Kagawa H (1991) Worm Breeder's Gazette "C. elegans Scientists visit to Japan in 1990"

Kagawa H

[Worm Breeder's Gazette, 1991]

Ed Hedgecock came to Tokyo in January for the 2nd seminar on Molecular and Developmental Neurobiology and his cell migration work impressed scientists of other animal field. Sydney Brenner won the Kyoto Prize and came to Japan on 23 of October for receiving the Prize on his fourth visit in this year. He talked with his excellent joke about telescope in Astronomy and microscope in Biology. Sydney is the man who gave the main lecture in the First Meeting of Japan Molecular Biology Society. Iva Greenwald came to Japan for attending the Naito Foundation International Workshop on Morphogenesis Program in early November together with her fiance Gary Struhl. It is true that the relation between Drosophila and Caenorhabditis is very good in U.S. and Japan. Some nematode scientists in Japan got grant from Drosophila project. John Sulston gave the lecture entitled 'The Genome of Caenorhabditis' on 28th of November at the main invited lecture of the Thirteenth Meeting of Japan Molecular Biology Society in Kyoto International Congress Hall. The lecture impressed many Japanese scientists especially his 'The Logical Next Step: Genome Sequencing'. On the 27th night, 34 worm people assembled to the worm party for welcoming to him. Sixteen papers from five labs were presented at the meeting.

Kondo K et al. (1988) Worm Breeder's Gazette "unc-51 and tRNA-Trp amber suppressors."

Hedgecock EM, Brenner S, Kondo K, Hodgkin JA

[Worm Breeder's Gazette, 1988]

unc-51(e369) was found to be an amber allele by Ed Hedgecock. Throughout life time, unc-51 animals are severely uncoordinated, especially in the posterior part of the animal. Cross suppression tests and reversion experiments (by EMS) were carried out. Revertants isolated by Sydney Brenner (EMS) and by Ed Hedgecock (EMS) were also analyzed by oligomer Southern to detect the single base change at the anticodon of tRNA(Trp) genes. For cross suppression tests, an unc-51; as constructed for each suppressor (except *'s), and evaluated for the extent of restoration of movement. Results are summarized below: [See Figure 1] These results show that the suppression pattern of unc-51 by the eight known suppressors is very similar to that of unc-13: it is suppressed effectively by only sup-5 and sup-7, weakly by sup-24 and not detectably suppressed by others. unc-13 affects the nervous system (I. Maruyama, WBG vol. 9, #3, 33). An amber allele of unc-52 which is presumably expressed in muscle cells is suppressed to a similar extent by each of seven tRNA(Trp) suppressors (sup-29 was the only one not active). The suppression pattern might suggest that unc- 51 is not a muscle-gene but a neuronal-gene, consistent with the observation that the unc-51 mutant animal has normal muscle structure. To date about 130 revertants from unc-13, dpy-20, n analyzed. Eight out of 12 tRNA(trp) genes have been converted to amber suppressors (same as WBG vol.10, #1, 54), but four are still silent (two are probably pseudogenes).

Kariya K-i et al. (1995) Worm Breeder's Gazette "The Novel Ras-binding Protein Ach-l is Associated with Centrosomes"

Akasaka K, Shima F, Yamawaki-Kataoka Y, Shibatohge M, Kataoka T, Goshima M, Kariya K-i, Suzuki N, Okada T, Watari Y

[Worm Breeder's Gazette, 1995]

