Ryuji Hosono

The unc-18 Gene Encodes a Novel Protein Affecting the Kinetics of Acetylcholine Metabolism in the Nematode Caenorhabditis elegans

Abstract: Genes affecting acetylcholine (ACh) levels without influencing choline acetyltransferase activity have been identified in Caenorhabditis elegans. We have examined one such gene, unc‐18. We isolated a transposon‐insertion allele for unc‐18 and used it to clone a genomic region containing the unc‐18 locus. The unc‐18 location within this region was determined by rescuing the unc‐18 mutant phenotype in a germline transformation experiment and identifying transcripts affected by four independent unc‐18 mutations. A single‐sized poly(A)+ RNA was synthesized from the gene. Expression of the transcript appears to be stage specific: The transcript is found in abundance at the early larval stage but in decreased amounts at the fourth larval and the adult stages. These results show that the unc‐18 gene plays a role in development as well as in the kinetics of ACh metabolism.

The C. elegans unc-18 gene encodes a protein expressed in motor neurons.

The C. elegans unc-18 gene is required to maintain normal acetylcholine levels. We determined the complete structure of an unc-18 cDNA that encodes a protein of 591 highly charged and hydrophilic amino acids. The protein shows sequence similarity with elements of the secretory pathway in the yeast S. cerevisiae. Antibodies raised against a portion of the unc-18-encoded protein (UNC-18) detected a 68 kd soluble antigen on immunoblots and intensely stained all vertical cord motor neurons in situ. These findings suggest that UNC-18 participates in the axonal transport system and influences the acetylcholine flow in motor neurons.

Alterations of life span in the nematode Caenorhabditis elegans under monoxenic culture conditions.

The nematode Caenorhabditis elegans was cultured monoxenically with E. coli as a food source and the influence of the bacterial growth conditions on the life span was studied. When bacterial growth was restricted by reducing the concentration of bactopeptone, which was supplied as the energy source in nematode growth medium (NGM), the nematodes life span tended to be prolonged without a marked effect on postembryonic development. The effect of bactopeptone on the life span was clearly observed during the postreproductive period (that is, after the egg-laying stage of the wild-type C. elegans) rather than during the larval to young adult stage. Evidence is presented that this alteration of the life span was not brought about by any factor in the bactopeptone but by the concentration of bacteria.

Age-dependent changes in mobility and separation of the nematode Caenorhabditis elegans

Easy classification and separation of aged C. elegans was performed on the basis of the difference in the movement activity; that is, sluggish but rhythmically locomoting worms (type I), worms that locomote but are irregular in movement (type II), and worms that are defective in locomotion but responsible for stimulus for touch (type III). The residual life span after the separation was also followed, in addition to the pumping frequency, the sinusoidal movement activity in the liquid and the resistancy to nile blue of each type. These parameters were well correlated to the length of life span of each type, and aging of the nematode seems to proceed in the order of type I, II and III.

Sterilization and growth inhibition of Caenorhabditis elegans by 5-fluorodeoxyuridine

Abstract The effect of 5-fluorodeoxyuridine (FdUrd) on the growth and the reproductive system of Caenorhabditis elegans has been studied. The growth of the nematode with heat-killed E. coli as food was inhibited by the addition of 0·1 mM of FdUrd, while in monoxenic conditions with intact bacteria, much higher concentration of FdUrd was required for the inhibition. The growth inhibition was observed if FdUrd was given at early larval stage but not at fourth larval stage or adult. By removal of the inhibitor, the nematode immediately resumed the normal growth after 40 h exposure to FdUrd, through the extent of recovery progressively decreased with longer exposure than 40 h. The nematode fed FdUrd laid eggs which are defective in hatching. The sterilization was brought about at more lower concentration of FdUrd than that required for the growth inhibition. The nematode was sterilized by feeding FdUrd any time during the egg-laying period and fled from the state if FdUrd was removed from the medium.

Life span of the wild and mutant nematode Caenorhabditis elegans. Effects of sex, sterilization, and temperature.

