Worm Breeder's Gazette 11(3): 10
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Mup-2 is a gene required for muscle attachment and oviduct function in C. elegans. The muscle phenotype and temperature sensitive period of mup-2(e2346ts) have been described in WBG 10(3), 104-105 and CSH 1990 meeting abstracts, page 83. RFLP mapping around mup-2: We mapped mup-2(e2346) between sup-7 and lev-9 on X. Cosmids from the contig between sup-7 and unc-4 were obtained from A. Coulson and John Sulston and used to probe genomic Southern blots of different wild type strains. Four RFLPs were identified between RC301 and N2. To map the RFLPs we constructed the strains RC301/mup-2(e2346) unc-6(e78) and RC301/dpy-8(e130) mup-2( e2346) and picked unc-6 recombinants and dpy-8 non mup-2 recombinants. Southern blots of DNA of these homozygosed strains were probed to determine the segregation of the RFLP relative to the mup-2 gene (see figure 1). Mup-2 maps between sup-7 and the RFLP on F14G9. A 16 kb BglII RFLP in T15G10 was not separated from mup-2 in a large number of recombinants and is therefore likely to be very close to mup-2.[See Figure 1] The alignment of the physical and genetic maps between sup-7 and unc- 4 (see figure 2) shows that recombination frequency is not linear between sup-7 and unc-4 and that there may be a recombination hotspot between K04G12 and sup-7. Extrapolating distances from the genetic to the physical map is thus not a reliable method to choose cosmids for transformation rescue. [See Figure 2] F2 transformation rescue of mup-2: We decided not to inject e2346b homozygous because e2346ts has a gonad defect at 25 C. Animals grown at 15 C have a small broodsize of about 40 or are sterile. Interestingly mup-2(e2346) unc-6(e78) hermaphrodites are about twice as fertile as mup-2(e2346) by itself. We co-injected the three overlapping cosmids T07F4, T15G10 and K04G12 and the rol-6 roller plasmid (cfr. WBG 11(1)) into the syncitial gonads of +mup-2(e2346) unc-6(e78)/lon-2(e678)++ hermaphrodites and grew the hermaphrodites at 25 C. Surviving unc-4 would be either rescued mup-2 unc-4 or + mup-2 unc-6/lon-2 + unc-6 recombinants. These recombinants segregate lon-2 thus be easily excluded. We have not obtained rescued mup-2 unc-4 in the F1 of injected hermaphrodites. However we have obtained F1 rollers of the genotype mup-2 unc-4/lon-2 which segregate apart from the expected offspring many sterile unc's, fertile unc's and rollers. The fertile unc's breed true and are homozygous mup-2 unc-4 (shown by outcrossing to N2). The sterile unc's are likely to be mup-2 unc-6 with insufficient levels of wild type product to rescue the sterility of mup-2. We know from the phenotype of mup-2(e2346); stDp2 that more than 33% wild type activity is required to rescue the gonad phenotype of mup-2(e2346) at 25 C. It is possible that we did not obtain rescue in the F1 because relatively high levels of wt product may be required for mup-2 rescue. Cosmids T15G10, C32C12 and F36F9 injected separately or as a mixture do not rescue mup-2 and serve as negative controls. We intend to narrow down the mup-2 gene by transformation rescue with overlapping lambda clones from the mup-2 region. General points: (1) The mup-2 rescue and the rol-6 phenotype need not segregate together in the rescued lines. (2) Compared to other wild type strains used, RC301 appears to be rich in RFLP's in both the unc-6 and the unc-53 region. (3) We have screened lambda 2001 genomic libraries directly with nick translated cosmid clones from A. Coulson and J. Sulston (lorist and PJB8 vector based) and had no problems recognizing positives over background although there is homology between cosmid vectors and lambda arms. (4) We have also obtained transformation rescue for unc-53 by injection in the gonad arm adding to the list of genes for which this method works. Acknowledgement: This approach to cloning mup-2 would not have been possible without the advice, physical map and cosmid clones from A. Coulson and J. Sulston.