Ras proteins have distinct downstream effectors, adenylyl cyclase in S. cerevisiae (1), Byr2 in S. pombe (2), and Raf-1, B-Raf, Ral GDS, and PI-3 kinase in higher organisms. Both yeast Ras2 and mammalian H-Ras proteins stimulate yeast adenylyl cyclase in a GTP-dependent manner in vitro, and post-translational modifications of Ras proteins and intact leucine-rich repeats (LRR) domain in cyclase are necessary for full stimulation (3,4,5). We have found that the GTP-Ras-stimulated cyclase activity was subject to competitive inhibition by the LRR domain of cyclase and N- terminal domains of Byr2 and Raf-1 (2,6). Kinetic analyses of the inhibition patterns showed that these molecules bound directly to GTP-Ras proteins with Kd values (about 10 nM) comparable to that of whole adenylyl cyclase. Strangely, these Ras-binding domains do not share structural homology. The absence of homology among Ras effectors suggest that a yeast adenylyl cyclase-type effector may exist in higher organisms. During the C. elegans genome sequencing project, a set of possible exons coding for a putative 380-amino acid residues polypeptide with a striking homology to the yeast adenylyl cyclase LRR domains were discovered (7). To determine the whole structure of the corresponding protein, we isolated overlapping cDNA clones which covered the entire protein-coding sequence. The protein termed Ach (adenylyl cyclase homologue)-1 consisted of a fusion between an N-terminal domain homologous to the yeast adenylyl cyclase LRR and a C- terminal domain bearing homology to gelsolin and villin. We have found that Ach-1 bound directly to the GTP-bound form of yeast Ras2 through its N-terminal domain in vitro with a Kd value (11 nM) similar to those of Byr2 and Raf-1 (8). Also, overexpression of Ach-1 suppressed heat shock sensitivity of yeast cells bearing RAS2(Val-l9) gene similarly to the LRR domain of cyclase and N-terminal domains of Byr2 and Raf-1. Ach-1 bound to actin-agarose through its C-terminal domain in vitro in a Ca2+-independent manner, and was co-purified with yeast actin when expressed in yeast. These results suggest that Ach-1 may represent a yeast adenylyl cyclase- type effector of Ras proteins in higher organisms involved in regulation of actin cytoskeleton. During the course of the characterization of Ach-1, Campbell et al. reported the cloning and structure determination of a Drosophila flightless-1 (fly-1) gene (9), and we immediately realized that the ach-1 gene is the C. elegans homologue of the fly-l gene. Mutations of the fly-1 gene result in abnormal cellularization, defects of gastrulation and flight muscle degeneration. Recently, deletion of one copy of human homologue of this gene has been demonstrated in patients with Smith- Magenis syndrome, which is associated with a variety of clinical features including developmental delay, dysmorphology and behavioral problems (10). These observations strongly suggest essential roles of Ach-1 in development. To address the roles of Ach-1 in development, we have determined the subcellular localization of this protein in dividing C. elegans embryos by immunofluorescence confocal laser scanning microscopy with an affinity- purified antibody. Strikingly, Ach-1 was associated with centrosomes in a cell-cycle-dependent manner (Figure 1). Ach-1 accumulated in the area of pericentriolar material during mitotic phase but was barely detectable during interphase. Accumulation of Ach may to be related to the initiation of astral microtubules rather than spindle microtubules, since Ach was found during the formation of sperm asters but not with meiotic spindles. Ach- 1 may be involved in initiation of astral microtubules or in signaling from centrosomes to cortical actin filaments through astral microtubules. Genetic studies in C. elegans and biochemical studies in Xenopus are underway to further clarify cellular functions of Ach-1 and their Ras- and cell-cycle-dependent regulation. References: 1. Kataoka et al., Cell 43, 493, 1985 2. Masuda et al., J. Biol. Chem. 270, 1979,1995 3. Suzuki et al., Proc. Natl. Acad. Sci. USA 87, 8711, 1990 4. Kuroda et al., Science 259, 683, 1993 5. Wang et al., Mol. Cell. Biol. 13, 4087, 1993 6. Minato et al., J. Biol. Chem. 269, 20485, 1994 7. Sulston et al., Nature 356, 37, 1992 8. Yamawaki-Kataoka et al., submitted 9. Campbell et al., Proc. Natl. Acad. Sci. USA 90, 11386, 1993 10. Chen et al., Am. J. Genet. 56, 175, 1995 See WBG for figure. Figure 1. A C. elegans embryo at metaphase of the first mitosis co-stained with an anti-tubulin antibody (left) and an ant-Ach-1 antibody (right).

Gengyo-Ando K et al. (1988) Worm Breeder's Gazette "head piece of paramyosin and progress of tropomyosin gene cloning."

