Andrews N et al. (1999) Worm Breeder's Gazette "Vulval Development-- Structure/Function Analysis of the Winged-Helix Transcription Factor LIN-31"

Andrews N, Doroquez DB, Hess H, Miller LM

[Worm Breeder's Gazette, 1999]

The lin-31 gene encodes a winged-helix transcription factor involved in specifying vulval cell fates (Miller et al., 1993, Genes and Development 7:933). The LIN-31 protein contains a DNA-binding domain, an acidic region, a serine-rich region, and a proline-rich region. To elucidate possible functions of these domains, the 4 kb genomic domain spanning the lin-31 coding region was sequenced in 16 lin-31 mutant alleles. This sequencing revealed 6 nonsense mutations, 3 deletions, 4 transposon insertions, 1 frameshift mutation, and 2 missense mutations. All six nonsense mutations were found in the DNA binding domain. No late nonsense mutations were observed, indicating that a LIN-31 protein truncated late in the protein may not result in a phenotype detectable by standard Muv and Vul screens. The two missense mutations are located at conserved residues in the DNA-binding domain. It is important to determine if the amino acid substitutions result in a destabilized protein or if the encoded mutant proteins are defective because they are no longer able to bind their DNA target. We have ruled out the first possibility by using antibody-binding experiments to show that the LIN-31 protein is present in the proper cells at the proper stage of development. Because all (or almost all) existing lin-31 alleles display a similar phenotype, these studies show that (1) the null phenotype of lin-31 is the phenotype displayed by most, if not all, of the existing alleles, (2) direct screens for multivulva and vulvaless mutants will probably only yield null (or strong) alleles of lin-31, and (3) the DNA-binding domain plays a critical role in LIN-31 function.

De Stasio E et al. (1994) Worm Breeder's Gazette "Mutagenesis of C. elegans Using N-ethyl-N-nitrosourea."

De Stasio E, Stanislaus D, Lephoto C

[Worm Breeder's Gazette, 1994]

Mutagenesis of C. elegans using N-ethyl-N-nitrosourea Elizabeth De Stasio, Dinesh Stanislaus and Catherine Lephoto. Department of Biology, Lawrence University, Appleton, Wl 54911

Bakaev VV (1995) Worm Breeder's Gazette "THE EFFECT OF INTERMITTENT TEMPERATURE ON LONGEVITY AND FECUNDITY OF C. ELEGANS"

Bakaev VV

[Worm Breeder's Gazette, 1995]

It is well known that low temperatures can prolong longevity of different animals. In this study the experimental worms were mantained in liquid medium with E. coli in +21 C during the day (8-20 hrs) and in +4 C during the night, in darkness. One control group was mantained in +21 C and other control group was mantained in +4 C constantly. The obtained results are presented in the following table. ......................................................................... Control group Experimental Control group (+21C) group (+4C) Mean +/- S.D. Mean +/- S.D. Mean +/- S.D. .......................................................................... Mean longevity (days) 19,86 +/- 1,63 22,96 +/- 1,57 38,30 +/- 2,72 (n = 22) (n = 24) (n = 22) Maximal longevity (days) 34 35 50 Minimal longevity (days) 6 10 5 Mean fecundity 76,91 +/- 4,54 54,33 +/- 3,32 4,45 +/- 2,07 (n = 22) (n = 24) (n = 22) Maximal fecundity 118 95 46 Minimal fecundity 33 25 0 .................................................................... It can be concluded that such intermittent temperature is not able to prolong the life-span of C. elegans significantly, in comparison with constant cold, as well as fecundity. Acknowledgment: The author wishes to express his thanks to CGC for providing C. elegans (wild line) and E. coli OP50.

Ray C et al. (1995) Worm Breeder's Gazette "Evidence for Proteolytic Processing of a Cuticle Collagen in a Plant-Parasitic Nematode"

Hussey RS, Ray C

[Worm Breeder's Gazette, 1995]

