180 Z. Wen, J.G. Scott Insect Biochemistry and Molecular Biology 31 2001 179–187
of P450s in herbivore–plant interactions, insecticide resistance, insect development and physiology.
P450s have been implicated in insect reproduction Hodgson, 1985; Yu and Terriere, 1974. However, no
individual insect P450 has yet been associated with reproduction. Herein, we report cloning of the full-length
cDNA of the first sex-specific insect P450, CYP6L1, which is exclusively expressed in the reproductive sys-
tem of male adult German cockroaches. This finding has important implications for understanding the roles of
P450s in insect reproduction and may offer a potential target for the development of novel insect control agents.
2. Materials and methods
2.1. Insects Two strains of German cockroaches were used: Bay-
gon-R Siegfried and Scott, 1991 and CSMA Scott and Matsumura,
1981. Cockroaches
were reared
as described previously Siegfried and Scott, 1991.
2.2. Dissection To examine the expression of CYP6L1 in different
body regions of male and female adult cockroaches the abdomens were separated from the remainder of the bod-
ies on dry ice using forceps. The expression of CYP6L1 in the abdomens of adult male cockroaches was also
examined. For this, cockroaches were anesthetized on ice and their wings removed. Then cockroaches were
pinned in a Sylgard dish, opened dorsal-ventrally and the testes andor accessory glands Cornwell, 1968;
Snodgrass, 1937 were quickly removed under a binocu- lar dissection scope at 24
° C. The heads and thoraces
were then cut off and the dissected abdomens put on dry ice for subsequent RNA isolation.
2.3. RNA isolation Total RNA was isolated using 5 M guanidine thio-
cyanate solution based on the method of Chirgwin et al. Chirgwin et al., 1979. Messenger RNA was isolated
from total RNA using Oligotex suspension QIAGEN or directly from tissues using a QuickPrep mRNA puri-
fication kit Pharmacia as described by the manufac- turers.
2.4. cDNA synthesis Superscript II GIBCOBRL was used to synthesize
the first strand cDNA following the manufacturer’s instructions. For the first strand cDNA synthesis, C3PT
Danielson and Fogleman, 1997 Table 1 was used as primer and |500 ng mRNA from abdomens of male
adult Baygon-R strain of German cockroaches as tem- plate. After RNAase H 2 units treatment at 37
° C for
30 min, the first strand cDNA was isolated using a QIA- quick PCR purification kit QIAGEN to remove the pri-
mers and short cDNA eluted with 100 µ
l H
2
O. The purified cDNA was used as a template for 3
9 and 59 RACEs Frohman et al., 1988 as described below.
2.5. 3 9 RACE
The first strand cDNA 5 µ
l was used as a template. A degenerate primer based on the P450 heme signature
motif GPRNCIG Danielson et al., 1999 and a C3 primer Danielson and Fogleman, 1997 Table 1 were
used to amplify putative 3 9 cDNA sequences of P450
genes by PCR. The PCR reaction 100 µ
l was heated to 95
° C for 3 min, followed by 35 cycles of amplifi-
cation 95 °
C, 50 °
C and 72 °
C sequentially each for 30 s and a final extension at 72
° C for 10 min. After 1.5
agarose gel fractionation of the PCR products, a region corresponding to about 300–700 bp based on DNA size
markers 100 bp ladder, GIBCOBRL was cut and the DNA purified using a QIAquick gel extraction kit
QIAGEN. The purified PCR products were cloned into pCR
2.1 vector Invitrogen following the manufac- turer’s instructions. Plasmids were isolated from positive
clones white colonies and the inserts sequenced by the DNA Sequencing Facility at Cornell University. Putative
P450s were identified based on BLAST analysis Altschul et al., 1997 using the deduced amino acid
sequences. The inserts of six clones MCH1, MCH6, MCH11, MCH45, MCH46 and MCH75 had identical
sequences with a length of 371 bp excluding poly A. The gene these sequences represented was named
MCHA.
