Purification, characterization, cDNA cloning and nucleotide sequencinghilaris Masahiro Sugimura1, Hirofumi Watanabe1, Nathan Lo1and Hitoshi Saito2 1 National Institute of Agrobiological
Trang 1Purification, characterization, cDNA cloning and nucleotide sequencing
hilaris
Masahiro Sugimura1, Hirofumi Watanabe1, Nathan Lo1and Hitoshi Saito2
1
National Institute of Agrobiological Sciences, Ibaraki, Japan;2Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Japan
A cellulase (endo-b-1,4-glucanase, EC 3.2.1.4) was purified
from the gut of larvae of the yellow-spotted longicorn beetle
Psacothea hilarisby acetone precipitation and elution from
gels after native PAGE and SDS/PAGE with activity
staining The purified protein formed a single band, and the
molecular mass was estimated to be 47 kDa The purified
cellulase degraded carboxymethylcellulose (CMC), insoluble
cello-oligosaccharide (average degree of polymerization 34)
and soluble cello-oligosaccharides longer than cellotriose,
but not crystalline cellulose or cellobiose The specific
activity of the cellulase against CMC was 150 lmolÆ
min)1Æ(mg protein))1 TLC analysis showed that the
cellu-lase produces cellotriose and cellobiose from insoluble
cello-oligosaccharides However, a glucose assay linked with
glucose oxidase detected a small amount of glucose, with a
productivity of 0.072 lmolÆmin)1Æ(mg protein))1 The optimal pH of P hilaris cellulase was 5.5, close to the pH in the midgut of P hilaris larvae The N-terminal amino-acid sequence of the purified P hilaris cellulase was determined and a degenerate primer designed, which enabled a 975-bp cDNA clone containing a typical polyadenylation signal to
be obtained by PCR and sequencing The deduced amino-acid sequence of P hilaris cellulase showed high homology
to members of glycosyl hydrolase family 5 subfamily 2, and,
in addition, a signature sequence for family 5 was found Thus, this is the first report of a family 5 cellulase from arthropods
Keywords: cDNA cloning; cellulase; endoglucanase; insect; purification
Cellulase (endo-b-1,4-glucanase) is a widespread enzyme in
micro-organisms such as bacteria and fungi [1,2] Until
recently it was believed that cellulose digestion in animals
was mediated by microbial cellulase activity in their
intestine, and that no animals possessed endogenous
cellulase This traditional view of cellulase activity in
animals was challenged by two reports of endogenous
animal cellulase genes from plant-parasitic nematodes and
a termite [3,4] Since these discoveries, a number of other
animal cellulase genes have been reported (summarized in
[5])
Glycosyl hydrolases are categorized into 90 families
according to amino-acid sequence similarity and
hydropho-bic cluster analysis, and among them, cellulases are found
in 14 families [6,7] (refer also to: http://afmb.cnrs-mrs.fr/
CAZY/index.html) Known animal cellulases belong to
three glycosyl hydrolase families (GHFs): GHF 5
(plant-parasitic nematodes), GHF 9 (termites, cockroaches and
crayfish) and GHF 45 (mussel and beetle) These three
families are structurally unrelated and their evolutionary origins are likely to be independent
Larvae of the yellow-spotted longicorn beetle, Psacothea hilaris, feed on mulberry and fig trees, tunneling inside the stems and ingesting the living wood The major constituent
is cellulose (44.6%), followed by hemicellulose (28.5%); soluble sugars constitute only 4.7% of the dry weight of the wood [8] The habitat of P hilaris larvae suggests that they possess the ability to digest cellulosic materials In fact, a variety of carbohydrase activities, including endo-b-1, 4-glucanase and b-glucosidase, have been detected in the gut of P hilaris larvae and adults [8]
To clarify further the cellulase activity in P hilaris, we purified, characterized and obtained the cDNA sequence of
a protein from this species with cellulolytic activity
Materials and Methods
Measurement of the pH in the gut juice ofP hilaris larvae
P hilarislarvae were anesthetized by immersion in ice water for 10 min and their guts, including their contents, were removed by dissection The removed guts were washed in ice-cold distilled water to prevent contamination by body fluid and blotted on filter paper Then, the guts were cut into three parts (anterior midgut, posterior midgut and hindgut) and transferred into 1.5 mL plastic centrifuge tubes The samples in the tubes were centrifuged, and insoluble materials were discarded The pH values of the recovered
Correspondence to H Watanabe, National Institute of
Agrobiological Sciences, Tsukuba, Ibaraki 305-8634, Japan.