Collagen is the major structural component of nematode cuticles. In Caenorhabditis elegans, the N-terminal regions of cuticle collagen proteins contain the highly conserved motif, R-X-X-R, which has been predicted to contain a potential subtilisin-like proteolytic processing site required for cleavage of the collagen proteins and normal cuticle collagen function (Kramer, 1994, FASEB 8:329-336). Cuticles of adult females of the plant-parasitic nematode Meloidogyne incognita contain a 76 kDa collagen which comprises over 50% of the B- mercaptoethanol-soluble cuticular proteins (Reddigari et al., 1986, J. Nematol. 18:294-302). We determined the N-terminal amino acid sequence of this major collagen and found it to be identical to the predicted amino acid sequence, starting at amino acid number 66, of the M. incognita Lemmi 5 cDNA clone (Van der Eycken et al., 1994, Gene 151:237-242) (Fig.1). A putative subtilisin-like protease recognition site was found immediately upstream of the region of amino acid homology between LEMMI 5 and the N-terminal sequence of the 76 kDa collagen (Fig.1). Our data support previous speculation about the existence of this novel method of collagen maturation and provide further evidence that this mechanism has been conserved during nematode evolution. In addition to protein processing, the expression of the Lemmi 5 gene was transcriptionally regulated: Lemmi 5-specific transcripts were present in adult females but not in eggs or second-stage juveniles. Also, Lemmi 5 analogs were present only in four Meloidogyne species, but not in C. elegans, Heterodera glycines, or tomato. .. PREDICTED LEMMI5 AMINO ACID SEQUENCE .. 1M A T L V V M P Q L Y S Q I N D L N L R V R D G V Q A .. F R V N T D S A W N D L M E L Q V A V T P Q S K P R S * N P F Q S L YR Q K RS L P D Y C I C Q P L E I N79 S L P D Y C I C Q P L E I N ............................................................ CONFIRMED N-TERMINAL AMINO ACID SEQUENCE .............................................................. Figure 1. The partial predicted amino acid sequence (residues 1-79) of the Lemmi 5 collagen gene is shown in roman letters. The putative subtilisin-like protease recognition sequence, R-Q-K-R, is boxed and the first amino acid after the proteolytic cleavage site is indicated by an *. The empirically determined N-terminal sequence of the 76 kDa from adult female M. incognita is italicized.

Ouazana R (1979) Worm Breeder's Gazette "some enzymatic comparisons between Bergerac and Bristol strains of C elegans."

Ouazana R

[Worm Breeder's Gazette, 1979]

To compare enzymatic activities of Bergerac and Bristol strains of C. elegans, we used the microsystem ' Api Zym ' focused by D. Monget ( 1978) and produced by the french firm API SYSTEM. Some of these tests, not yet commercialized, were a gift of API SYSTEM. The technique consists in microdishes which are impregnated with substrate and buffer ( PH is given in the table ). We introduced 0.1 ml of biological suspension ( a known number of adult worms sonicated in buffer and resuspended in distilled water after homogenization ) in the microdishes which were incubated at 37 C for 4 hours. Bacteria were grossly eliminated by washing; Afterwards their concentration was too low to give response with Api Zym. The reactions were revealed colorimetrically by 4 /oo ' Fast blue BB '. The intensity of the reaction is proportional to enzymatic activity and is noted from - to +++++ . A reaction noted +/- refers to a low but detectable response. To give reliable results, all the tests were read by the same experimenter and enzymatic activities in Bergerac and Bristol strains were performed at the same time. To date, the tests were carried out on 64 enzymatic activities. Most of the enzymes tested were hydrolases ( phosphatases,esterases, aminopeptidases, glycosidases, arylsulfatases ) and some were dehydrogenases. Some of the enzymatic activities tested seem to take a prominent part in postembryonic development. It's the case of Leucine arylamidase, which plays an important part in molting ( Rogers 1965, 1977 ), and could be also the case of glycyl-L-proline arylamidase which is implicated in the metabolism of collagen. The results are summarized in the following table. The two strains show similar enzymatic spectra, but differences can be noted, which are localized essentially on : - Trypsin ( n 8 ) - gamma L-glutamate transpeptidase ( n 38 ) - Glycyl-glycine arylamidase ( n 42 ) - L-threonine arylamidase ( n 54 ) - N-CBZ-glycyl-glycyl-L-arginine arylamidase ( n 56 ) Other differences, in a less degree, but yet detectable, are observed on: - Alkaline Phosphatase ( n 1 ) - L-histidine arylamidase ( n 33 ) - L-leucyl-glycine arylamidase ( n 45 ) - L-ornithine arylamidase ( n 51 ) - L-serine arylamidase ( n 53 ) All the differences observed in this table have been verified in two repetitions, which have similar results. But it could be argued that these differences could be due to nutritional adaptation, because these two strains, xenically cultured on the A1 medium ( for thirty years in case of Bergerac, and one year in the case of Bristol ) are associated with a different bacterial complex. [See Figure 1]

Wood WB (1976) Worm Breeder's Gazette "maternal effects among ts zygote-defective (zyg) mutants."