Kagawa H, Sugimoto K, Gengyo-Ando K

[Worm Breeder's Gazette, 1988]

Complete nucleotide sequence data of paramyosin gene, unc-15 will appear in the EMBL/GenBank/DDBJ Nucleotide Sequence Databases under the accession number X08068. By the friendly information from St Louis, Larry Shriefer and Bob Waterston, a head piece of paramyosin was extended a little bit. Finally, the gene is composed of 10 exons encoding a protein of 866 amino acid with molecular mass of 99,890. The gene had no TATA-box and promoter like sequences at 5' upstream as other muscle genes of the worm. Manuscript entitled 'The paramyosin gene (unc-15) of Caenorhabditis r cloning, nucleotide sequence and models for thick filament assembly' collaboration with Andrew D. McLachlan, Sydney Brenner and Jonathan Karn was submitted to J. Mol. Biol. cDNA clone of tropomyosin gene was cloned from a cDNA library (48hr sample) which was constructed by Julie Ahringer and Judith Kimble by using a tropomyosin gene fragment of C. elegans as a probe. The fragment of probe was cloned by screening an exon expression plasmid library (1985 C. elegans meeting abstract) with anti-tropomyosin antibody. Cloned 1.6kb fragment encoded the 6th to 241th amino acid residue of tropomyosin. The amino acid sequence was very conserved. To clone a complete fragment of the gene of genomic lambdoid phage library (kindly supplied by Claudia Cummins and Phil Anderson) was screened by using 1.6kb of cDNA clone as a probe. Ten positive clones were processed and mapped on the cosmid library by Alan Coulson and John Sulston. Unfortunately, all clones was contained the identical sequence to other part of unrelated cDNA (Worm gazette, Vol. 10, No. 1) which located the upstream of the cDNA clone of tropomyosin. The junction sequence of the both fragment was as follows. We are not sure why tropomyosin gene was not contained in the genomic library. [See Figure 1] Tropomyosin gene will be cloned from a genomic library which will construct with the genomic restriction fragment of the correct size. Original gene of cloned fragment of other part leaves in question.

Lobel L et al. (1993) Worm Breeder's Gazette "Mapping the Alpha1 and Beta Subunits of the C. elegans Voltage-Gated CalciumChannel"

Glossmann H, Lobel L, Horvitz HR, Grabner M

[Worm Breeder's Gazette, 1993]

We recently reported (1993 C. elegans meeting) the cloning of unc-36 ,a gene that encodes a protein similar to the alpha2 subunit of the voltage-gated calcium channel from mammals. Since then we isolated DNA clones by PCR encoding homologous sequences to portions of the alpha1 and beta subunits of the calcium channel. The alpha1 clone is 1 kb in length and has 52% identity and 74% similarity to a rodent alpha1 subunit. The beta clone is only 250 bp in size and has 68% identity and 76% similarity to a human beta clone. We have used these clones to probe the YAC polytene filters (provided by Alan Coulson). Our results indicate that there are at least two loci encoding an alpha1 homologous peptide, and at least five loci encoding beta homologous peptides. Our results are summarized below. There are at least two loci encoding alpha1 subunits: a) LGIV near eat-12 /egl-19 .Positive YACs Y51C8 and Y49F12 .Positive cosmids C11E10 ,K11C12 ,B0496 ,and C48A7 .b) LGII between fer-15 and rol-6 .Positive YAC Y24H1 . There are at least five loci encoding beta subunits: a) LGI near ces-2 .Positive YACs Y62C9 and Y65E10 .b) LGIV near let-280 ,let-281 ,let-282 ,let284 .Positive YACs Y53H3 and Y38A7 .c) LGV near ric-4 .Positive YACs Y47H11 ,Y22G9 and Y54B9 .Positive cosmid C14H1 .d) LGX near vit-3 ,vit-4 .Positive YAC Y60B6 .e) LGX between unc-6 and dpy-7 .Positive YACs Y51D11 and Y53D9 . The identification of multiple loci encoding alpha1 and beta homologous peptides agrees well with the variety of voltage-gated calcium channels being isolated from mammals. It has been demonstrated, for example by Campbell's laboratory (ref. 1 and personal communication), that the physiological classification of voltage-gated calcium channels (i.e. L and N) corresponds to slight antigenic variation between the subunits of these channel types. Our identification of multiple alpha1 and beta subunit loci may be a manifestation of the variety of voltage-gated channels in C. elegans. We plan to screen a cDNA library with these clones and use the cDNAs to produce subunit specific antibodies. We are also testing previously isolated mutants that map near the above loci for defects in these calcium channel subunits. l) Witcher D., De-Waard M., Sakamoto J., Franzini-Armstrong C., Pragnell M., Kahl S.D., Campbell K.P. Science, 261, 486,1993.

