Worm Breeder's Gazette 10(2): 105
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.
The deg-1(u38) mutation causes the PVC interneurons to degenerate and die during the late L3-early L4 stage. Thus, such mutants become insensitive to tail touch as they mature (other neurons, in the head, degenerate during the Ll stage). The phenotype conferred by u38 is dominant and temperature sensitive. We have obtained 26 pseudo-wild type revertants following EMS and transposition mutagenesis, including one deletion allele. These results suggest that the wild type function of deg-1 is dispensable, and that the u38 mutant gene product somehow 'poisons' specific neurons at specific times during development. We wish to learn more about the deg-1 gene product in order to understand the molecular basis of the u38 phenotype. As reported in the last gazette, we have cloned the gene using Tc1 tagging. Since then we have l) localized two insertions and one deletion genetically linked to deg-1 to two cosmids within a contig mapping in the center of X (cosmids and map kindly provided by A. Coulson and J. Sulston), 2) localized three different types of cDNA clones to the same two cosmids (cDNA library kindly provided by J. Ahringer and J. Kimble), and 3) begun sequencing our genomic and cDNA clones. A part of the relevant contig map is reproduced below. Our conclusions are based on Southern blots of DNA from various u38 revertants probed with the cosmids shown. We have subcloned three regions of cosmid C47C12 corresponding to three deg-1 linked DNA rearrangements. Two different Tc1 insertions map to adjacent fragments near the center of C47C12: a 3 Kb EcoRI-XbaI fragment, and a 2.3 Kb EcoRl fragment. Our deletion strain is missing DNA from an approximately 8.5 Kb EcoRl fragment hybridizing to both cosmids C47C12 and R02A8. We used our 3 deg-1 subclones to screen an approximately Ll-staged cDNA library (we have not yet detected deg-1 sequences on Northerns). Three different types of CDNA's were obtained. All three hybridize to the 2.3 Kb EcoRl fragment which is the site of one of the Tc1 insertions. Additional hybridizing sequences are found in the center of R02A8 for two cDNAs which differ in the amount of R02A8 sequences they contain (they also differ in that only one of the two contains the repetitive element of the C47C12 2.3 Kb EcoRl fragment described below. None of the cDNA's bind detectably to sequences outside the two cosmids. Thus, the R02A8 hybridizing cDNA's delineate a probable new region of deg-1 not previously identified by mapping DNA rearrangements in our revertant strains. We have begun sequencing our deg-1 cDNA and genomic clones. The largest cDNA is about 2.5 Kb, comprised of mostly R02A8 sequences. More of the C47C12 2.3 Kb EcoRl fragment is represented on another cDNA clone approximately 2 Kb in length. Our study of the structure of deg-1 has so far yielded three points of interest. l) The gene is large, extending from the center of C47C12 to the center of R02A8. It appears to be sandwiched between the vitellogenin and tubulin genes. 2) One type of deg-1 cDNA contains sequences separated on the cosmid map by more than 10 Kb, suggesting a possible large intron. 3) The 2.3 Kb EcoRl fragment from C47C12 hybridizes to 5 additional bands on high-stringency Southern blots of XbaI-EcoRI cut genomic DNA. The extra bands amy correspond to members of a gene family related to deg-1. The hypothesis of a deg- 1 gene family is a possible explanation for the non-essentiality of the gene. [See Figure 1]