Wood WB

[Worm Breeder's Gazette, 1976]

We have studied maternal effects in 23 zyg ts mutants to estimate the times of expression of genes whose products are required in embryogenesis. We have used the following three tests, called arbitrarily A, B, and C. A test: Heterozygous (m/+) L4's are shifted to 25 C and allowed to self-fertilize. If 100% of their eggs yield larvae (25% of which express the mutant phenotype as adults), then the mutant is scored as maternal (M). If 25% of the F1 eggs fail to hatch, then the mutant is scored as non-maternal (N). An M result indicates that expression of the + allele in the parent allows m/m zygotes to hatch and grow to adulthood. A result of N indicates the opposite: that the + allele must be expressed in the zygote for hatching to occur. Out of 23 zyg mutants tested, 3 were scored N and 20 were scored M in the A test. Therefore, for most of the genes defined by these mutants, expression in the parent is sufficient for zygote survival, even if the gene is not expressed in the zygote. B test: Homozygous (m/m) hermaphrodites reared at 25 C are mated with N2 (+/+) males. If eggs fail to hatch at 25 C, but mated hermaphrodites shifted to 16 C produce cross progeny to give proof of mating, then the mutant is scored M. If cross progeny appear in the 25 C mating, then the mutant is scored N. An M result indicates that expression of the + allele in the zygote is not sufficient to allow m/+ progeny of an m/m hermaphrodite to survive. Conversely an N result indicates either that zygotic expression of the + allele is sufficient for survival, or that a sperm function or factor needed for early embryogenesis can be supplied paternally (see C test below). Out of the 23 zyg mutants tested, 11 were scored M and 12 were scored N. The combined results of A and B tests and their simplest interpretation are as follows. Ten mutants are M,M; the genes defined by these mutants must be expressed in the hermaphrodite parent for the zygote to survive. Ten mutants are M,N; these genes can be expressed either in the parent or in the zygote. Two mutants are N,N; these genes must be expressed in the zygote. One mutant is N,M; this gene must be expressed both in the maternal parent and in the zygote. C test: Homozygous (m/m) hermaphrodites reared at 25 C are mated with heterozygous (m/+) males. If rescue by a +/+ male in the B test depends on the + allele, then only half the cross progeny zygotes of a C test mating (m/+ male x m/m hermaphrodite) should survive. However, if rescue depends on a function or cytoplasmic component from the male sperm, then all the cross progeny zygotes in a C test should survive. Of the 10 M,N mutants, 6 have been C tested; one exhibited paternal rescue independent of the + allele. The A and B tests also were carried out on 16 mutants that arrest before the L3 molt (acc mutants). In the A test on 2 of these mutants, all m/m progeny of m/+ parents grew to adulthood at 25 C. Therefore, parental contributions are sufficient to overcome a progeny mutational block as late as the L2 stage. All 16 acc mutants scored N in the B test.

Schriefer LA et al. (1988) Worm Breeder's Gazette "paramyosin phosphorylation, part II."

Schriefer LA, Waterston RH

[Worm Breeder's Gazette, 1988]

In the vol.10, number 2 edition of The Wormbreeder's Gazette, we reported that paramyosin is phosphorylated in vitro under low salt conditions by an endogenous kinase in a reaction that is Ca++ independent. Thin layer chromatography indicated that paramyosin contained phosphoserine. Phosphate assays suggested that in vivo paramyosin had 1.8 moles of phosphate per mole of paramyosin. To determine the sites of phosphorylation, in vitro phosphorylated paramyosin was digested by NTCB. A 15,000 Da fragment was phosphorylated which corresponded to the N-terminal fragment according to Hiro Kagawa's sequence data. To obtain a more precise localization, in vitro labelled paramyosin was digested by endoproteinase-Lys-C. 2 labelled HPLC fractions were obtained and subjected to protein sequencing. One however yielded no sequence, and since the N-terminus of paramyosin is known to be blocked, probably by acetylation, we guessed that this peptide included the blocked N-terminus. Amino acid composition of the peptide was determined. The second peptide was sequenced with no difficulty. This sequence and the amino acid composition data of the blocked peptide were compared with H. Kagawa's DNA sequence of the paramyosin gene, unc-15. They appear to arise from, and together define the N-terminus of paramyosin. [See Figure 1] With three prolines, two glycines, and eight serines(*), the N- terminal region that we have determined is decidedly not alpha-helical coiled-coil in structure and is substantially different then the remainder of C. elegans paramyosin (Kagawa's sequence) and myosin rod sequences. It would be interesting to determine what role the phosphorylation of the non-helical N-terminus of paramyosin plays in the interaction between paramyosin and other proteins in C. elegans' thick filament formation and function.

