Etter A et al. (1989) International C. elegans Meeting "a transcribed single-copy sequence of A lumbricoides is eliminated."

Aboutanos M, Mueller F, Etter A, Tobler H

[International C. elegans Meeting, 1989]

Etter A et al. (1989) Worm Breeder's Gazette "chromatin elimination as an alternative possibility of gene regulation."

Etter A, Aboutanos M, Tobler H, Spicher A, Mueller F

[Worm Breeder's Gazette, 1989]

Ascaris des (var. suum) and a few other parasitic nematodes undergo chromatin diminution in the somatic precursor cells of the early embryo. In Ascaris, about 25% of the total germ line genome is lost from the somatic cells. Most of the eliminated material is composed of highly repetitive material, but also middle repetitive and single copy sequences are involved. Recently, we have isolated from an Ascaris germ line DNA library a lambda clone containing eliminated single copy DNA. This clone carries at least two different regions which are transcriptionally active during the early embryonic development of Ascaris. By using these DNA sequences as hybridization probes we have isolated two different cDNA clones from a 4 cell stage cDNA library. One of them, EPAL 1, has a length of 540 bp and contains an ORF coding for a 148 amino acid long protein. Sequence comparison with the genomic DNA revealed the existence of at least 3 introns which are flanked by consensus splice sites identical to those found in C. elegans. Northern blot analysis demonstrates that the RNA transcripts of EPAL 1 are specifically present in oocytes as well as in 4 cell embryos. In 4 cell embryos the transcripts are located in the cytoplasm of only two of the four blastomers, as indicated by our preliminary in situ hybridization data. The transcribed region crosshybridizes to three single copy DNA bands within the germ line genome of Parascaris equorum, which are also eliminated from the somatic cells during chromatin diminution. Interestingly, not only the sequence of this gene is conserved between this two parasitic nematodes, but also its behavior during the process of elimination. We therefore suggest, that the process of chromatin elimination serves as an alternative possibility of gene regulation.

Etter A et al. (1993) International C. elegans Meeting "A ribosomal protein is located in the germ-line specific material of Ascaris lumbricoides."

Tobler H, Keiser M, Bernard V, Etter A, Mueller F

[International C. elegans Meeting, 1993]

Etter A et al. (1991) International C. elegans Meeting "A gene located in the eliminated chromatin of Ascaris which codes for a putative ribosomal protein."

Tobler H, Aboutanos M, Mueller F, Etter A

[International C. elegans Meeting, 1991]

Chromatin diminution in the nematodes Parascaris equorum and Ascaris lumbricoides leads to the formation of somatic cells with less DNA than the germ line cells. We show here evidence for the coding potential of germ line limited DNA. We report on a cDNA clone which codes for a putative ribosomal protein (ALEP-1, for A. Iumbricoides eliminated protein No.1). The fact that the corresponding gene is located in the eliminated portion of the genome suggests a difference in germ line and somatic ribosomes of A. Iumbricoides and P. equorum. Our data furthermore indicate that elimination of the ALEP-1 gene from all somatic cells in its fully active state may represent an alternative way to gene regulation.

Etter A et al. (1999) J Neurochem "Picrotoxin blockade of invertebrate glutamate-gated chloride channels: subunit dependence and evidence for binding within the pore."

Arena JP, Liu KK, Reiss B, Schaeffer JM, Vassilatis DK, Etter A, Cully DF

[J Neurochem, 1999]

