Clandinin TR et al. (1992) Worm Breeder's Gazette "Unusual Interactions and Strange Smls"

Clandinin TR, Mains PE

[Worm Breeder's Gazette, 1992]

The maternal-effect gene mei-1 produces a product essential for meiosis. However, mei-1 alleles fall into no fewer than 4 distinct classes and a number of models can accommodate their genetic interactions. The dominant mutation mei-1 (ct46 )causes a defect in mitotic spindle formation in the l-cell embryo. Previous genetic screens for ct46 suppressors had primarily identified intragenic mutations lacking functional mei-1 activity which fell into two classes. While one of these classes contained alleles with properties consistent with null alleles, the second class contained alleles producing defective mei-1 product and were able to suppress ct46 both in cis and in trans. To extend this analysis, we designed a screen which we expected would be biased toward the isolation of extragenic suppressors. However, while 3/14 suppressors identified are apparently unlinked to mei-1 ,the remaining 11 mutations were defined as a fourth class of mei-1 alleles. In an effort to assess the relationship between this group of suppressors and the previously known mei-1 alleles, we undertook a detailed analysis of a series of intragenic trans-heterozygotes. Our evidence suggests that dominant mei-1 mutations may stabilize a multimeric complex containing mei-1 protein resulting in the persistence of mei-1 product into mitosis and allowing misincorporation of mei-1 activity into the mitotic spindle. Alleles of mei-1 which result in the presence of a non-functional product are capable of acting as trans-suppressors of the dominant mutation by acting in a weakly antimorphic (dominant negative) manner. That is, complexes which incorporate any non-functional product are rendered inactive but not before they function in meiosis. Taken together, the genetic evidence suggests that mei-1 gene activity may exhibit a sharp temporal threshold; a relatively high level of activity is required for meiosis but must be eliminated prior to mitosis. All three extragenic suppressors lack an apparent phenotype (other than dominant trans-suppression). Logically enough, therefore, 'we' have named the gene(s) defined by these alleles suppressors of mitotic lethality, creating an acronym that wins little favour from my supervisor. Two of these mutations map between dpy-17 and unc-93 on LG III, suggesting allelism; the third mutation has not yet been mapped. Interestingly, all three mutations are capable of dominantly transsuppressing both mei-1 (ct46 )(as expected) and a dominant mutation at a second locus, mei-26 (ct61),even though they were only selected to suppress ct46 . The biological implications of these genetic interactions are quite interesting. Meiotic and mitotic spindles are clearly morphologically (and presumably structurally) distinct yet must perform somewhat analogous functions. One might imagine that there are a relatively large number of functionally non-interchangeable but analogous structures involved in the two processes. If this is the case, it serves to emphasize the importance of proper inactivation of meiotic structures after meiosis during worm embryogenesis. Extragenic suppressors of mei-1 dominant alleles could identify components of this 'disassembly' system.

Diana McCulloch et al. (2001) Worm Breeder's Gazette "N2 (ancestral) is a mutant with reduced fertility and longevity"

David Gems, Diana McCulloch

[Worm Breeder's Gazette, 2001]