Christensen M et al. (1999) Worm Breeder's Gazette "Identification of a Novel Mechanosensitive Anion Current in Embryonic Cells of C. elegans*"

Strange K, Christensen M

[Worm Breeder's Gazette, 1999]

Mechanotransduction plays a central role in fundamental physiologic processes such as detection of touch and sound, regulation of cell volume, and control of motility. Mechanosensitive channels have been studied extensively using electrophysiology. Little is known, however, about the molecular structure of mechanosensitive channels or about how mechanical stress is transduced into altered channel gating. We developed techniques to patch-clamp and study ion channels in C. elegans embryonic cells. In cell-attached and inside-out patches, application of gentle suction activated a mechanosensitive current. Suction caused an immediate increase in current amplitude of 6.4 2.8 fold (n = 20) at +100 mV in inside-out patches. The current rapidly inactivated when suction was discontinued and could be repeatedly reactivated by additional suction. When membrane voltage was ramped from +100 to -100 mV at 100 mV/second, the current showed moderate outward rectification (outward:inward current = 2.0 0.05 at 100 mV). Current amplitude was largely unaffected when bath Na+ was replaced with NMDG+ (n = 10). However, replacement of bath Cl- with either gluconate or glutamate reduced inward and outward currents by 46 10% and 39 7%, respectively (n = 9). Replacement of 120 mM bath Cl- with a mixture of 60 mM Cl- and 60 mM SCN- increased the inward current at -100 mV by 3.0 0.4 fold and shifted Erev by 16 2 mV (n=7). We conclude that membrane stretch activates a novel mechanosensitive anion current in inside-out patches from C. elegans embryonic cells.

Wolf N et al. (1978) Worm Breeder's Gazette

Wolf N, Hirsh DI

[Worm Breeder's Gazette, 1978]

We have examined the early cleavages of our ts zyg mutants by Nomarski optics and measured the number of nuclei in the embryos as a function of time at restrictive temperature using Hoechst stain. We find that all of the strict maternal mutants (M,M) either have abnormal early cleavages or never progress beyond 200 nuclei or both. Two of the other zyg mutants display abnormal early cleavages. Interestingly these are the paternal mutant B235 (M,N,P) and the anomalous mutant B261 (M,N,?). None of the other mutants displays abnormal early cleavages or stops before 200 nuclei. The first cleavages can be schematically diagrammed as follows for N2 and for some of the mutants: [See Figure 1]

Otsuka AJ et al. (1991) Worm Breeder's Gazette "Comparison of the unc-104 Protein 'Motor' Domain with Members of the Kinesin Family of Proteins"

Tang LZ, Whiteaker K, Boontrakulpoontatwee P, Otsuka AJ, Zhang Y

[Worm Breeder's Gazette, 1991]

The unc-104 gene is capable of encoding a kinesin-related protein ( Otsuka et al., Neuron, in press). Comparison of the presumed unc-104 'motor' domain to the ATPase and microtubule-binding region of members of the kinesin family demonstrates the greatest sequence identity in the putative ATP-binding domain [GYN-TIFAYGQTGSGKTYTM-G] and in conserved elements of unknown function, some of which may be involved in microtubule-binding [GIIPR, VK-S(Y/F)-E(I/L)YNE, DLL, R-VA-T--N-- SSRSH-VF-I, SGKL-LVDLAGSE, GAEG-R--E--NINKSL--LG-VI-AL, HIPYR(D/E)SKLT- LLQ-SLGG, N--ET-STL-(F/Y)A-RAK--(R/K)]. In the figure below, residues that are identical in 5 or more sequences are listed below the compilation and asterisks mark residues that are identical in all sequences. [See Figure 1]