2.6. 5 9 RACE
To obtain the 5 9 sequence of MCHA, the purified first
strand cDNA was tailed with dCTP by terminal deoxyn- ucleotidyl transferase GIBCOBRL following the
manufacturer’s instructions with the following modifi- cation. Instead of using the manufacturer’s 5
× reaction
buffer, we used a 5 ×
tailing buffer 500 mM sodium cacodylate, 1 mM
β -mercaptoethanol, 20 mM magnes-
ium chloride, pH 7.2 as suggested by P. Danielson University of Denver, Colorado. The tailed cDNA was
purified using a PCR purification kit as described above eluted with 100
µ l H
2
O and 5 µ
l was used as template for 5
9 RACE. An MCHA specific antisense primer MCHAA1
Table 1 and Fig. 1 was designed based on the 3 9
sequence information of MCHA. Two rounds of PCR were performed for the 5
9 RACE. The first round PCR
181 Z. Wen, J.G. Scott Insect Biochemistry and Molecular Biology 31 2001 179–187
Table 1 Primers used for cloning of CYP6L1 cDNA
Primer Sequence
C3PT 5
9-GACTCGAGTCGGATCCATCGTTTTTTTTTTTTTTTT-39 C3
5 9-GACTCGAGTCGGATCCATCG-39
Heme 5
9-GGICCIACGIAACTTGCTATIGG-39 AAP
5 9-GGCCACGCGTCGACTAGTACGGGGGGGGGGGGGGGG-39
AUAP 5
9-GGCCACGCGTCGACTAGTAC-39 MCHAA1
5 9-AACAAACATCAGAGGAAGTCGTCC-39
MCHAA9 5
9-CTCTGAACCCTTCAGTTTCACC-39 MCHAS3
5 9-CAACGTCAAGAAGTAGCCAACC-39
MCHAS4 5
9-TTCAGTCCTGTGGTTCCTTTC-39
100 µ
l reaction used MCHAA1AAP GIBCO, Table 1 as the primer set and dCTP tailed first strand cDNA
see above as template. The conditions for the first round PCR were 95
° C for 3 min followed by 32 cycles
in the order of 95 °
C for 1.5 min, 50 °
C for 1.5 min and 72
° C for 4 min. The PCR reaction had a final extension
for 15 min at 72 °
C. After electrophoresis 1.5 agarose gel of the first round PCR products, a region corre-
sponding to 1–2 kb was cut and the PCR products pur- ified eluted with 100
µ l H
2
O. The purified first round PCR products 5
µ l then served as templates for the
second round PCR using MCHAA1AUAP GIBCO, Table 1 as the primer set. Products of the second round
PCR conditions were the same with that of the first round PCR, except the annealing temperature was raised
to 55
° C and cycles increased to 35 corresponding to a
region of 1–2 kb on agarose gel was again purified and cloned into pCR
2.1 vector. Positive clones were screened by colony PCR Gussow and Clackson, 1989
using single putatively positive colonies white colonies as templates and heme primerMCHAA1 as the primer
set. The insert lengths of those positive clones were further checked by colony PCR using MCHAA1AUAP
as the primer set. Plasmids were isolated from two clones A11 and A180 with the longest inserts and the
inserts were sequenced. The newly obtained sequence information from A11 and A180 was used to design pri-
mers for another round of 5
9 RACE. After two rounds of 5
9 RACE, the 59 sequence beyond the translational start codon was obtained. All the adjacent sequences
obtained by 3 9 and 59 RACEs had at least 150 bp overlap
with each other. The putative full-length MCHA sequence MCHAMER was obtained by integrating over-
lapping sequences from the 3 9 RACEs and 59 RACEs.
