Hsu DR et al. (1992) Worm Breeder's Gazette "Dosage Compensation and a Very Small Protein"

Hsu DR, Meyer BJ

[Worm Breeder's Gazette, 1992]

dpy(y130 )is required for the hermaphrodite mode of X-chromosome dosage compensation. Homozygous y130 animals display a maternal-effect, XX-specific lethality characteristic of dosage compensation upsets. This lethality is temperature sensitive. dpy(y130 )differs from the other dosage compensation dpy genes in several respects. Unlike the other dpy genes involved in the control of dosage compensation, the dpy(y130 )maternal-effect lethality is completely penetrant at the restrictive temperature. In addition, dpy(y130 )appears to function in XO animals. y130 XO animals are viable, however, they are scrawny, slow growing and mating defective. In order to better understand how dosage compensation functions at a molecular level, we cloned dpy(y130 ).Groups of cosmids near myo-3 (0.1 map units away from y130 ),were assayed for their ability to rescue the maternal-effect lethality in germline transformation assays. Rescuing activity was localized to cosmid C09F3 .Subsequent rescue experiments narrowed the rescuing region to a 1.2 kb region of this cosmid. Northern blot analysis using this region as a probe detects a highly abundant 0.7kb transcript expressed throughout all stages of development. Using the minimal rescuing fragment as a probe, we isolated cDNA's corresponding to this transcript (Libraries from Stuart Kim and Serge Lichtsteiner). Sequence analysis of these cDNA's and the corresponding genomic region identifies a 372 nucleotide open reading frame encoding a 13kd protein (Figure 1). This protein appears to have no significant homology to anything in the protein databases. To confirm that this small ORF was indeed coding for the dpy(y130 )protein, frameshift mutations were introduced in the coding region. These frameshift mutations were not able to rescue the y130 maternal-effect lethality. Sequence analysis of the y130 mutation reveals a Gly to Arg change in the protein. Much to our dismay, sequencing of new alleles obtained in a non-complementation screen (y185 ,y186 )revealed that they result in the same nucleotide change. Thus the null phenotype of dpy(y130 )remains unclear. Current efforts are being directed at the isolation of new alleles in order to better define the null phenotype. [See Figure 1]

Hsu DR et al. (1988) Worm Breeder's Gazette "an autosomal gene translocated to the X chromosome acquires dosage compensation."

Hsu DR, Meyer BJ

[Worm Breeder's Gazette, 1988]

Hsu DR et al. (1987) Worm Breeder's Gazette "some X-linked deficiencies masculinize her-1(n695) XX animals."

Hsu DR, Meyer BJ

[Worm Breeder's Gazette, 1987]

n695 is a semi-dominant allele of her-1 which partially masculinizes XX animals. In an n695 homozygous strain, 100% of the XX hermaphrodites are egg-laying defective (Egl). A smaller subset of these animals are further masculinized and exhibit a partial transformation of the tail towards a more male fate (Tra) (Carol Trent, personal communication). These weak masculinizing effects of n695 are sensitive to small perturbations in the rest of the sex determination pathway. For example, n695 in combination with weak alleles of tra-2 masculinizes XX animals much more than either single mutation alone. In addition, duplications of X which have no phenotype in a her-1(+) background can suppress the masculinization caused by n695 (Anne Villeneuve, personal communication). Using the phenotype of n695 as an assay, we are attempting to identify regions of the X chromosome which interact with the sex determination pathway in a dose-dependent fashion by looking for X- linked deficiencies which increase the masculinization caused by n695. These deficiencies could interact with the rest of the sex determination pathway by removing a region of X that is important in the assessment of the X/A ratio. As a consequence, the apparent X/A ratio would be reduced and the animal would be directed toward a more male developmental fate. Alternatively, they might uncover a haplo insufficient gene that is required for hermaphrodite development in n695 XX animals, thus revealing a function that is likely to be important for proper sex determination in wild-type animals. Thus far, the n695-deficiency assay has localized several regions of the X chromosome, which when present in one copy, cause n695 XX animals to be highly masculinized. One masculinizing region, defined by the deficiency uDf1, results in the production of some n695 XX males which are able to mate. This deficiency, uDf1, has been shown by L. Miller to uncover the gene xol-1. This result is in agreement with the previous finding that some n695; xol-1(y9)/+ XX animals are transformed into males capable of mating (L. Miller, personal communication). Other masculinizing regions have been identified on the right arm of X. We have looked at the nested set of deficiencies on the right arm of X and have found that a subset of these also have a dominant masculinizing effect when placed in combination with n695 (Table 1, Figure 1). In one copy, these deficiencies increase the number of transformed animals. These deficiencies define two small regions which when present in one copy appear to result in further masculinization of n695 XX animals. [See Figure 1] [See Figure 2]

