Moerman DG (1986) Worm Breeder's Gazette "Tc1 transposition occurs in both germlines."

Moerman DG

[Worm Breeder's Gazette, 1986]

I have been attempting to determine if transposition occurs in both the oocyte and sperm lines, and if it does to compare the relative rates of movement in these two tissues. To do this I have placed the following mutant construct, [See Figure 1], from strain CB3640 (kindly provided by Jonathan Hodgkin) in a mixed Bristol/Bergerac background. This male/female strain can be continuously propagated by crossing. The key to the analysis is that fem-1 and tra-3 are on chromosome IV and flank unc-22. By picking single spontaneous males that twitch in nicotine, mating them to unc-5 tra-3 females and scoring in the next generation whether males or females twitch, it is possible to determine in which germline the transposition event occurred. If the male progeny twitch in nicotine the unc-22 gene on the tra-3 chromosome must have been hit by Tc1 because this chromosome determines maleness. This means that the original transposition event must have occurred in the sperm line since this chromosome can only come from the father. On the other hand, if the female progeny twitch in nicotine then the unc-22 gene on the unc-5 tra- 3 chromosome must have been hit by TC1. In this case the original transposition event must have occurred in the oocyte line since this chromosome is inherited from the mother. In principal, this experiment is analogous to the Drosophila attached X mating scheme. The data: The strain constructed has a rather low activity which is useful to insure independent events but means that I have a small number of positive events. Only males which came from plates with no other twitching worms were analyzed to insure that I was monitoring the initial event. To date I have analyzed 10 events; 4 occurred in the sperm line, 4 occurred in the oocyte line and 2 are indeterminate. These latter two events are puzzling because in the test cross male and female progeny twitched in nicotine. I do not have an explanation for this observation although the time during development that transposition occurred, or recombination are two possibilities that need to be explored. I think the present data is sufficient to conclude that transposition occurs in both germlines. Taking into consideration the low numbers I would also suggest that the oocyte and sperm lines are not markedly different in their transpositional activities.

Moerman DG et al. (1986) Worm Breeder's Gazette "activation of Tc1 excision."

Waterston RH, Moerman DG, Mori I

[Worm Breeder's Gazette, 1986]

In the process of mapping mut-6(st702) we have observed a remarkable enhancement or activation of Tc1 excision from unc-22. The observation is as follows: Fl hybrids of the genotype mut-6(st702) 192::Tc1) / daf-11 136::Tc1) ntly yield 5 - 20 revertants per 100 progeny: a reversion rate 10 to 40 times higher than we have previously observed with any Bergerac derived strains. The Fl hybrids were derived from a cross of daf-11 136::Tc1) males with mut-6 192::Tc1) hermaphrodites. The revertants represent germ-line events ( whether these events are premeiotic and/or meiotic in origin is not clear ) and both st136::Tc1 and st192::Tc1 revert at similar high frequencies often in the same brood. The parent strains revert at significantly lower frequencies: mut-6 0-3 and daf-11 dpy-4 at << 10-6. This high frequency reversion can be propagated as long as the mut-6 activity is maintained either in a heterozygous or homozygous state. That is daf-11 dpy-4 F2 progeny did not yield revertants in any of 18 broods, but about 50 % of either the heteroallelic combination or the unc-22(st192::Tc1) homozygotes will revert within a single brood. Curiously, during further propagation of a derived mut-6 192::Tc1) homozygous line we consistently observed that only 50 % of the progeny in turn yielded the high proportion of revertants in the next generation. Further propagation of the animals that did not give a high proportion of revertants showed that they remained relatively stable. These animals had not simply lost mut-6 because they could still revert at the lower frequency ascribed to the parental mut-6 192::Tc1) strain. We are currently investigating the basis of this phenomenon and its generality; i.e., is it unique to this pair of strains, and does it apply to other genes and to transposition as well as excision? Our results on these important questions are as yet too preliminary to bother to pass on. But the phenomenon is so impressive we will leave you with a specific example to stimulate thoughts of dysgenesis etc. In propagating a Tc1 excision activated mut-6 192::Tc1) line these are some of the numbers we have observed ( wild / total progeny 2/43, 6/59, 2/20, 5/93 and 4/26.

