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
3.1. cDNA cloning and characterization The
full-length cDNA
sequence accession
: AF227531 of MCHA is shown in Fig. 1. MCHA was
named CYP6L1 by the P450 Nomenclature Committee. CYP6L1 has an open reading frame of 1509 nucleotides
with a deduced protein of 503 amino acids and a molecu- lar mass of 57 kDa. A BLAST search Altschul et al.,
1997 with the deduced amino acid sequence shows that CYP6L1 is most similar to the members of P450 family
6 from insects. A comparison of CYP6L1 with some members of family 6 is shown in Table 2. CYP6L1 has
the highest percent amino acid identity 37 with CYP6H1, a putative ecdysone 20-hydroxylase from the
grasshopper, Locusta migratoria Winter et al., 1999.
CYP6L1 is a typical microsomal P450 and its deduced
Table 2 Comparison of CYP6L1 from B. germanica with other related P450s
a
Isoforms Species
identity
b
Life stage specific expression Sex specific expression
Source of tissues
d
Embryos Immature
c
Adults Males
Females CYP6L1
B. germanica 100
– –
+ +
e
– Many tissues examined
see text CYP6H1
L. migratoria 37
– +
+ NI
NI Malpighian tubules
CYP6A1 M. domestica
28 +
+ +
+ +
Gut, fat body CYP6A2
D. melanogaster 31
NI NI
+ NI
NI Fat body and other tissues
CYP6A9 D. melanogaster
29 NI
NI +
NI NI
NI CYP6B1
P. polyxenes 28
NI +
NI NI
NI Midgut
CYP6B2 H. armigera
28 –
+ –
NI NI
Midgut, fat body, headforegut
CYP6D1 M. domestica
26 –
– +
+ +
Many tissues
a
References: CYP6A1 Carino et al., 1992, 1994; CYP6A2 Brun et al., 1996; CYP6A9 Maitra et al., 1996; CYP6B1 Cohen et al., 1992; CYP6B2 Ranasinghe et al., 1997; Xiao-Ping and Hobbs, 1995; CYP6D1 Scott, 1999 and CYP6H1 Winter et al., 1999 NI
= not investigated.
b
The per cent amino acid identity was calculated based on the Clustal method of MEGALIGN program DNASTAR Inc., Madison, Wisconsin.
c
Nymphs for B. germanica and L. migratoria, larvae for all others.
d
Tissues where the expression was investigated or from which the cDNA was cloned.
e
Specifically in testes and accessory glands see text.
amino acid sequence shares a number of common characteristics with other members of the P450 super-
family. The N-terminal region is strongly hydrophobic, with hydrophobic residues accounting for |70 16 out
of the first 23 amino acid residues, which is important for membrane-anchoring Wachenfeldt and Johnson,
1995. The sequence FGDGPRNCIG at positions 431– 440 matches the signature motif FxxGxxxCxG, which is
the heme-binding region and is highly conserved among P450s. AGFET at positions 298–302 is highly conserved
consensus AGGxxT within the I helix where T
302
may function as a proton donor andor an acid-base cata- lyst in oxygen scission Wachenfeldt and Johnson,
1995. The charge pair at positions 356–359 is highly conserved EVLR; consensus ExxR within the K helix
which is assumed to be hydrogen-bonded to the ‘meander’, a region N-terminus to the heme-binding
region Peterson and Graham-Lorence, 1995. Other conserved
regions or
residues include:
WRQIR consensus WxxxR at positions 129–133 in the C helix,
P
318
exactly 16 residues from T
302
and YPDP consensus aromaticxxPD at positions 404–407 followed five
residues later by PERF at positions 412–415 Nelson, 1998.
3.2. Expression in adults CYP6L1 mRNA expression was examined in six life
stages mixed sexes: eggs egg cases, first and second instars, third and fourth instars, fifth instars, sixth instars
and adults. CYP6L1 mRNA ~1.8 kb long signal was detected only in adults, but not in eggs nor in any nym-
phal instars Fig. 2.
184 Z. Wen, J.G. Scott Insect Biochemistry and Molecular Biology 31 2001 179–187
Fig. 2. Life stage-specific expression of CYP6L1. Total RNA was prepared from approximately 1 g of cockroaches mixed sexes for each life
stage and 10 µ
g of total RNA was loaded for each lane. Northern hybridization with the CYP6L1 cDNA probe is shown on the top panel. RNA loading was standardized by ethidium bromide staining of 18S rRNA bottom panel.
3.3. Expression in adult male abdomens CYP6L1 mRNA expression was examined in the
abdomens A versus the remainder of the bodies H +
T in adult males or females. CYP6L1 mRNA was detected
in the abdomens of males, but not in the abdomens of females nor in the remainder of the bodies of males or
females Fig. 3.
3.4. Expression in reproductive tissues Given that CYP6L1 mRNA was expressed exclus-
ively in the abdomens of adult males, expression in the
Fig. 3. Sex-specific expression of CYP6L1. Total RNA was prepared from abdomens A or the remainder H
+ T of the bodies excluding
abdomens of 15 male or female adult German cockroaches. Ten micrograms of total RNA was loaded for each lane. Northern hybridization with the CYP6L1 cDNA probe is shown on the top panel. RNA loading was standardized by ethidium bromide staining of 18S rRNA bottom panel.
male reproductive system was examined. Fig. 4 shows that CYP6L1 mRNA was detected in the abdomens after
either testes or accessory glands were removed, but could not be detected when both the testes and accessory
glands were removed. These results indicate that CYP6L1 was specifically expressed in the testes germ
cells and accessory glands somatic cells of male adult German cockroaches. The greater reduction in CYP6L1
signal following the removal of accessory glands com- pared to removal of testes may simply be a reflection of
the greater mass of the accessory glands. CYP6L1 mRNA was also detected by Northern blot using total
RNA from testes, but could not be detected using total
185 Z. Wen, J.G. Scott Insect Biochemistry and Molecular Biology 31 2001 179–187
Fig. 4. Tissue-specific expression of CYP6L1. Total RNA was pre-
pared from abdomens of 15 male adult German cockroaches excluding certain tissues. Ten micrograms of total RNA was loaded for each
lane. Lane 1: Abdomens with testes removed; Lane 2: Abdomens with accessory glands removed; Lane 3: Abdomens with both testes and
accessory glands removed; Lane 4: Whole abdomens. Northern hybridization with the CYP6L1 cDNA probe is shown on the top
panel. RNA loading was standardized by ethidium bromide staining of 18S rRNA bottom panel.
RNA from guts or abdomen remains excluding testes, accessory glands and guts results not shown. For
unknown reasons, we were unable to isolate enough RNA from dissected accessory glands for analysis.
3.5. Southern blots Genomic DNA digested with BamHI, NotI, SmaI, and
XhoI was used in a Southern blot to evaluate the speci- ficity of our CYP6L1 probe. A single band was observed
in all cases data not shown, suggesting that the probe was specific for CYP6L1. The relatively large fragments
that were detected ranging from approximately 10–20 kb make it impossible to conclude whether CYP6L1
exists as a single copy gene or not.
4. Discussion