Tel./Fax: + 81 29 8386108,
E-mail: hinabe@affrc.go.jp
Abbreviations: GHF, glycosyl hydrolase family; CMC,
carboxy-methylcellulose; CBB, Coomassie Brilliant Blue.
Enzymes: endo-b-1,4-glucanase (EC 3.2.1.4).
(Received 30 January 2003, revised 22 May 2003,
accepted 30 June 2003)
Trang 2supernatants were measured with a pen-type pH meter
(model B-212; Horiba, Kyoto, Japan)
Enzyme assay
A carboxymethylcellulase (CMCase) assay was performed
by measuring the amount of reducing sugars after
incuba-tion of 100 lL 1% (w/v) CMC (standard molecular mass,
250 kDa; degree of carboxymethyl substitution, 0.7;
Sigma-Aldrich) in 0.1M sodium acetate (pH 5.5) with 20 lL
sample at 37C for an appropriate time period Reducing
sugars were measured with tetrazolium blue
(Sigma-Aldrich) as a chromogenic reagent, with glucose as a
stand-ard, as described by Jue & Lipke [9] Glucose production
from insoluble cello-oligosaccharide by the P hilaris
cellu-lase was investigated with a commercial glucose assay kit
(Glucose C-test; Wako Pure Chemicals), which utilizes
glucose oxidase in an enzyme-linked colorization step,
according to the instructions Insoluble
cello-oligosaccha-ride was prepared by the method of Sawano et al [10], and
the average degree of polymerization was 34 To test the
degradation activity of P hilaris cellulase against crystalline
cellulose, Avicel (Merck) was used under the same
condi-tions as the CMCase assay Optimal pH for P hilaris
cellulase activity against CMC was determined with 0.1M
sodium acetate (4–6), sodium phosphate (5.5–7.5) or Tris/
HCl (7–9.5) Hydrolytic products from
cello-oligosaccha-rides (cellobiose, cellotriose, cellotetraose and cellopentaose)
were analyzed by TLC
Electrophoresis and activity staining
SDS/PAGE was performed as described by Laemmli [11],
and native PAGE was carried out in the same way except
that SDS was excluded Proteins were stained with CBB,
and cellulase was visualized by activity staining on the gel In
the case of activity staining, the gel contained 0.1% (w/v)
CMC and the conditions of sample treatment for SDS/
PAGE were changed from boiling for 5 min to incubating at
33C for 30 min After being run, the gel was twice rinsed in
distilled water with gentle shaking for 5 min, and soaked in
0.1Msodium acetate buffer (pH 5.5) for 5–20 min The gel
was briefly rinsed with distilled water before staining with
0.2% (w/v) Congo red (Sigma-Aldrich) in water for 30 min
The excess dye was removed in 1M NaCl with gentle
shaking and replacement of the solution several times
Purification procedure
P hilarislarvae reared on a commercial artificial diet (Insecta
LF; Nihon Nosan Kogyo, Yokohama, Japan) were
dissec-ted, and whole guts were obtained The guts from six larvae
were homogenized in 4.5 mL sodium acetate buffer (0.1M,
pH 5.5) using a glass homogenizer, and centrifuged at
10 000 g for 10 min The supernatant was mixed with 3 vol
cold ()20 C) acetone by gentle stirring and then kept in a
freezer ()35 C) for 15 min The supernatant was decanted
after centrifugation at 20 000 g for 5 min, and the pellet was
dissolved in a minimum volume of 62.5 mM Tris/HCl
(pH 6.8) The suspension was centrifuged at 20 000 g for
5 min to remove insoluble materials, and then the
super-natant was loaded on a gel for native PAGE and activity
staining After electrophoresis, the gel was rinsed twice in distilled water with gentle shaking for 5 min and divided into two parts: (a) a gel strip for activity staining; (b) the remaining gel sheet for elution of proteins The gel sheet was stored at
4C until use, and the gel strip was subjected to activity staining The position of cellulase activity was identified, and the corresponding position of the gel sheet was cut out, sliced into small sections ( 1 mm cubes) and transferred to 1.5 mL centrifugal micro tubes The micro tubes were filled with distilled water and kept at 4C overnight to allow elution of the proteins from the gel sections The tubes were centrifuged at 10 000 g for 5 min to sediment the gel sections, and the supernatants were transferred to a commercial disposable device for ultrafiltration (Ultrafree-MC 10 000 NMWL centrifugal filter unit, Millipore), which had a polysulfone membrane with a 10-kDa exclusion size The filter unit was centrifuged at 5000 g until the remaining volume decreased below 40 lL The remaining solution, in which proteins were concentrated, was mixed with an equal volume of Laemmli Sample Buffer (Bio-Rad) without the addition of 2-mercaptoethanol before incubation at 33C for 30 min Then, proteins were subjected to SDS/PAGE After electrophoresis, proteins including the cellulase activity were eluted from the gel and concentrated as for native PAGE The purity of the eluted proteins was checked by further SDS/PAGE with CBB staining