Hsu DR et al. (1990) Worm Breeder's Gazette "dpy(y130): A New Maternal-Effect Gene Essential for the XX Mode of Dosage Compensation"

Meyer BJ, Hsu DR

[Worm Breeder's Gazette, 1990]

The genes dpy-26, viously been shown to be required for proper dosage compensation in XX animals. Mutations in any of these genes cause an incompletely penetrant maternal-effect XX-specific lethality due to disruption of the hermaphrodite mode of dosage compensation (Hodgkin MGG 192: 452 1983, Meyer and Casson Cell 47:871 1986, Meneely and Wood Genetics 117:25 1987, DeLong, Casson and Meyer Genetics 117:657 1987. Plenefisch, DeLong, and Meyer Genetics 121:57 1989). We have recently isolated yet another gene, dpy(y130), that appears to be a member of this family of dosage compensation genes. dpy(y130) was isolated in a xol-1 suppressor screen (Miller, et al Cell 55:167 1988) looking specifically for mutations that result in a maternal effect rescue of the xol-1 XO-specific lethality. dpy(y130) maps to the sma-1 on LGV. Homozygous dpy(y130) XX progeny of heterozygous mothers exhibit a temperature-sensitive phenotype. At 25 C, these animals are generally slightly Dpy, Egl, and Pvul. At 15 C, they appear wild type. The zygotic phenotype at 25 C appears to be similar for y130/Df1 animals. When y130 him-5(e1490) males are mated with ctDf1/nT1 unc(n754dm) hermaphrodites at 25 C, the non-Unc outcross progeny are viable and appear slightly Dpy, Egl, and Pvul. In contrast, homozygous dpy(y130) XX progeny of homozygous mothers display a temperature-sensitive lethality (see table). At 25 C or 20 C, most of these animals die before hatching or arrest as L1 larvae, with a small number of animals persisting through later larval stages before dying. The 25 C lethal phenotype is also seen in the progeny of y130 him-5(e1490)/ctDf1 mothers. This complete lethality is unusual: animals mutant in dpy-26, dpy-27, or dpy-28, always give rise to small number of dpy escapers. This does not appear to be the case for dpy(y130). At 15 C, the same terminal phenotypes are seen. In addition, a small number of fertile Dpy escapers are present. In order to determine the null phenotype of dpy(y130) we have isolated new alleles on the basis of their failure to complement the y130 zygotic phenotype at 25 C. This screen should allow us to obtain null alleles since y130/ctDp animals are viable in this generation. We have screened approximately 6500 haploid genomes and have recovered three new EMS-induced alleles of this gene. These alleles, y185, y186, and y187, also fail to complement ctDf1 and exhibit an XX-specific, maternal-effect lethality similar to y130. Interestingly, each of the new alleles appears to be temperature-sensitive for both the zygotic and maternal phenotypes. None of the alleles tested appears to be suppressible by the tRNA amber suppressor sup-7(st5) at 20 C or at 22. 5 C. [See Figure 1] In order to better characterize the nature of the dosage- compensation defect associated with dpy(y130 etc), we have performed reconstruction experiments to determine the degree to which these alleles will rescue xol-1 XO dead animals. Preliminary results indicate that fewer than 1% of dpy; xol-1 XO homozygotes are rescued if their mother is dpy/+. A far greater number of dpy; xol-1 XO progeny are rescued if the mother is homozygous for the dpy mutation. The rescued animals exhibit a broad range of sexual phenotypes extending from fertile hermaphrodites to males. In contrast to the other maternal-effect dpy genes, dpy(y130, y185, y186, y187) appears to rescue xol-1 XO animals well only if both the mother and her progeny are mutant. This may reveal a qualitative or quantitative difference in this gene's role in the dosage compensation process. We have also begun to clone this new gene. dpy(y130) lies on the her-1 contig between the cloned loci act-1, 2, 3 and myo-3. We have identified RFLPs in wild C. elegans strains between these markers, and we are in the process of mapping these polymorphisms with respect to dpy(y130) in order to pinpoint its position on the contig.

Fire A et al. (1998) Worm Breeder's Gazette "On the generality of RNA-mediated interference"

Fleenor J, Fire A

[Worm Breeder's Gazette, 1998]

