Northern blot and RT-PCR analysis of Toll-related gene
expression. (A) Poly(A)+ RNA extracted from
embryos (Emb.), third-instar larvae [L(3)], pupae (P, 0–2 or 2–4
days), adult male or female Drosophila, and the cell
line S2 were submitted to Northern blot analysis and hybridized to
probes derived from Toll, 18w, and
Toll-3 to Toll-8 gene sequences. No
signal could be observed for the Toll-3 and
Toll-4 probes. A probe recognizing RNA coding for
ribosomal protein 49 (rp49) was used to ensure that comparable amounts
of RNA were loaded in all lanes. uc, unchallenged; p.i., postinfection
with a mixture of Gram-negative and Gram-positive bacteria; and d,
days. (B) RT-PCR analysis of Toll-3 and
Toll-4 expression. Primers specific for the
Toll-3 gene were designed to flank a 104-bp intron
sequence in the genomic DNA, resulting in the amplification of a 342-bp
cDNA (c)-derived fragment (arrow) or a 446-bp genomic DNA (g)-derived
fragment (*). Primers for the Toll-4 gene were
designed to amplify a 480-bp intronless fragment. In this case, RT was
omitted in a control reaction to ensure that the amplified band is
derived from RNA. cDNA was prepared from mRNA derived from pupae
(Toll-3) or third-instar larvae (Toll-4). L,
100-bp ladder.