Glutamate-gated chloride channels have been described in nematodes, insects, crustaceans, and mollusks. Subunits from the nematode and insect channels have been cloned and are phylogenetically related to the GABA and glycine ligand-gated chloride channels. Ligand-gated chloride channels are blocked with variable potency by the nonselective blocker picrotoxin. The first two subunits of the glutamate-gated chloride channel family, GluClalpha and GluClbeta, were cloned from the free living nematode Caenorhabditis elegans. In this study, we analyze the blockade of these novel channels by picrotoxin. In vitro synthesized GluClalpha and GluClbeta RNAs were injected individually or coinjected into Xenopus oocytes. The EC50 values for picrotoxin block of homomeric GluClalpha and GluClbeta were 59 microM and 77 nM, respectively. Picrotoxin block of homomeric GluClbeta channels was promoted during activation of membrane current with glutamate. In addition, recovery from picrotoxin block was faster during current activation by glutamate. A chimeric channel between the N-terminal extracellular domain of GluClalpha and the C-terminal membrane-spanning domain of GluClbeta localized the higher affinity picrotoxin binding site to the membrane-spanning domains of GluClbeta. A point mutation within the M2 membrane-spanning domain of GluClbeta reduced picrotoxin sensitivity >10,000-fold. We conclude that picrotoxin blocks GluCl channels by binding to a site accessible when the channel is open.

Etter A et al. (1996) J Biol Chem "An amino acid substitution in the pore region of a glutamate-gated chloride channel enables the coupling of ligand binding to channel gating."

Cully DF, Arena JP, Liu KK, Etter A, Schaeffer JM

[J Biol Chem, 1996]

Many of the subunits of ligand-gated ion channels respond poorly, if at all, when expressed as homomeric channels in Xenopus oocytes. This lack of a ligand response has been thought to result from poor surface expression, poor assembly, or lack of an agonist binding domain. The Caenorhabditis elegans glutamate-gated chloride channel subunit GluClbeta responds to glutamate as a homomeric channel while the GluClalpha subunit is insensitive. A chimera between GluClalpha and GluClbeta was used to suggest that major determinants for glutamate binding are present on the GluClalpha N terminus. Amino acid substitutions in the presumed pore of GluClalpha conferred direct glutamate gating indicating that GluClalpha is deficient in coupling of ligand binding to channel gating. Heteromeric channels of GluClalpha+beta may differ from the prototypic muscle nicotinic acetylcholine receptor in that they have the potential to bind ligand to all of the subunits forming the channel.

Spicher A et al. (1991) International C. elegans Meeting "THE TRANSCRIPT OF AN ELIMINATED GENE REMAINS STABLE UNTIL THE FIRST STAGE LARVAE: IN SITU LOCALIZATION."

Etter A, Tobler H, Mueller F, Spicher A

[International C. elegans Meeting, 1991]

The process of chromatin diminution in Ascaris lumbricoides leads to an irreversible determination of the somatic cell lineage during the first cleavages of the embryonic development. This differentiation does not only include a quantitative but also a qualitative loss of genetic information.We found the eliminated chromatin to contain at least two genes; one of them, ALEP-l (see Etter et al. in this volume), is coding for a putative ribosomal protein. The second gene, 2B 12, is strongly transcribed in early developmental stages. On Northern blots, three different transcripts can be detected. The two larger RNA species disappear from the somatic cells soon after elimination has occurred. The smallest transcript (SSObp), however, is more stable and can still be detected in first stage larvae (8 days later than the 4- cell stage), in which its intensity, compared to the 4-cell stage, is only slightly diminished. In situ hybridization on whole mount embryos with digoxigenin-labelled probes demonstrates that these transcripts accumulate in the nuclear region of the blastomeres. The transcription of this gene is switched on already in the l-cell stage. At the 4- cells stage embryos, all of the four nuclei are stained, but the presomatic nuclei A and B show stronger coloration than the EMS and the P2 nuclei.

Spicher A et al. (1992) Worm Breeder's Gazette "Transcriptional activity in Ascaris embryos begins just afterfertilization"

Spicher A, Tobler H, Etter A, Mueller F

[Worm Breeder's Gazette, 1992]