Moerman DG et al. (1984) Worm Breeder's Gazette "germline transposition of transposon Tc1."

Waterston RH, Moerman DG, Benian GM

[Worm Breeder's Gazette, 1984]

In the last newsletter we reported on a HindIII Tc1 polymorphism, now called stP1, that is 0.5 to 1.0 map unit left of unc-22 on the genetic map. We cloned this polymorphic site into lambda 590 and then subcloned a HindIII/EcoRV adjacent unique DNA segment into pBR322. Using this unique DNA sequence as a probe we found that the stP1 site is empty in the parental Bristol and Bergerac strains, but is occupied by Tc1 in 2 independent unc-22 mutant derivative strains, RW7002 and RW7008 (see last newsletter for strain descriptions). As well, while outcrossing RW7002 to Bristol males to establish the RW7012 strain, a second site, stP2, became occupied by Tc1 and the entire region underwent duplication. The stP2 polymorphism is also present in RW7008 but in this case we do not know the order of occurrence of stP1 and stP2. These observations indicate that Tc1 can transpose in Bergerac or Bergerac/Bristol hybrids. In a third unc-22 mutant strain neither polymorphism is present so we do not think stP1, or stP2, has any causative role in regard to Bergerac spontaneous twitchers. However, we have now identified a third Tc1 polymorphism, called stP3, in 2 independently derived strains which is solely associated with the mutated state of the unc- 22 locus. This Tc1 polymorphism is not genetically separable from these unc-22 mutations even by intragenic mapping. Both mutant strains have Tc1 in the same small (2kb) BglII fragment. We have now examined five intragenic revertants of one strain and all five lack stP3. To investigate the relationship of stP3 to unc-22 we have cloned the stP3 BglII fragment into pBR322 and subcloned a unique 1kb EcoRV fragment from this region. Rehybridization of our Southern's with this unique piece of DNA confirms our earlier result that stP3 is strictly associated with the mutated state of unc-22. Our preliminary conclusion is that this site is part of the unc-22 locus itself. We have used this 1kb fragment to isolate lambda clones from our lambda 1059-N2 library and are using these clones as probes against known deficiencies for the region. A dosage response, or the identification of a fusion fragment due to a deficiency breakpoint would be an independent confirmation that we have molecularly cloned unc-22.A few further comments: Our Bergerac spontaneous twitchers are weak alleles when compared to s32, an unc-22 amber allele. We think there are two possible explanations for this observation, neither of which can be ruled out at the present time. One possibility is that Tc1 may have preference for an intron in unc-22. However, it is also possible that somatic excision of Tc1 may allow enough transcription of unc-22 to allow these mutants to mimic leaky EMS alleles like e105, or s12. The origin of stP1 in RW7002 is clearly by transposition, but its mode of occurrence in RW7008 is unclear. It could be due to transposition, or possibly recombination between the Bristol and RW7002 fourth chromosomes when the RW7008 parental strain was constructed. The 2 stP3 events although independent, are selected transposition events, because they occur simultaneously with the twitching phenotype. The 2 stP2 events, in RW7008,and RW7012, are definitely independent events which have occurred in the same restriction fragment and these events were unselected. This independent recovery of unselected insertion events into the same restriction fragment suggests Tc1 may have strong target preference. Although we have found that germline excision of Tc1 is sensitive to genetic background, i.e. our spontaneous twitchers revert in a Bergerac background, but are stable in a predominantly Bristol background, this is not true for somatic excision of Tc1. The stP1, stP2, and stP3 Tc1 polymorphisms have similar somatic excision frequencies in Bristol and Bergerac. This suggests that excision in these tissue types is controlled differently. Finally, the observation of somatic excision of stP3 led us to examine the muscle cells in our mutants for mosaics. We have used phalloidin-rhodamine staining of F-actin as well as polarized light microscopy to examine cells. To date we have found no evidence for mosaicism but the screen is difficult because of the relatively mild structural disorder of the muscle cells in these animals. Of course, we also do not know whether unc-22 is cell autonomous.

Moerman DG et al. (1983) Worm Breeder's Gazette "spontaneous twitchers and Tc1 - no correlations yet."