Analysis of N-terminal amino-acid sequence The purified cellulase from P hilaris larvae was subjected
to SDS/PAGE and transferred to a poly(vinylidene difluo-ride) membrane (Bio-Rad) in transfer buffer [48 mM Tris,
39 mM glycine, 0.0375% (w/v) SDS, pH 9.2] by using a TRANS-BLOT SD Semi-dry Transfer Cell (Bio-Rad) Proteins were visualized with CBB, cut out, and subjected
to gas phase protein sequencing (model LF-3400 DT; Beckman)
cDNA cloning, genomic PCR and sequencing
A QuickPrep Micro mRNA Purification Kit (Amersham Bioscience) was used for isolation of mRNA from P hilaris larval midguts First-strand cDNA synthesis from the isolated mRNA and the following amplification of the target cDNA were performed with a SMARTTM RACE cDNA Amplification Kit (Clontech) according to the manufacturer’s instruction except that SuperScript II reverse transcriptase (Invitrogen) was used A degenerate oligonucleotide primer (5¢-GTICARGGIGTITGYATHG TIGAYG-3¢) was designed based on the N-terminal amino-acid sequence to amplify the cDNA for P hilaris cellulase RACE amplification of the 3¢-end was performed with this degenerate primer and an anchor primer corresponding to the anchor sequence combined to the 3¢-end of the
oligo-dT primer for first-strand synthesis of cDNA [12], and 5¢-RACE amplification was performed with a gene-specific primer based on the sequence of the 3¢-fragment The nucleotide sequence was determined by using a BigDyeTer-minator cycle sequencing kit and an ABI3700 automated DNA sequencer (Applied Biosystems) Sequence similarities were determined by aBLASTsearch (http://www.ncbi.nlm nih.gov/BLAST/) Forward (F) and reverse (R) primers,
Trang 31–24 (F), 209–230 (F), 289–311 (R), 446–469 (R), 651–680
(R), 667–688 (F), 713–740 (F), 781–807 (R) and 995–1013
(R), were designed from the cDNA sequence and used in
various combinations of genomic PCRs of P hilaris fat
body tissue, extracted as described previously [13] The
conditions for PCR were 35 cycles of 94C for 1 min, 52 C
for 1 min, 72C for 3 min Care was taken with the
solutions, and pipettes were used to avoid potential
contamination from previously prepared cDNAs
Protein assay
Protein concentration was determined by using a protein
assay kit (Coomassie Plus Protein Assay Reagent; Pierce)
with BSA as a standard
TLC analysis
TLC was performed with silica gel 60 (Merck) in a solvent
system of butan-1-ol/acetic acid/water (2 : 1 : 1, by vol.),
and sugars were visualized by a heat treatment at 120C for
10 min after the spraying of 50% (v/v) H2SO4in methanol
Results
Purification ofP hilaris cellulase from larval guts
The gut homogenate of P hilaris larvae was precipitated
with acetone The acetone treatment was effective in
reducing sample volume while minimizing loss of cellulase
activity On native PAGE, although a number of proteins
from the acetone-treated samples were detected by CBB
staining, only one band was detected by activity staining
(data not shown) After elution and concentration of the
protein solution containing the cellulase activity after native
PAGE, the solution was mixed with sample buffer for SDS/
PAGE without reducing agent and incubated at 33C for
30 min to load an SDS/polyacrylamide gel The SDS/
PAGE analysis detected three proteins bands by CBB
staining and one activity band by activity staining Proteins
including cellulase activity were eluted and concentrated
from the unstained gel, and the protein solution was
incubated with sample buffer for SDS/PAGE, adding
reducing agent at 100C for 5 min Then, the protein
solution was again subjected to SDS/PAGE A single
47-kDa protein band was detected by CBB staining, which
indicated a successful purification of cellulase (Fig 1)
Optimal pH forP hilaris cellulase activity and pH
in the gut juice ofP hilaris larvae
The effect of pH on P hilaris cellulase activity was tested
with 0.1Msodium acetate, sodium phosphate and Tris/HCl
buffers For a particular pH, the use of different buffers did
not markedly alter enzyme activity, nor did different
