Kinchen et al, Nature submission 2004-07-22085
Supplementary Methods
Supplementary Methods
Characterization and cloning of ced-10(t1875)
The ced-10(t1875) allele was isolated in a genetic screen for linked lethal genes on LGs
IV and V. t1875 was originally mapped close to the ced-10 locus and failed to
complement ced-10(n1993). The embryonic lethality of t1875 is maternally rescued since
98% of progeny (n=100) from heterozygous mothers survive. t1875 shows a non-strict
maternal effect, as lethality can be zygotically rescued. In addition to maternal
contribution, phagocytosis of apoptotic corpses in t1875 mutants is also subject to zygotic
effects, since homozygous embryos from heterozygous mothers showed some persistent
apoptotic corpses (wild-type, 0 corpses vs. t1875m+z-, 5.0  3.8, n=10, as scored in the
L1 head).
To identify the molecular lesion in t1875, the ced-10 locus was amplified by PCR off
wild-type (Bristol N2) and t1875 genomic DNA prepared by ‘single worm lysis’ (worms
were picked into 50 mM KCl, 10 mM Tris pH 8.3, 2.5 mM MgCl2, 0.45% NP-40
(IGEPAL), 0.45% Tween-20, 20 g/mL proteinase K and incubated at 65 C for 1 hour,
then 95 C for 10 minutes). For primer information please see Supplementary Table 6.
Amplified fragments were gel-purified and sequenced; a single point mutation, T2A, was
identified, which removes the initiator methionine and disrupts a HpyCH4 V restriction
site in the mutant (AGCA) versus wild-type (TGCA), which was used to confirm the
mutation.
Plasmid Construction
The act-5 genomic fragment was amplified by PCR off N2 genomic DNA using primers
(Supplementary Table 6) that added an Asc I site upstream and an Fse I site downstream
of the coding sequence. The PCR product was then cloned under the control of the lim-7
promoter (gift of O. Hobert), which is primarily expressed in the somatic sheath cells1, in
a construct that had been modified with Asc I and Fse I sites within the polylinker to
create pLB.lim-7p. YFP (gift of A. Fire) was amplified by PCR as an Asc I cassette and
then cloned upstream of act-5 in the pLB.lim-7p vectors. act-5 coding sequences were
sequenced and found to be wild-type.
The ced-1 locus was amplified from pZZ6102 by PCR (for primer information, see
Supplementary Table 6); the ced-1 promoter was also amplified from this plasmid and
approximately 1 kb of sequence 3’ to ced-1 was amplified from wild-type genomic DNA
with Fse I added upstream and Apa I added downstream of the 3’ sequence, and cloned
into the vector. The construct was then C-terminally tagged with yfp or cfp as an Fse I
cassette to create pJMK261 and pJMK263. ced-1 coding regions were sequenced and
found to be wild-type.
The ced-6 minigene was amplified from N2 genomic DNA using PCR (primers,
Supplementary Table 6); the pieces were assembled in the pLB.eft-3 vector3. yfp or cfp
(gift of A. Fire) was then fused to the N-terminus of ced-6 as an Asc I cassette to generate
Kinchen et al, Nature submission 2004-07-22085
pJMK303 and pJMK304; the ced-6 coding sequence was sequenced and determined to be
wild-type.
Co-localization of reporter constructs
All YFP/CFP transgenes were checked for bleed-through into YFP or CFP filter sets
prior to analysis; no bleed-through was identified at the exposure times used.
In the germ line, the somatic sheath cells are very thin; when cells aren’t engulfed, they
push against the sheath and raise an area, much the way a muffin underneath a sheet of
Saran WrapTM will cause a localized distortion. This can be seen as an area of fainter
staining (Figure 2T, arrowhead) that is much wider and morphologically distinct from the
discrete halo around cells being actively engulfed (Figure 2R, arrow).