Moerman DG, Waterston RH

[Worm Breeder's Gazette, 1983]

We have been investigating the possibility that the spontaneous unstable unc-22 alleles induced in the BO strain of C. elegans may be caused by Tc1. Our conclusion, based on two types of analysis, is that Tc1 is probably not the causative agent of unstable twitchers. To investigate the causative role of high Tc1 copy number in generating twitcher mutations we have tested several strains in addition to N2 and BO for the induction of twitcher mutations. These strains include the 'high' copy number strains, FR, BL1, and EPC-4; the 'low' copy number strains, Ga-5, PA-1, and PA-2 (Emmons et al., Cell 32:55-65, 1983; Liao et al., P.N.A.S. 80:3585-3590, 1983); and HA-8 which has not been examined for Tc1 copy number. Of these nine stains, only the BO strain exhibits the high frequency of spontaneous induction of twitcher mutations. These observations suggest that high Tc1 copy number per se is not sufficient to induce unstable unc-22 mutations. Rather there is something unique in the Bergerac background resulting in the high spontaneous twitcher frequency which, for example, could be an altered Tc1 or other factor. Our second approach to this problem consisted of examining Tc1 copy number and position in DNA from a number of different genetic strains with and without spontaneous twitcher mutations. The strains used were as follows: N2, RW7012 (contains unc-22(st136), a spontaneous twitcher isolated on a Bergerac IV chromosome in the BO strain and then outcrossed to an N2(male) strain via 8 cross intercross mating cycles), RW7018 (contains unc-22(st137), a spontaneous twitcher isolated on a Bristol IV chromosome in a BO background and then outcrossed as described for RW7012), Rw2370 (a spontaneous revertant to wild type from RW7018), RW1461 (a triple containing daf-14(m77) 136) 66),st136 derived from RW7012), RW1462 (an unc-43 ant from daf-14 136) -43(e408) 4)), RW1463 (a daf-14 recombinant from daf-14 1363 -43 )), RW1464 (a triple of daf-14 137) rived from RW7018), RW1465 (an unc-43 ant from daf-14 137) -43 4)), RW1466 (a daf-14 recombinant from daf-14 137) -43 )). In DNA from the various strains we could detect about 30 bands that hybridized to the Tc1 probe after HindIII, EcoRI, or XbaI digestion. However, we could not identify a Tc1 that was strictly associated with the mutated state of the unc-22 locus. In particular, we saw no difference between the digestion patterns of RW7018 and RW2370 for these three restriction enzymes. The strains derived from three factor crosses showed some banding heterogeneity, but again, we did not detect a band that was present in those strains containing a twitcher mutation (RW1461 and RW1464), and absent in those strains that did not (RW1462, RW1463, RW1465, and RW1466). These observations are negative in that we failed to observe a difference in Tc1 location consistent with Tc1 as the cause of the unc- 22 mutations. Of course, we cannot rule out the possibility that the critical Tc1 element is in a band comigrating with other Tc1 containing segments. Although failing to identify a Tc1 at the unc-22 site, our analysis of the recombinants has revealed two putative unc-22 linked Tc1 polymorphisms, one between daf-14 and unc-22 (a 2 m.u. interval), and one between unc-22 and dpy-4 (a 4.5 m.u. interval). The putative polymorphism to the left of unc-22 is a 2.1Kb HindIII fragment containing Tc1 which we have cloned into lambda 590. At present we are trying to isolate a unique sequence segment from this HindIII fragment so that we can use it to position the polymorphism with greater certainty. If the fragment is suitably close to unc-22 we may be able to use it to walk to the unc-22 gene. In conclusion, we feel that Tc1 is unlikely to be responsible for generating the unc-22 mutations, but linked Tc1 polymorphisms may be useful in providing us with a molecular probe for the region.

Moerman DG et al. (1984) Worm Breeder's Gazette "multiple Tc1 insertion sites in unc-22."