concentrations of these buffers The optimal pH for cellulase
activity against CMC was 5.5 Although the cellulase
activity was greatly reduced at pH values above 7,
some activity remained at pH 9.5 (data not shown) No
cellulase activity was observed at pH values less than 4.0
The digestive tract of P hilaris larvae was found to be
folded, and its total length was 1.5 times its body length
The boundary between the midgut and hindgut was indistinct, and no specialized hindgut structure such as that
of symbiont-possessing insects such as termites and scarab-aeid beetles was observed The gut contents were semisolid and scarcely flowed out upon dissection The pH values estimated in the anterior midgut, posterior midgut and hindgut were 5.7, 5.9 and 7.7, respectively
Enzymatic degradation of cellulose and its derivatives
byP hilaris cellulase
To investigate the ability of P hilaris cellulase to degrade cellulose and its derivatives, CMC (soluble in water but carboxymethylated), insoluble cello-oligosaccharide (aver-age degree of polymerization 34), Avicel (crystalline cellu-lose), cellobiose, cellotriose, cellotetraose and cellopentaose were used as substrates P hilaris cellulase readily degraded CMC, and the specific activity was determined to be
150 lmolÆmin)1Æ(mg protein))1 Insoluble cello-oligosaccha-ride was also readily degraded, and the degradation products were investigated by TLC Cellobiose and cello-triose were detected but not glucose (Fig 2A) However, with the use of a glucose assay kit, a small amount of glucose was detected as a degradation product from insoluble cello-oligosaccharide, and the glucose productivity was 0.072 lmolÆmin)1Æ(mg protein))1 Both cellotetraose
Fig 1 SDS/PAGE analysis of the purified cellulase fromlarval gut juice of P hilaris Lane 1, molecular mass standards consisting of myosin (200 kDa), b-galactosidase (116 kDa), phosphorylase B (97.4 kDa), BSA (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), trypsin inhibitor (21.5 kDa), lysozyme (14.4 kDa) and aprotinin (6.5 kDa) Lane 2, purified cellulase Proteins were stained with CBB R-250.
Trang 4and cellopentaose were degraded to cellobiose and
cellotri-ose (Fig 2B) Production of cellobicellotri-ose and cellotricellotri-ose from
cellopentaose by degradation is natural but production of
cellobiose and cellotriose from cellotetraose is abnormal
Cellotetraose should be degraded to produce two cellobiose
molecules or a combination of a cellotriose and glucose
To obtain information about the missing glucose,
glucose-digesting activity was tested No glucose-glucose-digesting activity
was detected in the purified P hilaris cellulase, suggesting
that contamination by enzymes that digest glucose was not
responsible for the missing glucose Transglycosylation
activity, known as the reverse reaction of some
endo-glucanases, was examined Very low activity was detected in
the purified P hilaris cellulase (data not shown) Avicel and
cellobiose were not degraded by P hilaris cellulase, and
cellotriose was partially degraded to cellobiose
Analysis of N-terminal amino-acid sequence, cDNA
sequence and deduced amino-acid sequence
The N-terminal amino-acid sequence of the purified P
hi-laris cellulase was analyzed The sample blotted to a
poly(vinylidene difluoride) membrane after SDS/PAGE
was used, and 30 amino acids from the N-terminus were
determined as follows: KDAAL ETVSK HGQLS VQGVD
IVDES GEKVQ A degenerate primer was designed based
on the amino acids determined, and an 0.9-kbp
3¢-frag-ment was amplified The flanking region for the 5¢-end of
the cDNA was obtained by 5¢-RACE with a gene specific
primer based on the sequence of the first fragment A
full-length of cDNA clone encoding a cellulase gene was
obtained and sequenced The nucleotide sequence was
deposited in GenBank (accession number is AB080266)
The cDNA encoding P hilaris cellulase contained an ORF
975 bp long, starting with an ATG codon at position 38 and
ending with a TAA codon at position 1014 A poly(A) tail
and typical polyadenylation signal were found Two
poten-tial N-glycosylation sites, N270 and N300, were detected in
the deduced amino-acid sequence The ORF consisted of a
protein of 325 amino acids The molecular mass of P hilaris
cellulase was calculated to be 36.0 kDa, and the first 21 amino acids were predicted to be a signal sequence for secretion The molecular mass of the mature enzyme was deduced to be 33.8 kDa The P hilaris cellulase consisted