Hodgkin JA (1986) Worm Breeder's Gazette "observations on strains with high Tc1 copy number."

Hodgkin JA

[Worm Breeder's Gazette, 1986]

In order to identify Tc1 polymorphisms close to the sex-determining gene tra-1 III, congenic strains carrying the interval vab-7 - dpy-18 ( markers that flank tra-1) from several different high copy number strains have been constructed. The first of these, using the Bergerac strain N62, revealed several polymorphisms mapping between tra-1 and dpy-18 (see WBG 8-1: 45). However, neither of the strains DH424 and TR403 appear to contain any extra Tc1 sites in the vab-7 - dpy-18 interval. Also, unique probes derived from the two of the N62 polymorphisms were used to show that the N62 insertion sites are vacant in another Bergerac strain, RW7000. Thus, different high copy number strains may have markedly different Tc1 patterns in a given interval. Carol Trent reported at the 1985 C. elegans Meeting that a strain carrying Sma-1(e30), derived from N2 by Sydney Brenner, had considerably more than thirty copies of Tc1 (the normal N2 number). The Cambridge working stock of Sma-1(e30) (designated CB4000) has been grown continuously at 15 C for more than fifteen years, by a succession of workers at the MRC. DNA from this stock was compared with DNA from the oldest available stock of e30 (designated CB30, formerly E30), which has been stored frozen since approximately 1969. CB30, which is the ancestor of CB4000, has a Tc1 pattern (EcoRI digest) identical to N2, but CB4000 has many more copies of Tc1 (more than 100, i.e. a smear resembling that seen with DH424 or TR403). The CB4000 strain differs from CB30 in other ways: it grows poorly and has a slightly different small phenotype, males are unable to mate, the strain is slightly Him, and it has a mutator activity (twitchers and other mutants are occasionally segregated). Therefore it seems that a Tc1 mobilization event took place at some point during the maintenance of e30 as a 15 C stock, before 1975 but after the original stock, CB30, had been frozen. So, who was Prince Charming, and how did he wake up Sleeping Beauty?

William C Campbell (2001) Worm Breeder's Gazette "Thiabendazole (with dimethyl sulfoxide and methylene blue) - as a possible aid in the 'limited contamination' culture of Caenorhabditis - elegans."

William C Campbell

[Worm Breeder's Gazette, 2001]