Moerman DG, Benian GM, Waterston RH

[Worm Breeder's Gazette, 1984]

Using a Tc1 transposon tagging procedure we have cloned the unc-22 gene (WBC 8:2) and 50kb of DNA in the unc-22 region. This includes DNA from over 20 lambda clones and 2 cosmid clones provided by J. Sulston. The deficiency sDf19 has a breakpoint within unc-22 (T. Rogalski, personal communication) and was used to confirm that we had isolated DNA from unc-22 and also to orientate the lambda clones relative to the genetic map. We have examined 14 spontaneous twitchers isolated in Bergerac B0 and in all 14, a 1.6kb insertion, presumably Tc1, is present in the region cloned. These inserts occur at multiple sites in unc-22. The limited restriction mapping done so far resolves these elements into at least 10 sites spanning about 20kb. Two spontaneous mutations, st136 and st137, which map to the central region of unc-22 and have identical morphological phenotypes have been shown by cloning to be Tc1 elements inserted about 40bp apart in opposite orientations.

Moerman DG et al. (1981) Worm Breeder's Gazette "another internal deletion mutant of unc-54."

Moerman DG, Weidman D, Waterston RH

[Worm Breeder's Gazette, 1981]

A rarely commented on and not understood phenotype of unc-54(e675) mutants is that along with being paralyzed, these worms display a slight surface twitch. Another mutant allele of unc-54 called s291 exhibits this same phenotype. This mutant was isolated after a mutagenesis screen in which N2 hermaphrodites were mutagenized with 0. 07% formaldehyde and their F1 progeny were examined to see if any twitched in 1% nicotine. Unlike the majority of mutants from this type of screen, which are either alleles of unc-22IV or deficiencies for the unc-22 region (Moerman, Rogalski and Baillie, unpublished results), mapping of s291 demonstrates that it is an allele of unc-54, a structural gene for a myosin heavy chain (mhc). Using the techniques developed by MacLeod et al. (1977) in their analysis of e675 we have done a similar structural analysis of the s291 mhc. One-dimensional sodium dodecyl sulfate (SDS)/polyacrylamide gels run on s291 whole worms and s291 purified myosin reveal a new mhc that is considerably shorter than N2 mhc's. Cyanylation fragments from the rod portion of this heavy chain molecule of s291 are also shorter than N2 cyanylation fragments (on a 1-D SDS/polyacrylamide gel the s291 alpha and beta fragments bracket paramyosin). To examine whether the shortened s291 mhc is due to an internal deficiency we ran two-dimensional isoelectric focusing and SDS/polyacrylamide gels on complete cyanogen bromide (CnBr) digests of whole myosin from N2 and s291 worms. The gel pattern is more complicated from whole myosin than the pattern observed by MacLeod et al. (1977) for CnBr digests of cyanylation fragments from rods, but the three carboxy-terminal fragments of interest (COOH-38K, 8K, 40K), and the 48K partial digestion product (8K + 40K) are easily discernable in gels from CnBr digested N2 whole myosin. The overall gel pattern of CnBr treated s291 myosin is similar to the N2 pattern except that the 38K, 8K, 40K, and 48K digestion products are all missing. Instead, a new peptide of 41K (approx.) is present. This new peptide lies between the 38K and 40K peptides in the isoelectric focusing dimension. Our interpretation of these observations is that the s291 deficiency is an internal deficiency that takes out the 8K peptide and part of the 38K and 40K peptides. The 41K peptide observed is the fusion product of the remainder of the 38K and 40K peptides. This gives an estimate of the size of the deficiency as 45K. Fine structure mapping studies show that s291 maps to the left-hand side of the gene between e1300 and e1301. The heteroallele s291/e675 appears to be lethal. The s291 mhc is much less stable than the e675 mhc. This is not surprising since it has a much larger deficiency, perhaps including as much as two thirds of the light meromyosin. Why worms homozygous for e675 or s291 twitch is not clear but we suspect that it is related to the fact that both mutants produce intact myosin heads that are not part of the myofilament lattice.

Moerman DG et al. (1977) Worm Breeder's Gazette "lethal mutations generated at different doses of EMS."