The survival of Caenorhabditis elegans was studied comparing animals of different sexes, sterilized animals, and animals grown at different temperatures as a prelude to more detailed cytological and genetic analysis of aged nematodes. Temperature-sensitive sterile mutants, animals sterilized by 5-fluorodeoxyuridine treatment, and wild-type males showed little difference in life span from that of wild-type hermaphrodites, although slight changes in P (time of beginning of the dying phase) or T1/2 (half-life of the population in the early dying phase) values were observed. At higher temperatures, P and T1/2 values markedly decreased, indicating a shortened life span. Temperature shift between 16 degrees C and 25 degrees C revealed that an increase in life span always involved low temperatures after the adult phase. High temperature treatment during the growing phase or after the adult phase caused an earlier start of the dying phase, but a downward temperature during the adult phase resulted in a great increase in the half-life of the population (T1/2). The results suggest that the life span of C. elegans is rigidly determined by somatic cells and markedly influenced by the effects of temperature on the cells during the post-mitotic state.

Memory in Caenorhabditis elegans is Mediated By NMDA-Type Ionotropic Glutamate Receptors

Learning and memory are essential processes of both vertebrate and invertebrate nervous systems that allow animals to survive and reproduce. The neurotransmitter glutamate signals via ionotropic glutamate receptors (iGluRs) that have been linked to learning and memory formation; however, the signaling pathways that contribute to these behaviors are still not well understood. We therefore undertook a genetic and electrophysiological analysis of learning and memory in the nematode Caenorhabditis elegans. Here, we show that two genes, nmr-1 and nmr-2, are predicted to encode the subunits of an NMDA-type (NMDAR) iGluR that is necessary for memory retention in C. elegans. We cloned nmr-2, generated a deletion mutation in the gene, and showed that like nmr-1, nmr-2 is required for in vivo NMDA-gated currents. Using an associative-learning paradigm that pairs starvation with the attractant NaCl, we also showed that the memory of a learned avoidance response is dependent on NMR-1 and NMR-2 and that expression of NMDARs in a single pair of interneurons is sufficient for normal memory. Our results provide new insights into the molecular and cellular mechanisms underlying the memory of a learned event.

Additional genes which result in an elevation of acetylcholine levels by mutations in Caenorhabditis elegans

Four mutant genes (unc-17, unc-18, unc-41 and unc-13) have been identified that result in abnormal accumulation of acetylcholine (ACh). We have now identified 3 more such genes (unc-63, unc-11 and unc-64). In addition to the abnormal accumulation of ACh, mutants in these 7 genes possess common phenotypes in locomotion, resistance to inhibitors of acetylcholinesterase (AChE) and in post-embryonic development. These results suggest that the 7 genes are involved in some related functions.

Mutations Affecting Acetylcholine Levels in the Nematode Caenorhabditis elegans

Gene cha‐1 · unc‐17 of the nematode Caenorhabditis elegans is a complex gene, consisting of at least two complementation groups. One part (cha‐1 region) of the gene encodes the enzyme choline acetyltransferase (ChAT), but the function of the other part (unc‐17 region) is still unclear. We measured the ChAT activity and ACh levels of the cha‐1 and unc‐17 complex gene mutants. We show here that alterations in ACh levels, rather than the ChAT activity, reflect abnormal phenotypes accompanying cha‐1 · unc‐17 mutations, that is, the decreased ACh levels in cha‐1 mutations and abnormal accumulation in unc‐17 mutations. Our results suggest that the unc‐17 region may encode functions necessary for storage and/or release of ACh at the presynaptic level.

Age dependent changes in the behavior of Caenorhabditis elegans on attraction to Escherichia coli.

Abstract A method to analyze the response of Caenorhabditis elegans to Escherichia coli as food was devised. Age dependency of the response was purchased with this method chronologically. Although the activity of sinusoidal movement and the utilization of bacteria as food began to decrease rapidly in previous to maximum growth, the attraction potency in young adults was maintained irrespective of advancing in age.