356 H. Endo et al. Insect Biochemistry and Molecular Biology 30 2000 355–361
L has not been elucidated Macins et al., 1999. cDNAs encoding ITP and ITP-L were also cloned in Locusta
migratoria Macins et al., 1999. It is an intriguing question whether insects, like crus-
taceans, make use of multiple CHH-family peptides to regulate physiology, development andor reproduction.
It also remains unknown whether the ITP is unique to orthopterans, or common to other insect orders. To
address these questions, the authors set out to search for cDNAs encoding CHH-family peptides in the silkworm
Bombyx mori order Lepidoptera. Here we report the first isolation of a cDNA encoding a CHH-family pep-
tide in non-orthopteran insects.
2. Materials and methods
2.1. Experimental animals Fourth instar B. mori larvae were purchased from
Kanebo Silk Elegance, Co., and grown at 25 °
C until sac- rificed during fifth instar.
2.2. PCR amplification of B. mori genomic DNA B. mori genomic DNA was generously provided by
Dr Haruhiko Fujiwara. The polymerase chain reaction PCR reaction with degenerate oligonucleotide primers
was carried out as previously reported Watanabe et al., 1996, except that denatured genomic DNA was added
to 1 ng
µ l.
2.3. Preparation of RNA The brain [to which the CC and corpus allatum CA
were attached] and the suboesophageal, prothoracic and mesothoracic ganglia, were collected from 200 fifth
instar larvae. The mesothoracic ganglion was only occasionally included. The mixture of tissues was homo-
genized in 6 ml of Isogen Wako, and total RNA was prepared according to instructions from the manufac-
turer. PolyA
+
RNA was prepared using Oligotex-dT30 super Roche Japan.
2.4. 5 9 Rapid amplification of cDNA ends RACE
To 100 ng of the total RNA, 100 pmol of primer CHHR Fig. 1 was annealed, and cDNA was synthe-
sized and polyA-tailed. The cDNA was used as a tem- plate in a PCR reaction with the following three primers:
a 3 9 primer 59-GCAGATTCTGTCGAGACG-39, and
two 5 9 primers 59-GAGTCGACTCGAGAATTCT
17
-3 9
and 5 9-GAGTCGACTCGAGAATTC-39. The product
of this reaction was subcloned in pCR2.1 Invitrogen, and analyzed for the nucleotide sequence.
2.5. Construction and screening of a cDNA library cDNA was synthesized with 1
µ g of the polyA
+
RNA using
a Time
Saver cDNA
synthesis kit
Pharmacia Biotech, and ligated to the λ
ZAPII vector Stratagene. The ligation product was packaged using
Gigapack Gold Packaging Extract Stratagene. The 5
9 RACE product was radio-labeled to be used as a probe to screen the library. Procedures for library
screening have previously been described Watanabe et al., 1996.
2.6. Nucleotide sequence analysis Nucleotide sequences of cDNA clones were determ-
ined, and conceptual translation performed as has been described Watanabe et al., 1996. In order to reveal
similarities between the BmCHHL protein sequence and previously reported protein sequences, a homology
search was performed in ‘Protein All’ version 3.0t84 databases including PIR and SWISS-PROT of DNA
Databank of Japan using the FASTA program Pearson and Lipman, 1988.
2.7. Northern hybridization and reverse transcription RT-PCR
PolyA
+
RNA 0.5 µ
g prepared as described above was electrophoresed and blotted on to a nylon mem-
brane, and hybridization was performed as previously described Watanabe et al., 1996. A HindIII fragment
of 515 bp [A
622
–A
1137
nucleotide positions based on Fig. 2A] was used as the hybridization probe. After
hybridization the filter was washed under a low strin- gency condition 2
× SSPE, 37
° C.
For RT-PCR analysis, total RNA was isolated from staged fifth instar larvae. An anti-sense probe, the
sequence of which was inverse-complementary to A
247
– C
264
[nucleotide positions based on Fig. 2A], was annealed to the total RNA samples and cDNA was syn-
thesized as previously described Watanabe et al., 1996. The cDNA samples were used as templates in a PCR
reaction 24 or 27 cycles of 94 °
C for 30 s, 55 °
C for 30 s and 72
° C for 30 s using the following two primers: the
5 9 primer corresponding to C
75
–A
95
[Fig. 2A] and the 3
9 primer inverse-complementary to A
247
–C
264
. PCR products were run on 6 polyacrylamide gels, and
detected with ethidium bromide. A control experiment in which the reverse transcriptase step was omitted was
also performed.
2.8. In situ hybridization The above HindIII fragment was subcloned in the
pBluescript II SK vector Stratagene. The digoxigenin- 11-dUTP Boehringer-Mannheim labeled anti-sense and
357 H. Endo et al. Insect Biochemistry and Molecular Biology 30 2000 355–361
sense riboprobes were generated with T7 and T3 RNA polymerases PROMEGA, respectively. The latter
probe was used as a negative control. The whole mount in situ hybridization was performed on the brain with
attached CC and CA, and suboesophageal and protho- racic ganglia, from day 4 fifth instar larvae as previously
reported Tsuzuki et al., 1997, except that non-fat dry milk was used as the blocking agent instead of sheep
serum and that the methyl salicylate clarification step was omitted.
3. Results