Riekki KRC, Baillie DL, Moerman DG, Rose AM

[Worm Breeder's Gazette, 1977]

Moerman DG et al. (1977) Worm Breeder's Gazette "Isolation, Mapping and Characterization of Lethals Closely Linked to the unc-22 Gene in Caenorhabditis elegans"

Baillie DL, Riekki KRC, Rose AM, Moerman DG

[Worm Breeder's Gazette, 1977]

Using ethylmethane sulfonate (.05M) several lethals linked to the unc-22 mutant have been isolated (14 lethals in 320 tested fourth chromosomes within 5 map units i.e. 4.4%). Worms heterozygous for the mutant unc-22 show a different behavior in a solution of 1% nicotine than do wild-type worms. This observation has allowed us to keep lethals linked to the unc-22 mutant for further study. Ten of the lethals have been found to map within two map units either side of the twitcher gene. Several of these have now been given left/right positions. As well, the time of lethality for eight of these mutants has now been determined. All eight are post-embryonic lethals. One dies in the egg just prior to hatching. Six die during various larval stages. One grows to the adult size but it is sterile. The evidence of linkage data, complementation analysis, left/right positioning and time of lethality lead us to the conclusion that we have identified ten separate lethal genes around the unc-22 gene. This work is supported by a grant from the National Research Council of Canada.

Johnsen RC et al. (1986) Worm Breeder's Gazette "formaldehyde mutagenesis: dose response curve and analysis of recessive lethals recovered over eT1(III;V)."

Johnsen RC, Baillie DL

[Worm Breeder's Gazette, 1986]

We have established a dose-response curve for the formaldehyde induction of recessive lethals in the region balanced by eT1(III,V). We used Moerman and Baillie's (Mutation Research, 80(1981) 273-279) method of formaldehyde mutagenesis. The lethal induction frequencies that we found were: {Figure 1} We found that 65% of all the mutations lie on LGIII and 35% lie on LGV which is very similar to our EMS results, see Johnsen and Baillie WBG Vol.9 #2. There was no significant difference in the ratio of lethals on the two Linkage Groups for the different formaldehyde doses.

Rogalski TM et al. (1982) Worm Breeder's Gazette "a deficiency on the left half of unc-22 IV causes dominant twitching."

Baillie DL, Rogalski TM

[Worm Breeder's Gazette, 1982]

The mutation s290 was picked up as a dominant twitcher after formaldehyde mutagenesis of N2 worms (Moerman and Baillie, 1981). This mutation is lethal when homozygous, blocking at an early larval stage (D. Moerman, personnel communication). Although allelism tests could not be done, it was possible to show that the twitcher phenotype is due to a defective unc-22 gene. s290 was positioned with respect to two alleles of the unc-22 gene by intragenic recombination mapping. These two alleles, s8 and s12, represent respectively the left and right boundaries of the unc-22 fine-structure map (Moerman and Baillie, 1979). s290 maps approximately 0.004 map units (6 recombinants/565,500 total progeny) to the left of s12. This result places s290 near the middle of the unc-22 fine-structure map. However, no recombinants were observed ( 283,650 total progeny) from unc-5(e152)unc-22(s8)dpy-4(e1166)/s290 hermaphrodites, placing s290 near the left end of the gene. These mapping results can be explained if s290 is a deficiency breaking in the middle of the unc-22 gene and extending to the left end of this gene. The lethality of s290 homozygotes can be explained if this mutation is a deficiency extending into essential genes adjacent to the unc-22 gene. Complementation tests were done between s290 and four essential genes, let-56, let-65 and let-59, which map 0.01 0. 2, 0.4 and 0.6 map units respectively to the left of unc-22 (see map below). Hermaphrodites with the genotype, s290 IV/ +; dpy-11 V, were mated to let-x s. Since s290 is dominant, the outcrosss Unc-22 worms had to be progeny tested to determine whether their genotype was s290/ +; dpy-11/ + or s290/ let-x dpy-11/ +. If none of the outcrossed Unc-22 progeny from a particular cross carried the let-x me, then that let gene is uncovered by s290. s290 failed to complement only let-56, the closest known let gene ( 68/68 Unc-22 progeny were s290/ +; dpy-11/ +). Therefore s290 appears to be a deficiency breaking inside the unc-22 gene and extending into at least one essential gene to the left of unc-22.If the dominant effect of s290 is due to the presence of an incomplete unc-22 peptide, then the deficiency hypothesis indicates that it is the right half of the gene that is being transcribed and translated. The most appealing explanation to account for this is that the unc-22 gene is transcribed from right to left on the genetic map. [See Figure 1]