In the routine propagation of C. elegans, steam sterilization of the culture medium is standard practice, but in some circumstances it is inconvenient, impractical, or prone to error. In preliminary experiments in this lab, thiabendazole (TBZ) was added to C. elegans cultures in the expectation that the life cycle would be interrupted because of the well-known efficacy of the compound in preventing the development and hatching of nematode eggs. Surprisingly, the propagation of C. elegans appeared to be unaffected. Subsequent investigation showed that TBZ is without ovicidal efficacy against C elegans at concentrations that are fully effective against the eggs of the parasitic nematode Haemonchus contortus (Fasiuddin and Campbell, 2000). The qualitative observations here reported suggest the possibility of exploiting this finding to achieve a useful degree of microbial control in laboratory cultures. TBZ was added to modified Nematode Growth Medium (Avery and Horowitz, 1990) at 20 microg/ml -- a concentration known to be ovicidal for nematodes (Egerton, 1969) and to have a broad spectrum of antifungal activity (Robinson et al., 1969). Methylene Blue, at 0.0002%, was added as a marker to prevent inadvertent mix-up in routine transfer operations (qualitative observations in this lab had indicated that such a concentration did not suppress the growth of the Escherichia coli seeded onto agar plates as food for C. elegans ). The medium was boiled briefly to dissolve and clarify the agar, but it was not autoclaved. TBZ and Methylene Blue were added while the medium was still hot. TBZ was prepared as a solution of 10 mg/ml in 100% dimethyl sulfoxide and added to the medium so as to give a 500 fold dilution. An aqueous solution of Methylene Blue, 0.1%, was similarly added to give a 500 fold dilution. After dispensing the medium into 90-mm petri dishes, a suspension of E. coli (OP 50) was spread on the solidified medium and allowed to form a bacterial lawn in the usual way. Three days after the initial (October 8, 1999) preparation of the plates, 2 plates were inoculated with C. elegans ; and single plates were inoculated with worms on day 28, 46, 55, 63 and 88. The plates were held in a humidity chamber at room temperature, without further addition of bacteria. Periodic microscopic examination of the dishes revealed prolific propagation of C. elegans , followed by the persistence of low numbers of motile worms (presumably dauer larvae) at intervals up to day 367 after inoculation of the first 2 plates. At that time, medium from each dish was transferred, without sterile technique, to conventional agar plates, resulting in abundant propagation of worms in all cases. Another batch of 17 culture plates was prepared in a similar non-aseptic way, but without the addition of Methylene Blue. This batch has remained in a refrigerator for 9 months, during which time small compact colonies (presumably bacterial) have appeared, but there has been no trace of the mycelial fungal contaminants so commonly observed when sterilization and handling procedures have been imperfect. The preparation of these two batches of non-sterilized culture plates was not accompanied by the simultaneous preparation of autoclaved plates, or plates with dimethyl sulfoxide or Methylene Blue as the only additive; but throughout the test period conventional plates, without additives, were being routinely made and used. Nor was any attempt made to expose the plates to known air-borne contaminants or to inoculate them with bacteria or fungi. Nevertheless, the absence of visible mycelial growth was in marked contrast to the degree of contamination customarily seen when sterility measures have been less than rigorous, and the year-long persistence of C. elegans was also striking. If these findings can be confirmed in quantitative trials, supplementation of a medium with TBZ in a dimethyl sulfoxide vehicle (with an antibacterial adjunct) may provide a convenient 'limited contamination' medium for the routine maintenance of C. elegans . Other antimycotic agents are available for use in culture systems. In the present context, the most useful characteristics of TBZ are its very long shelf life at room temperature, its stability in media when boiled or even autoclaved, and the degree to which this otherwise antinematodal agent is tolerated by the nematode C. elegans . Methylene Blue has some degree of antibacterial efficacy and, at the concentration used here, was tolerated by both the worm and the bacterium on which it feeds. Other compounds with selective antibacterial activity may be more effectively combined with TBZ to suppress unwanted bacteria in C. elegans cultures. References. Avery, L and H. R. Horvitz. 1990. Effects of starvation and neuroactive drugs of feeding in Caenorhabditis elegans . J. Experimental Zoology 253: 263- 270. Egerton, J. R. 1969. The ovicidal and larvicidal effect of thibendazole on various helminth species. Texas Reports on Biology and Medicine (Suppl. 2) 27: 561 - 580. Fasiuddin, A. and W. C. Campbell. 2000. Haemonchus contortus (Nematoda: Trichostrongylidae) is much more sensitive than Caenorhabditis elegans to the ovicidal action of thiabendazole. Journal of Parasitology 86: 628-630. Robinson, H. J., R. H. Silber and O. E. Graessle. 1967. Thiabendazole: toxicological, pharmacological and antifungal properties. Texas Reports on Biology and Medicine (Suppl. 2) 27: 537-560.

Miller DM et al. (1993) Worm Breeder's Gazette "Dominant Mutations in unc-37 Suppress the unc-4 Neuronal Wiring Defect(or bkn-1 Bites the Dust)"

Miller DM, Niemeyer CJ, Chitkara P

[Worm Breeder's Gazette, 1993]

