TABLE 1.
ceh-39 is an XSE in region 2
| yDp14/yDp14; him-8; fox-1 XO + RNAi of genea | Male viability (%)b | nc |
|---|---|---|
| A. RNAi of ceh-39 suppresses the XO-specific lethality caused by the increase in XSE dose from two copies of yDp14 | ||
| No RNAi vector or gene | 6 | 1225 |
| RNAi vector with no gene | 18d | 1236 |
| fox-1(RNAi) | 84d | 1050 |
| ceh-39(RNAi) | 84d | 857 |
| ceh-21(RNAi) | 20d | 776 |
| ceh-41(RNAi) | 28d | 923 |
| ceh-39(RNAi), ceh-21(RNAi), ceh-41(RNAi) | 76e | 946 |
| RNAi of X ORFs not in region 2 | 19 ± 9f (13 genes) | NA |
| RNAi of ORFs on autosomes | 18 ± 7f (14 genes) | NA |
| XO genotypeg | ceh-39 and fox-1 dose | Male viability (%) | nc |
| B. ceh-39 and fox-1 are not the only XSEs in yDp14 | |||
| Wild typeh | 1 | 100 | 1632 |
| yDp14/+i | 2 | 61 | 724 |
| yDp14/+; ceh-39(y414) fox-1(y303)j | 1 | 98 | 1369 |
| yDp14/+; ceh-39(gk296) fox-1(y303)j | 1 | 102 | 1566 |
| yDp14/yDp14; him-8(e1489)b | 3 | 0 | 929 |
| yDp14/yDp14; ceh-39(y414) fox-1(y303)k | 2 | 8 | 863 |
| yDp14/yDp14; ceh-39(gk296) fox-1(y303)k | 2 | 1 | 716 |
Candidate genes were tested for XSE activity. yDp14/yDp14(X;I); him-8(e1489) IV; dpy-3(e27) fox-1(y303) unc-2(e55) X hermaphrodites were treated with RNAi against the indicated gene, and the viability of progeny males was assessed. In all cases except as described in footnote e, the RNAi was achieved through feeding. RNAi-mediated knockdown of an XSE should decrease the male lethality caused by the increase in XSE dose from yDp14. Animals were fed bacteria that produced dsRNA to the listed gene (see materials and methods). yDp14 is an X duplication attached to LG I and can exist in one copy (yDp14/+) or two copies (yDp14/yDp14) (Akerib and Meyer 1994). him-8 XX animals produce 37% XO males, 57% XX hermaphrodites, and 6% Dpy XXX hermaphrodites (Hodgkin et al. 1979).
Male viability was calculated by the following formula: (no. of adult males)/(expected no. of males) × 100. The number of expected males was (0.37)n.
n is the total number of embryos from six independent sets of progeny counts.
Male viability is significantly higher only for fox-1(RNAi) and ceh-39(RNAi), both P ≤ 0.01, when compared to male viability of the true control: yDp14/yDp14; him-8; fox-1 animals grown on bacteria carrying an RNAi vector with no gene insert. Male viability for neither ceh-21(RNAi) (P = 0.70) nor ceh-41(RNAi) (P = 0.02) was significantly different from the true control. Male viability due to RNAi of these genes instead was equivalent to that due to RNAi of random X ORFs not in region 2 or of autosomal ORFs. The unexpected observation that RNAi against any C. elegans gene, or even the introduction of double-stranded RNA not similar to C. elegans RNA, rescued some XO lethality caused by yDp14/yDp14 suggests that the RNAi machinery may affect the sex-determination and dosage compensation pathway. This RNAi effect appears to be weak since it was observed only in the sensitized XO genetic background and only when RNAi was achieved through dsRNA feeding. Induction of RNAi in XX animals did not cause a notable dosage compensation disruption, and thus the observed effects of RNAi in XX animals (Table 3) are due to the reduced function of the XSE genes targeted.
RNAi was achieved by simultaneously injecting double-stranded RNA from ceh-39, ceh-21, and ceh-41. Injection RNAi against fox-1 resulted in 87% male viability (n = 1217); against ceh-39, 85% male viability (n = 759); and against dsRNA made from the vector with no cloned gene, 6% male viability (n = 1002).
The numbers presented include the average and the standard deviation of male viability for RNAi against 13 X ORFs not in region 2 (C05D9.5, F49E7.1, C05D9.7, R193.2, R193.3, R193.1, T13G4.3, F09E10.3, F09E10.6, F09E10.7, F09E10.8, K06A9.1, K06A9.2) and 14 ORFs on autosomes (F44E8.2, C31H1.1, C31H1.2, C31H1.5, C31H1.6, C31H1.7, C31H1.8, C10G6.1, T10B9.3, T10B9.4, T10B9.5, T10B9.7, T10B9.8, ZK938.1). Approximately 200 embryos were scored per ORF tested.
These animals also carry a dpy-3(e27) mutation, except for yDp14/+ and yDp14/yDp14; him-8(e1489) animals, which carry unc-2(e55) instead.
Males were generated by mating wild-type males and hermaphrodites. Male viability was calculated by the following formula: [adult males]/[expected no. of males, (0.5)n] × 100. The number of hermaphrodites was 0.5(n), implying a viability of 100% and a mating that produced only cross progeny.
Males were produced by mating wild-type males with yDp14/yDp14; unc-2(e55) hermaphrodites. The number of hermaphrodites was (0.5)n, indicating that the hermaphrodite viability was 100% and the cross went to completion.
yDp14/+; ceh-39 fox-1 males were generated from a cross of mIs11 males with yDp14/yDp14; ceh-39 fox-1 hermaphrodites. mIs11 is a dominant, integrated transgenic marker that expresses GFP from pes-10 and myo-2 promoters and a gut-specific enhancer. It was used to identify cross progeny. Male viability was calculated by the following formula: [adult males)]/[expected no. of males, (0.5)n] × 100. All progeny were gfp(+), indicating that the cross went to completion.
yDp14/yDp14; mIs11/+; ceh-39(y414 or gk296) fox-1(y303) males were generated by crossing yDp14/+; mIs11; ceh-39(y414 or gk296) fox-1(y303) males with yDp14/yDp14; ceh-39(y414 or gk296) fox-1(y303) hermaphrodites. Fifty percent of the XO (male) cross progeny should be of genotype yDp14/yDp14; ceh-39(y414 or gk296) fox-1(y303). Since another 50% of the XO progeny are yDp14/+; ceh-39(y414 or gk296) fox-1(y303), which are ∼100% viable, the viability of yDp14/yDp14; ceh-39(y414 or gk296) fox-1(y303) XO males was calculated by the following formula: [no. of males − (0.25)n]/[expected no. of yDp14/+ males, (0.25)n] × 100.