Worm Breeder's Gazette 12(4): 53 (October 1, 1992)
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.
xol-1 (XO lethal) is required in XO animals for proper sex determination and dosage compensation. In XO animals xol-1 mutations cause lethality, under expression of X-linked genes and some feminization. In otherwise wild-type XX hermaphrodites, xol-1 mutations show no effects, but they further masculinize XX animals already partially masculinized by mutations in other sex determination genes. For example, tra-2 ( e1095 )XX animals are males with shrunken rays and fan, while tra-2 ( e1095 ); xol-1 ( y9 )XX animals are wild-type mating males. Previous molecular analysis of xol-1 in our lab by Leilani Miller (1991 Meeting Abstracts p. 217) indicates that xol-1 produces three transcripts of sizes 1.5 kb, 2.2 kb, and 2.5 kb. These transcripts are alternatively spliced, sharing their first six exons, but differing in their ultimate exon. They each can encode a protein with a unique carboxy terminus. The 2.2 kb transcript is about 10-fold more abundant than either of the other transcripts. Roughly the same pattern and level of transcripts are seen in XX and XO animals. None of the open reading frames show any significant homology to any proteins in the current data bases.
Analysls of the xol-1 uslng In vltro mutagenized constructs:
To determine which transcripts are involved in the XO- and XX- specific phenotypes, we created constructs that mutated or eliminated different transcripts. Each transcript was mutated individually by introducing two adjacent stop codons so as to truncate the unique part of each open reading frame. In addition, the 1.5 and 2.5 kb transcripts were both eliminated by replacing part of the genomic sequence with the 2.2 kb cDNA such that the construct could produce only the 2.2 kb transcript. These constructs were injected into him-8 ( e1489 ); xol-1 ( y9 )animals ( y9 is a deletion that removes the entire xol-1 locus) and assayed for their ability to rescue the XO-specific lethality. The transgenic arrays were then crossed into a tra-2 ( e1095 )/ dpy-10 ( e128 ); xol-1 ( y9 )background to assess their ability to rescue the XX-specific phenotype. Our results indicate that, at least in an extra-chromosomal array, the 2.2 kb transcript is necessary and sufficient to rescue both the XO and XX-specific phenotypes. Among the constructs that rescue the two phenotypes, there is no obvious difference in the extent of rescue. A hint to the possible function of xol-1 was the highly acidic nature of the final 33 amino acids encoded by the 2.2 kb transcript (19/33 58% are acidic), reminiscent of a transcriptional activator. Alas, constructs in which these 33 amino acids have been truncated still rescue the XO-specific phenotype. The effect of this construct on rescue of the XX phenotype is being determined.
Consistent with the results of the transformation rescue experiments, we have found that the mutation in xol-1 ( y138 )is within the 2.2 kb transcript specific exon. It introduces a translational stop in the 87th codon from the end of the ORF, ironically one of the same codons that was changed in the in vitro 2.2 kb mutant. Another xol-1 mutation, y95 ,disrupts the fifth and final splice acceptor site common to all three transcripts.
Creating new xol-1 alleles:
Leilani had noticed that when she injected him-8 ( e1489 ); xol-1 ( y9 )animals with low concentrations of xol-1 genomic DNA (lug/ml, co-injected with rol-6 (d)[ pRF4 ])she saw more F1 Rol hermaphrodites than Rol males, but when she used high concentrations (20ug/ml) she saw more F1 Rol males than Rol hermaphrodites. Using the same injection conditions. we were able to create xol-1 arrays that were transmitted exclusively from male to male. This hermaphrodite-specific lethality is presumably caused by the over expression of xol-1 invoking the male mode of dosage compensation in hermaphrodites. Unfortunately after a few generations the arrays seemed to break down and hermaphrodites began to appear. To make an allele of xol-1 that would have a stable dominant hermaphrodite-specific lethal phenotype, we constructed a fusion between the promoter of dpy-30 and the xol-1 genomic coding region. dpy-30 is a dosage compensation gene that is expressed at high levels early in development and probably in both sexes (Dave Hsu, personal communication). We hoped that this construct would sufficiently over express xol-1 in hermaphrodites to kill them; and it does. By co-injecting the fusion with a plasmid that rescues unc-76 (thanks to Laird Bloom) into him8 ( e1489 ); unc-76 ( e911 )we have established a mating strain in which the only hermaphrodites are Unc and fail to carry the array, whereas the array-bearing males are wild type. We hope to use this strain to isolate suppressors of the hermaphrodite-specific lethality.
We also created a condHional allele wHh the hopes of being able to determine when xol-1 transcription is required. We fused xol-1 to the two hsp-16 promoters (courtesy of Dennis Dixon via Andy Fire, WBG 12:2 p17 ),injected each of them into him-8 ( e1489 ); xol-1 ( y9 )and established transmitting arrays. When a two hour heat shock at 30° is applied around the time that the embryos are laid some XO males survive. They range from healthy mating males to small slow-growing scrawny males, but all look fully masculinized. In this preliminary experiment, males were not rescued by heat shocks at any other time during embryogenesis. None of the two hour heat shocks caused significant hermaphrodite-specific lethality. We are in the process of integrating one of the arrays in order to be able to perform more synchronous heat shock experiments.
Dave Hsu, personal communication.
Dennis Dixon via Andy Fire, WBG 12:2 p17 .