Genetic variation among different laboratory lines of N2 affects fertility and lifespan. A previous study found that median lifespans of different lines ranged from 12.0+/-0.8 to 17.0+/-0.6 days (20 o C)(1). To attempt to establish which of these best resembles the original N2 isolate, the lifespans of F1 hybrids between N2 variants, and those of seven C. elegans wild isolates, were examined (1). The results suggested that the N2 male stock currently distributed by the CGC (here designated CGCM), is most similar to the original N2 isolate. This strain is the longest-lived N2 variant. An alternative approach to establish which variant best resembles wild type is to examine an N2 stock frozen at an early date. One such stock is N2 (ancestral), which is only 6 generations removed from a stock frozen in the Brenner lab in 1968. We compared the fertility and lifespan of N2 (ancestral) with 3 N2 variant lines: CGCM, CGCH (slightly short-lived) and JW (very short-lived). Our expectation was that it would most closely resemble CGCM. Table 1: Longevity and fertility of N2 (ancestral) and other N2 lines Strain Median lifespan (days) Maximum lifespan (days) N* Mean brood size N* N2 CGCM 20.5 +/- 1.0 24.4 +/- 0.7 5 (127) 292 +/-11 1 (18) N2 CGCH 18.3 +/- 0.8 21.9 +/- 0.7 7 (136) 270 +/- 9 1 (18) N2 JW 13.4 +/- 1.0 23.1 +/- 1.0 8 (194) 213 +/- 9 1 (15) N2 (ancestral) 13.6 +/- 0.7 21.2 +/- 1.0 10 (243) 260 +/- 7 1 (19) N2 CGCM/N2 (ancestral) 19.7 +/- 0.3 25.7 +/- 0.7 3 (53) ND - N2 JW/N2 (ancestral) 15.0 +/- 3.0 22.0 +/- 0.0 2 (46) ND - +/- Standard error. *Number of trials (number of animals scored). To our surprise, the lifespan of N2 (ancestral) was significantly shorter than CGCM and CGCH (median 20 o C lifespan, p < 0.001 in each case)(Student's t test) (Table 1), yet indistinguishable from JW ( p > 0.1). Furthermore, its fertility was significantly lower than that of CGCM ( p < 0.02) but not CGCH ( p > 0.1), but higher than that of JW ( p < 0.001). Does this mean that the longer-lived line CGCM is a mutant variant after all? We favour an alternative explanation: that as early as 1968 there existed a number of genetic variants of N2 in the Brenner lab (Cambridge), and N2 (ancestral) is a short-lived one. In 1975, when Don Riddle moved from Cambridge to Missouri, he took with him CGCH, CGCM and JW (obtained from John White), and froze them upon arrival. These three strains have different lifespans (1). Thus, by 1975 a number of variants certainly did exist. One possibility is that N2 (ancestral) and JW are related. To test this, the lifespans of F1 hybrid hermaphrodites issuing from crosses between CGCM, JW and N2 (ancestral) were examined. The lifespan of CGCM/N2 (ancestral) was indistinguishable from that of CGCM, and significantly longer than that of N2 (ancestral) ( p < 0.001). A reduction in median lifespan was seen in JW/N2 (ancestral) hybrids relative to CGCM/N2 (ancestral) hybrids, but due to the large standard error this did not reach significance. However, the maximum lifespan of JW/N2 (ancestral) was significantly reduced ( p < 0.02). These preliminary results support the view that N2 (ancestral) and JW are related, short-lived mutant variants, and that the mutations concerned are recessive. N2 (ancestral) thus appears to be another short-lived N2 variant, related to JW. We therefore advise that N2 (ancestral) not be used as wild type; instead, we recommend CGCM. (1) D. Gems & D.L. Riddle (2000) Defining wild-type life span in Caenorhabditis elegans. J. Gerontol . 55A: B215-B219.

van Luenen HGAM et al. (1993) Worm Breeder's Gazette "Target Choice of the Transposable Elements Tc1 and Tc3"

Plasterk RHA, van Luenen HGAM

[Worm Breeder's Gazette, 1993]

Transposable elements insert at many different positions in the genome, but the integrations are not necessarily randomly distributed over the genome. A detailed analysis of the insertion specificity of a transposon may be revealing with respect to the transposition mechanism of the element, since the integration preference may reflect how the integration complex interacts with the target sequence. We have investigated the target choice of the related transposable elements Tc1 and Tc3 . The exact locations of 204 independent Tc1 insertions and 166 Tc3 insertions were determined. We used a PCR based approach which is sensitive enough to detect transposon insertions represented by a single DNA molecule and which does not involve a phenotypic selection for insertions (and is therefore unbiased for the distribution of insertions). The conditions were chosen such that insertions in an 1 kbp region of the genome were detected and that every insertion represents an independent somatic event. All 204 Tc1 insertions were into the sequence TA. Within the region investigated there are 82 TA's, but not every TA is used as often by Tc1 for integration (some TA's contain many insertions, others contain a few or no insertions; figure 1). The distribution of Tc1 insertions is independent of the orientation of the transposon. This implies that the integration reaction or the target sequence is symmetrical. We favour the first option since the flanking sequences of the TA's at the insertion sites have hardly any discernible symmetry. We considered several explanations for the target site preference. 1) The local DNA structure. If only the local DNA structure makes one TA more attractive that another, we might expect to find a similar distribution for the two related elements Tc1 and Tc3 .Therefore we determined the exact locations of 166 Tc3 insertions. All insertions were into the sequence TA and they are also non-randomly distributed among the TA's in this region. We compared the distribution of Tc1 and Tc3 insertions and found that they are completely different (figure 2). Simple DNA structures (such as bends and kinks) are therefore not very likely determinants in target site selection (unless the two elements recognize them differently) . 2) The donor site. The non-random distribution of insertions could be explained if the original insertion site of the element effects the target choice. To investigate this we compared the distribution of Tc3 insertions to the distribution of a marked Tc3 transposon that jumped from one specific donor site in a transgenic line. The distribution of normal Tc3 insertions is the result of transposition from approximately 15 different donor sites. There is no difference between the two distributions (figure 3) indicating that the donor site has no effect on the target choice. 3) The flanking sequence of the TA dinucleotides. The most likely explanation of local target site preference is the recognition of the sequence flanking the TA dinucleotide by the incoming transposon. We investigated whether we could recognize a pattern of preferred sequences in the close vicinity of the insertion site. No clear consensus sequence was found. These studies focused on somatic transposition. Assuming these studies are also relevant for germ line transposition (and on the basis of germ line insertions studied thus far that seems a reasonable assumption) we do not think it is useful to look for "consensus" Tc1 sites when planning e.g. reverse genetic experiments.