We are interested in the question of how neurons choose synaptic partners. In unc-4 mutants, VA motor neurons receive synaptic input normally reserved for their sister cells the VB motor neurons. As a result of the lost input to the VA's, unc-4 mutants are unable to crawl backwards (1). unc-4 encodes a homeodomain protein (2) that is expressed in VA but not VB motor neurons (WBG 12, #3, p80 )which suggests that unc-4 may regulate the expression of some feature of the VA's that allows presynaptic interneurons to distinguish them from their VB lineage siblings. We have previously described a selection strategy for isolating suppressors of the temperature sensitive allele unc-4 (e2322ts)(WBG 12, #3, pp 107-108) as a means of identifying the presumptive unc-4 target gene. [unc-4 (e2322ts)contains a Leu to Phe substitution in the unc-4 homeodomain.] Four dominant, extragenic suppressors and four dominant, intragenic revertants were isolated. All of these suppressors/revertants appear essentially wild type in an unc-4 (ts)background which suggests that the normal pattern of input to VA motor neurons has been restored. The extragenic suppressors are within 0.07 map units to the left of unc-87 suggesting that they are allelic. This interval includes the unc-37 locus. Sydney Brenner isolated unc-37 (e262)and described it as a "coiler." We have noticed that unc-37 (e262)is also a phenocopy of unc-4 (0)mutants; neither can crawl backwards and both coil dorsally (vulva on outside of loop) when touched on the head. This suggested that the unc-4 suppressors are likely to be unc-37 (gf)mutations. To test this idea, we screened for revertants of the suppressor unc-37 (wd17).EMS-mutagenized dpy-5 (e61)unc-37 (wd17 )I;unc-4 (e2322ts)IIhermaphrodites were mated at 25 C with unc-4 (e2322ts)males [grown at 16 C]. From ~10,000 F1 'swe obtained two revertants, unc-37 (wd17wd19)and unc37 (wd17 wd20 ).Both fail to complement unc-37 (e262).We propose therefore, that the unc-4 suppressor mutations are in fact, unc-37 (gf)alleles. unc37 (wd17 wd19 )and two other recently isolated unc-37 alleles (3) are recessive lethals which suggests that unc-37 (0)is lethal. Perhaps unc-37 corresponds to an unc-4 target gene encoding a cell surface receptor expressed in VA motor neurons and recognized by specific presynaptic interneurons. Alternatively, unc-37 could encode a cofactor that interacts with the unc-4 homeodomain. In either case, however, unc-37 is also likely to provide essential functions in other cell types as well. We are now microinjecting cosmids to rescue unc-37 (e262).

Riddle DL (1976) Worm Breeder's Gazette "genetic suppression in muscle mutants."

Riddle DL

[Worm Breeder's Gazette, 1976]

Suppressor mutations compensating for defects in the body musculature of C. elegans have been studied in collaboration with Sydney Brenner. Suppressor mutants were found among EMS-induced revertants of the unc-54 mutants, E1258 and E1315, and the unc-15 mutant, E73. Nine independent suppressor mutations were judged to lie at a single suppressor locus, sup-3. The sup-3 locus is located 3.8% to the right of dpy-11 on LG V. Suppression is dominant, but dose- dependent, and results in improved locomotion as well as an increased ability to lay eggs. Mutations in 6 genes known to affect muscle assembly or function ( unc 15, 22, 52, 54, 66, 87) were tested for suppression by three representative suppressor alleles. Mutations in unc 15, 54, and 87 are suppressed. There is no indication of allele-specific suppression, except that dominant or semi-dominant mutants are generally not suppressed. It seems that among unc-54 alleles at least, those mutants producing no unc-54 myosin are suppressed best, while those mutants producing the full amount of protein are not suppressed. Quantitative differences were detected between independent suppressor isolates based on locomotion and on egg laying. Additionally, suppression of E73 is invariably stronger than suppression of any unc- 54 or unc-87 allele tested. These factors, coupled with the dose- dependence of suppression, gives rise to the widest possible range of suppressed phenotypes. In the case of weak suppressor alleles like e1390, heterozygous-suppressed unc-54 mutants are hardly distinguishable from the unc-54 mutant itself. At the other extreme, E73 homozygous for a strong suppressor like e1407 is almost indistinguishable from wild-type in its movement. With one notable exception, suppressor mutations are non-lethal and produce no obvious phenotype when they are placed in a wild-type genetic background, although e1407 homozygotes exhibit a marginal (approx. 20%) decrease in brood size when compared with N2. The exceptional case is e1405, a suppressor allele which is lethal when homozygous. Genetic tests revealed that e1405 is a small deletion encompassing unc-41 and unc-42, two genes adjacent to the sup- 3 locus. The e1405 mutation fails to complement unc-41 or unc-42 mutations. While unc-41 and unc-42 recombine with each other at a frequency of 0.1%, they fail to recombine with e1405 at the level of 10+E-5 . The lethality of the e1405 suppressor may be due to deletion of these or other adjacent genes. Of the 9 suppressor mutants isolated, e1405 is the only one having the characteristics of a deletion. Whether e1405 was induced by EMS or arose spontaneously in the reversion experiment is unknown. However, no revertants have arisen spontaneously in unmutagenized cultures of unc-15 or unc-54 mutants. To my knowledge, this is the first deletion characterized in C. elegans.The significance of the e1405 mutation is that it implies that a 50% reduction in the level of sup-3 gene product ( in e1405/+heterozygotes ) leads to suppression. The high frequency of suppressor mutants (approx. 10+E-4 ) arising as E73 revertants supports the idea that suppression results from a general knock-out of sup-3 gene function rather than a specific alteration in gene activity.

