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
3.1. Isolation of a cDNA encoding a CHH-family peptide
As the first step in isolating a cDNA encoding a CHH- family peptide, degenerate oligonucleotide PCR primers
were designed based on amino acid sequences of regions conserved among CHH-family peptides [Fig. 1A]. The
amino acid sequences of six CHH-family peptides were aligned to find highly conserved regions. Two regions
Cys
7
–Asp
12
and Cys
23
–Asn
28
in the locust ITP were thereby selected, and two degenerate oligonucleotide pri-
mers CHHF and CHHR were designed by reverse translation [Fig. 1B].
Genomic DNA prepared from B. mori was used as the template in a PCR reaction using CHHF and CHHR.
Multiple bands were observed when the PCR product was separated by polyacrylamide gel electrophoresis and
detected with ethidium bromide data not shown. A band of about 65 bp, the size expected for a CHH-family
peptide, was excised from the gel, and DNA eluted from the gel was subjected to another round of PCR amplifi-
cation. The product of the second PCR was subcloned and analyzed for the nucleotide sequence. cDNA pre-
pared from the brain with CC and CA attached, and the suboesophageal, prothoracic and mesothoracic gang-
lia, was also used as the PCR template, but a product of the expected size was not detected.
One of the subclones analyzed contained a PCR pro- duct of 65 bp, including 34 bp derived from the two pri-
mers [Fig. 1C]. Its amino acid sequence, when concep- tually translated in one of the reading frames, exhibited
significant similarities to CHH-family peptides such as the ITP of the locust, S. gregaria Meredith et al., 1996,
and a CHH of the crayfish, Orconectes limosus Kegel et al., 1991 [Fig. 1C]. Thus, the PCR product was
likely to be derived from mRNA encoding a CHH-fam- ily peptide.
Next, the nucleotide sequence 253 bp of a 5 9 portion
of the cDNA was obtained by 5 9 RACE data not
shown, and this RACE product was used as a probe to screen a cDNA library about 200,000 clones of the
brain with attached CC and CA, and the suboeso- phageal, prothoracic and mesothoracic ganglia. Two
positive clones were isolated. One of them [1796 bp excluding the putative polyA tail] was analyzed for the
nucleotide sequence [Fig. 2A]. The clone contained a sequence of 31 bp [T
231
–C
261
in Fig. 2A] that was identical to the PCR product, and another [C
1
–C
236
in Fig. 2A] identical to a 3
9 part of the RACE product data not shown. As described below, this cDNA con-
tained an open reading frame ORF for a CHH-family peptide.
3.2. Inferred structure of the encoded protein In the clone analyzed, an ORF of 110 amino acids
was found near the 5 9 end [Fig. 2A]. This conceptual
protein was named BmCHHL due to its sequence simi- larities to CHH-famaily peptides. In order to reveal simi-
larities between the conceptual BmCHHL sequence and previously reported protein sequences, a computer-aided
homology search was performed. Five proteins with the highest similarity score were the ITP and ITP-L of the
locusts S. gregaria and L. migratoria Meredith et al., 1996; Macins et al., 1999, and the MOIH of the spider
crab Libinia emarginata Liu et al., 1997.
A computer-aided analysis Nielsen et al., 1997 sug- gests that the conceptual BmCHHL protein contains a
signal peptide with the likeliest signal cleavage site between Ala
23
and Leu
24
. The protein is likely to be further processed between Arg
35
and Ser
36
, since this putative processing site is preceded by a dibasic
sequence Arg
34
–Arg
35
, and the ITP precursor of S. gre- garia is processed at the corresponding site Meredith et
al., 1996. The three amino acids at the C-terminus of the ORF Gly
108
–Lys
109
–Arg
110
are likely to constitute the site of cleavage and amidation, as many CHH-family
peptides are amidated at the C-terminus Keller, 1992. Based on these results, we presume that the concep-
tual BmCHHL protein is a prepropeptide consisting of a signal peptide Met
1
–Ala
23
, a decapeptide Leu
24
– Glu
33
, a dibasic cleavage site Arg
34
–Arg
35
, a peptide of 72 amino acids Ser
36
–Val
107
, and an amidation sig- nal Gly
108
–Arg
110
. The putative mature BmCHHL Ser
36
–Val
107
dis- played 63 identity to the S. gregaria and L. migratoria
ITP at the amino acid level [Fig. 2B]. Sequence simi- larities to the ITP-L of S. gregaria and L. migratoria
were slightly lower 60 and 61 identity, respectively. The level of amino acid identity to crustacean CHH-fam-
ily peptides was much lower about 40 or less. In the putative mature BmCHHL peptide, six cysteine residues
were found at positions conserved in the CHH family [Fig. 2B], indicating that BmCHHL is a member of the
CHH family.
358 H. Endo et al. Insect Biochemistry and Molecular Biology 30 2000 355–361
Fig. 1. PCR amplification of a fragment of a cDNA encoding a CHH-family peptide in B. mori. A Designing PCR primers. Alignments of the
following CHH-family peptides are shown: a crab Carcinus maenas CHH Kegel et al., 1989, a crayfish Orconectes limosus CHH Kegel et al., 1991, a lobster Homarus americanus CHH Chang et al., 1990, a woodlice Armadillium vulgae CHH Martin et al., 1993, a prawn
Penaeus japonicus CHH Yang et al., 1995; Ohira et al., 1997 and a locust Schistocerca gregaria ITP Meredith et al., 1996. These peptides belong to ‘Type I’ subgroup of the CHH family see Discussion. Amino acids that are identical to those in the ITP sequence are indicated by