After suggestions from Mei Hsu and from Mike Nonet that some tissues might show reduced sensitivity to RNA-mediated interference (RNAi), we have investigated the generality of RNAi using a broadly expressed gfp-reporter transgene. As a target for these experiments, we needed a gfp fusion that was expressed in all somatic tissues. For this purpose, we fused the upstream region from the ribosomal protein L5 gene to gfp (with a nucleolar localization signal to facilitate cell identification). A transgenic line with this fusion expressed nucleolar GFP in all somatic tissues. Although GFP was stronger in younger larvae than in adults, some activity was retained in all cells throughout the life of the animals. Activity was comparable in different cell types (gut showed slightly more activity and neurons slightly less, with muscle and hypodermis intermediate, but these differences were not extreme). We injected young adult animals from this strain with a double-stranded RNA hairpin corresponding to the complete coding sequence for gfp. RNA was injected at a high concentration (~1mg/ml) to obtain maximal interference, and animals were then grown at 25!C under standard conditions. We then examined gfp activity patterns in the injected animals and in their progeny. *Injected animals ("I0"): Within three days, GFP was essentially eliminated in all muscle, somatic gonad, hypodermal, and intestinal cells. Staining in the nervous system appeared to decrease somewhat but was not fully eliminated: a high fraction of neurons (particularly in the head or tail) contained GFP even after the RNA injection. Control animals (uninjected) retained activity in all tissues. *Progeny Animals ("F1"): In young and mid-stage larvae, GFP was absent in the vast majority of muscle, hypodermal, and intestinal cells (although sporadic cells of these classes were GFP+). By contrast, a high fraction of cells in the nervous system retained GFP activity. We assume that these neuronal cells were at least partially able to resist or escape the the RNAi. This was particularly true for cells in the head and tail region, while the ventral cord motor-neurons were frequently affected by the RNAi. Even in the head and tail, some nervous system cells are clearly affected (GFP-) in each animal, while others continued to express at least a fraction of the GFP level seen in uninjected controls. It is not clear whether the general pattern of RNAi-resistant and RNAi-sensitive neuronal cells varies from animal to animal or is constant in the population. *F2 animals: All F2 animals that we examined showed a return to the fully GFP+ phenotype of the original strain.

Danielle Thierry-Mieg et al. (2003) Worm Breeder's Gazette "www.wormgenes.org , a new gene centered database"

Vahan Simonyan, Michel Potdevin, Mark Sienkiewicz, Yuji KOHARA, Jean Thierry-Mieg, Danielle Thierry-Mieg, Tadasu SHIN-I

[Worm Breeder's Gazette, 2003]

Wormgenes is a new resource for C.elegans offering a detailed summary about each gene and a powerful query system.

Edgar RS (1975) Worm Breeder's Gazette "the shipping and handling of nematodes."

Edgar RS

[Worm Breeder's Gazette, 1975]

Many of us have totally abandoned wooden sticks for transferring worms. Instead a short (1 - 1 1/2') piece of platinum wire (32 gauge) is sealed in a pasteur pipette holder. Platinum is soft and a sharp hook or spatular end can be shaped with a razor blade and forceps. Various 'tools' of varied shape can be manufactured. Such implements are readily sterilized in a micro-burner. I have had some success shipping worms in letters. Three or four small pieces of filter paper are placed on a piece of aluminum foil. The filter papers are saturated with a buffer washate from a starved plate. The foil is folded several times to create a seal. The recipient places the filter papers on a plate or washes them in buffer.

Gruidl ME et al. (1995) Worm Breeder's Gazette "The Adult Sterile Mutant, let-545, Is Likely To Be a Mutation in glh-1, a Putative P-Granule Component."

Gruidl ME, McKay S, Bennett KL, Rose AM

[Worm Breeder's Gazette, 1995]

The adult sterile mutant, let-545, is likely to be a mutation in glh-1, a putative P-granule component.

Lewis JA (1988) Worm Breeder's Gazette "referencing, crossreferencing, and creating reference lists with Microsoft Word."

Lewis JA

[Worm Breeder's Gazette, 1988]

I've written a little primer on how to use Word (version 3.0 or higher) to automatically generate references (reference number or author-year citation) and cross-references (to page number) within a document using a combination of Word's merge function and table of contents generation facility. Also included in the article are directions for using reference information stored in a database to automatically generate a reference list according to a given style using Word's merge function. The utility of all this is likely to be superseded by future versions of Word and commercially available add- on programs. However, if you are interested, drop me a line and I'll send you a copy of my article.

Way JC et al. (1986) Worm Breeder's Gazette "a simple method for decontaminating worm stocks."

Way JC, Chalfie M

[Worm Breeder's Gazette, 1986]

In the past, we have used the standard method for decontaminating stocks, which involves washing worms off a plate, spinning them in a centrifuge, etc The following procedure is much quicker and requires fewer worms. Also, it can be used to directly decontaminate the progeny of a mating and thus accelerate strain constructions. Using a platinum wire with a glob of bacteria on the bottom, pick up several gravid adults and/or eggs from the contaminated plate and transfer them to a seeded plate. Then put a drop of the usual sodium hypochlorite solution (2-4% NaOCl, .4M NaOH) onto the eggs. About half of the eggs will hatch. Occasionally, a very tough bacterial contaminant will survive to form a few colonies, so it is wise to transfer the survivors on the following day.