2.7. Cloning of full-length cDNA Two sense primers upstream of the start codon
MCHAS3 and MCHAS4, Table 1 and Fig. 1 and one antisense
primer downstream
of the
stop codon
MCHAA9, Table 1 and Fig. 1 were designed based on the sequence of MCHAMER. The first round PCR
products using first strand cDNA as template and MCHAS4MCHAA9 as primer set were used as tem-
plates to
perform the
second round
PCR with
MCHAS3MCHAA9 as primer set. The conditions for the first round PCR were 95
° C to denature for 3 min
followed by 32 cycles in the order of 95 °
C for 1 min, 50
° C for 1 min and 72
° C for 2 min. The PCR reaction
ended with a final extension for 15 min at 72 °
C. The conditions for the second round PCR were the same as
the first round PCR, except that the cycles were 35 instead of 32. A 1.6 kb band on agarose gel was purified
and cloned into pCR
2.1 vector. A positive clone FA13 was identified by colony PCR and its insert
entirely sequenced. 2.8. Northern blots
Northern blot analysis of CYP6L1 mRNA expression was performed as described by Sambrook et al.
Sambrook et al., 1989. Briefly, 10 µ
g of total RNA was fractionated on 1 denaturing formaldehyde agarose gel
containing ethidium bromide. After washing in distilled water for about 3–4 h with several changes, the RNA
was transferred to a GeneScreen Plus
hybridization transfer membrane NEN
Life Science Products, Inc..
Equal loading was monitored by comparing the density of the 18S ribosomal RNA rRNA band Savonet et al.,
1997; Spiess and Ivell, 1998 on the agarose gel before transfer andor on the membrane after transfer under UV.
A 1.6 kb CYP6L1 fragment was amplified by PCR using plasmids isolated from FA13 as templates and
MCHAS3MCHAA9 as the primer set, labeled with [
α -
32
P] dCTP using the RadPrame
labeling system GIBCOBRL, and used as a hybridization probe. The
membrane was hybridized to the probe in QuickHyb sol- ution Stratagene at 68
° C. Washing was done at high
stringency i.e. three 15 min washes with 2 ×
SSC +
0.1 SDS at room temperature, followed by a 30 min wash
with 0.2 ×
SSC +
0.1 SDS at 60 °
C. The membrane was air dried and exposed to a BioMax MR film Eastman
Kodak. All Northern analyses were repeated at least three times using independent preparations of RNA.
182 Z. Wen, J.G. Scott Insect Biochemistry and Molecular Biology 31 2001 179–187
Fig. 1. CYP6L1 full-length cDNA and deduced amino acid sequence. Amino acids AA are numbered on the left and nucleotides NT, on the
right. Primers Table 1 are thickly underlined and conserved regions thinly underlined. Putative polyadenylation signals are indicated by bold font.
2.9. Southern blots Genomic DNA was isolated from the abdomens of
male adult German cockroaches of Baygon-R strain using standard methods Sambrook et al., 1989. South-
ern blots
were performed
by standard
methods Sambrook et al., 1989 with some modifications. Four
restriction enzymes BamHI, NotI, SmaI, and XhoI, hav- ing no cutting site within the CYP6L1 cDNA sequence
were used to digest 2.5 µ
g of genomic DNA overnight as described by the manufacturer GIBCOBRL. Fifty
units of each enzyme was used for each reaction in a volume of 100
µ l. The digested DNA was then ethanol
precipitated and fractionated by electrophoresis on 1
183 Z. Wen, J.G. Scott Insect Biochemistry and Molecular Biology 31 2001 179–187
agarose gel containing ethidium bromide. After electro- phoresis, the gel was placed in 0.25 M HCl with gentle
shaking until 10 min after the dyes had changed color. After being rinsed in H
2
O, the gel was denatured in a solution containing 1.5 M NaCl and 0.5 M NaOH with
gentle shaking for 30 min. The gel was then rinsed with H
2
O and neutralized in a solution containing 1.5 M NaCl, 0.5 M Tris–HCl pH 7.2 and 0.001 M EDTA.
After rinsing in H
2
O, the DNA was transferred to a GeneScreen Plus
membrane NEN
Life Science Pro- ducts, Inc.. Probe preparation, membrane hybridization
and film exposure were performed exactly as described in Section 2.8. Southern analyses were repeated three
times.
3. Results
