsae"

Shahla Gharib, Bhagwati P Gupta, Paul W Sternberg, Jennifer X Li

[West Coast Worm Meeting, 2002]

To understand the evolution of developmental mechanisms, we are doing a comparative analysis of vulval patterning in C. elegans and C. briggsae. C. briggsae is closely related to C. elegans and has identical looking vulval morphology. However, recent studies have indicated subtle differences in the underlying mechanisms of development. The recent completion of C. briggsae genome sequence by the C. elegans Sequencing Consortium is extremely valuable in identifying the conserved genes between C. elegans and C. briggsae.

Oomura, S. et al. (2019) International Worm Meeting "Early embryogenesis of C. inopinata, a sibling species of C.elegans."

Matsumura, Y., Haruta, N., Hoshi, Y., Sugimoto, A., Oomura, S.

[International Worm Meeting, 2019]

C. inopinata is a newly discovered sibling species of C. elegans. Despite their phylogenetic closeness, they have many differences in morphology and ecology. For example, while C. elegans is hermaphroditic, C. inopinata is gonochoristic; C. inopinata is nearly twice as long as C. elegans. A comparative analysis of C. elegans and C. inopinata enables us to study how genomic changes cause these phenotypic differences. In this study, we focused on early embryogenesis of C. inopinata. First, by the microparticle bombardment method we made a C. inopinata line that express GFP::histone in whole body, and compared the early embryogenesis with C. elegans by DIC and fluorescent live imaging. We found that the position of pronuclei and polar bodies were different between these two species. In C. elegans, the female and male pronuclei first become visible in anterior and posterior sides, respectively, then they meet at the center of embryo. On the other hand, the initial position of pronuclei were more closely located in C. inopinata. Also, the polar bodies usually appear in the anterior side of embryo in C. elegans, but they appeared at random positions in C. inopinata. Therefore, we infer that C. inopinata may have a different polarity formation mechanism from that in C. elegans. We are also analyzing temperature dependency of embryogenesis in C. inopinata, whose optimal temperature is ~7 degree higher than that in C. elegans.

Karin Kiontke et al. (2008) Development & Evolution Meeting "New Phylogeny Of Caenorhabditis Including Recently Discovered Species"

David Fitch, Karin Kiontke, Marie-Anne FELIX

[Development & Evolution Meeting, 2008]

Recently, seven new Caenorhabditis have been discovered, bringing the number of Caenorhabditis species in culture to 17, 10 of which are undescribed. To elucidate the relationships of the new species to the five species with sequenced genomes, we have used sequence data from two rRNA genes and several protein-coding genes for reconstructing the phylogenetic tree of Caenorhabditis. Four new species (spp. 5, 9, 10, 11) group within the so-called Elegans group of Caenorhabditis, with C. elegans being the first branch. Whereas none of them is likely to be the sister species of C. elegans, we now know of two close relatives of C. briggsae-C. sp. 5 and C. sp. 9. C. sp. 9 can hybridize with C. briggsae in the laboratory [see abstract by Woodruff et al.]. Of the remaining new species, C. sp. 7 branches off between C. elegans and C. japonica. This species is easier to cultivate than C. japonica and may be a better candidate for comparative experimental work. Two of the new species branch off before C. japonica as sister species of C. sp. 3 and C. drosophilae+C. sp. 2, respectively. Only one of the new species, C. sp. 11, is hermaphroditic. The position of C. sp. 11 in the phylogeny suggests that hermaphroditism evolved three times within the Elegans group. Two of the new species were isolated from rotting leaves and flowers, and five from rotting fruit. Rotting fruit is also the habitat in which C. elegans has been found to proliferate (Barriere and Felix, Genetics 2007) and from which C. briggsae, C. brenneri and C. remanei were repeatedly isolated. This suggests that the habitat of the stem species of Caenorhabditis after the divergence of the earliest branches (C. plicata, C. sonorae and C. sp. 1) was rotting fruit. The rate of discovery of new Caenorhabditis species has steadily increased since the description of C. elegans in 1899, with a leap in the last two years. There is no indication that we are even close to knowing all species in this genus.

Schartner, Caitlin M. et al. (2015) International Worm Meeting "X-chromosome evolution: divergence of X-sequence motifs that drive dosage compensation across Caenorhabditis species."

Meyer, Barbara J., Lo, Te-Wen, Schartner, Caitlin M.

[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.