During animal embryogenesis, the genome becomes transcriptionally active and contributes increasingly to the control of development. C. elegans embryos were shown to start transcriptional activity prior to the approximately 30-cell stage at which gastrulation begins (Schauer and Wood, Dev. 110 (1990), 13031317). In Ascaris, by performing in vitro nun-off transcription assays with nuclei from synchronous populations of early embryonic stages, transcriptional activity was found as early as the 4-8-cell stage (Cleavinger et al., Dev. Biol. 133 (1989), 600-604). These findings suggest that both, Ascaris and C. elegans embryos, are transcriptionally active very early in development. Recently, we have identified and isolated an Ascaris gene (Fert-1), which is expressed even earlier in development, namely just after fertilization. Fert-1 is a single copy gene that is expelled during the process of chromatin diminution from all somatic precursor cells. It yields a 560 nt long, polyA+ transcript of unknown function, which is composed of two exons and carries a spliced leader at its 5'end. A short ORF encodes a putative polypeptide. No Fert-1 RNA is detected in spermatids, oocytes and in mature germ cells prior to fertilization, which are located in the oviduct near the junction to the uterus. Transcripts are first found in fertilized eggs and are then persisting in increasing amounts throughout the early embryonic development until the stage where chromatin diminution occurs. After the elimination of the gene, its poly A+ transcription products disappear and are no longer detectable after the sixth day of embryonic development. Fert-1 provides a striking example of Ascaris early embryonic gene activity: Transcription of this non-maternal RNA species starts immediately after fertilization. Our data thus show that gene expression in Ascaris is switched on even in the earliest stages of embryonic development. Restriction of Fert-1 gene activity to early embryonic stages suggests a possible developmental function during initial embryonic differentiation. It should be interesting to see whether a homologous gene can be found in C. elegans and what its biological function might be.

Bernard V et al. (1994) Worm Breeder's Gazette "Cloning of the Ribosomal Protein S19 in Caenorhabditis elegans"

Mueller F, Etter A, Tobler H, Bernard V

[Worm Breeder's Gazette, 1994]

The nematode Ascaris lumbricoides undergoes chromatin diminution which causes a loss of about 25% of the total genomic DNA from all somatic cells. We have identified a ribosomal protein (rp) gene (coding for ALEP1 = Ascaris lumbricoides eliminated protein 1) which is located within the germ-line specific material. ALEP1 shows strong homologies with the eukaryotic ribosomal protein S19 family (rpS19 ).2D-gel electrophoresis on germ-line and somatic purified ribosome fractions shows that the ALEP1 protein (rpS19 )is present only in the germ-line ribosomes. In the rat, rpS19 has been localized at the tip of the small ribosomal subunit. What is the function of rpS19 ?Since Ascaris is not suitable for genetic analyses, we have isolated rpS19 from the nematode Caenorhabditis elegans. We used RT-PCR with degenerate oligonucleotides to amplify an 81 nucleotide long fragment. The Barstead cDNA library was screened with this fragment and positive clones were isolated and sequenced. The C. elegans rpS19 cDNA shows only 65% homology at the nucleic acid level. The transcript is trans-spliced with the splice leader SL1 .The deduced amino acid sequence predicts a protein of 150 amino acids with a molecular weight of 17 Kd. The C. elegans rpS19 protein shows perfect homology with other eukaryotic rpS19 proteins in the conserved regions. Overall, the RpS19 protein from C. elegans has 80% homology with Ascaris ALEPl. RpS19 is located on the C. elegans chromosome IV. Experiments are in progress to clone the C. elegans RpS19 gene. The identification of a rpS19 mutant in C. elegans might indicate its function.

Cully DF et al. (1996) Parasitology "Molecular biology and electrophysiology of glutamate-gated chloride channels of invertebrates."

Vassilatis DK, Cully DF, Arena JP, Etter A, Wilkinson HA

[Parasitology, 1996]

In this chapter we summarize the available data on a novel class of ligand-gated anion channels that are gated by the neurotransmitter glutamate. Glutamate is classically thought to be a stimulatory neurotransmitter, however, studies in invertebrates have proven that glutamate also functions as an inhibitory ligand. The bulk of studies conducted in vivo have been on insects and crustaceans, where glutamate was first postulated to act on H-receptors resulting from hyperpolarizing response to glutamate. Recently, glutamate-gated chloride channels have been cloned from several nematodes and Drosophila. The pharmacology and electrophysiological properties of these channels have been studied by expression in Xenopus oocytes. Studies on the cloned channels demonstrate that the invertebrate glutamate-gated chloride channels are the H-receptors and represent important targets for the antiparasitic avermectins.