White JG et al. (1985) Worm Breeder's Gazette "alterations in the synaptic connections made by VAn ventral cord motoneurons in mutants of unc-4."

White JG, Southgate E, Thomson JN

[Worm Breeder's Gazette, 1985]

Mutants in the gene unc-4 were first isolated by Sydney Brenner in 1969 and since that time they have become well known and loved genetic markers. The phenotype of these mutants is quite characteristic, they are healthy and grow well but have a striking locomotory defects They can move forward, albeit with a certain amount of difficulty, but, when provoked to move backwards by a tap on the head, the body coils up with the dorsal side innermost. The tail does not coil up in these circumstances and usually curves in the opposite sense We have reconstructed regions of the ventral cord from three unc- 4( e120) animals. The morphology and disposition of motoneurone processes is much the same as is seen in wild type animals There is a striking difference in the synaptic connections that are made onto certain VAn motoneurones however. Normally VAn motoneurones receive chemical synapses from AVA, AVU and AVE interneurones and also make gap junctions to AVA. In unc-4(e120) animals VA2, VA3 and VA10 motoneurones do not make these synapses, but instead make prominent gap junctions onto AVB interneurones, which is characteristic of the connections made by VBn and DBn motoneurones. Thus, although these neurones have the same morphological features as VAn motoneurones (eg. have anteriorly directed axones), they make synaptic connections that are appropriate to VBn motoneurones. Only certain VAn motoneurones are affected, neurones at either end of the cord (VAl, VAll and VA12) are normal (VA4 to VA9 have not been reconstructed). In wild type animals VAn and DAn motoneurones receive the same synaptic inputs and respectively provide excitatory input to ventral and dorsal body muscles during forward locomotion. Conversely, VBn and DBn motoneurones inervate the ventral and dorsal body muscles during backward locomotion (Chalfie et al. 1985, J. Neuroscience, 5: 956). The behavioral phenotype of unc-4 is consistent with its anatomical phenotype; forward locomotion is presumably mediated by the VBn and DBn neurones in the normal way however there will be a certain amount of interference from the transformed VA neurones which will presumably be also activated because they have the same synaptic inputs as VBn and DBn neurones. During backward movement the transformed VAn motoneurones will not be activated. This will have the consequence that only dorsal muscles will receive excitatory input in the middle regions of the body, giving rise to the dorsal coiling behavior that is observed. The tight coil of the body additionally suggests that VA4 to VA9 are also transformed. The reverse curve of the tail is presumably a reflection of the normal VA11 and VA12 neurones. It is interesting to consider why only certain VAn neurones are transformed. Leakiness is an unlikely explanation because the same behavioral phenotype as unc-4(e120) is seen in animals with two trans deficiencies that overlap in the unc-4 region (Sigurdson et al. 1984, Genetics 108: 331), suggesting that this represents the null phenotype. A possible clue is that all the transformed VAn neurones are sisters of VBn neurones whereas none of the untransformed VAn, or for that matter DAn neurones, are sisters of VBn or DBn neurones. The significance of this coincidence is not clear, but it does provide food for speculation.