of a single catalytic module only and no carbohydrate binding module was found.BLASTsearches with the deduced amino-acid sequence indicated that P hilaris cellulase was closely related to nematode cellulases and some bacterial cellulases, those belonging to GHF5 subgroup 2 The overall identities and similarities of P hilaris cellulase
to GHF 5 subgroup 2 members were: 49% and 67%
to Pseudomonas fluorescence CelE, 49% and 66% to Meloidogyne incognitaMI-ENG1, respectively A GHF 5 signature sequence, [LIV]-[LIVMFYWGA](2)-[DNEQG]-[LIVMGST]-x-N-E-[PV]-[RHDNSTLIVFY], was con-served in the deduced amino-acid sequence of P hilaris cellulase (Fig 3) The conserved potential proton donor and
Fig 2 Substrate specificity and degradation products of P hilaris cellulase TLC was performed with the solvent system butan-1-ol/acetic acid/water (2 : 1 : 1, v/v/v.) Sugars were visualized by incubating the plate at
120 C for 10 min after spraying with 50% (v/v) H 2 SO 4 in methanol (A) Lane 1, standard sugars: glucose (G1), cellobiose (G2), cellotri-ose (G3) and cellotetracellotri-ose (G4) Lane 2, insoluble cello-oligosaccharides Lane 3, purified P hilaris cellulase Lane 4, insoluble cello-oligosaccharides treated with P hilaris cellulase at 37 C for 8 h (B) Lane 1, standard sugars: G1, G2, G3, G4, cellopentaose (G5) and cellohexaose (G6) Lanes 2–5, G2–G5 treated with P hilaris cellulase at 37 C for
2 h.
Fig 3 Comparison of amino-acid sequences for potential catalytic proton donor and nucleophile regions of P hilaris cellulase (AB080266), Globodera rostochiensis ENG1 (GR-ENG1, AF004523), Meloidogyne incognita ENG1 (MI-ENG1, AF100549), Pseudomonas fluorescence CelE (CelE, X86798) and Erwinia chrysanthemi CelZ (CelZ, Y00540) Alignment was performed with the computer program CLUSTAL X (version 1.81) using the catalytic module of each cellulase Residue numbers are given on the left of the sequences Amino acids with similar groups of side chains and identical amino acids in sequences are indicated by : or *, respectively, above the sequence P and N below the sequence indicate potential catalytic proton donor and nucleophile amino acids, respectively.
Trang 5nucleophile amino acids are also found in the sequence
(Fig 3) Genomic PCR experiments with a variety of
different primers designed from the cDNA sequence
resulted in the amplification of bands in each case
Sequencing of these bands showed that they matched the
sequence of the cellulase cDNA completely; however, no
introns were found in any case
Discussion
Although optimal pH values for cellulase activity vary from
acidic to alkaline [14–17], all animal cellulases reported until
now have optimal activity under weak acidic conditions
[4,18,19] The optimal pH for the purified cellulase from the
larval gut of P hilaris against CMC was also 5.5 This is
reasonable for the physiological function of cellulase activity
in larval guts as the pH values in the anterior midgut,
posterior midgut and hindgut were 5.7, 5.9 and 7.7,
respectively These results suggest that P hilaris cellulase
functions mainly in the midgut, which is generally thought
of as a digestive and absorptive organ in the insect
alimentary canal
The purified P hilaris cellulase showed no degradation
activity against crystalline cellulose, which suggests that
P hilarislarvae may utilize only the amorphous parts of
cellulose materials ingested The elongated (about 1.5 times
its body length) digestive tract of P hilaris presumably
means that the ingested cellulose material is exposed to
enzymatic digestion for long periods
Animals in general absorb sugars in monomeric forms
such as glucose and fructose [20,21] Although the
degra-dation products of the P hilaris cellulase were found to be
cellotriose and cellobiose, glucose would be produced by
b-glucosidase activity in the larval gut, which has been
previously demonstrated in P hilaris [8]
TLC analysis detected cellotriose and cellobiose after
cellotetraose was treated with the purified P hilaris cellulase
(Fig 2B) A molecule of cellotetraose should be degraded
into two cellobiose molecules, or a cellotriose molecule and
a glucose molecule, by a single catalytic event Therefore,
after cellotetraose is degraded, glucose equivalent to
cello-triose should be detected However, only cellocello-triose was
detected by TLC Similarly, after cellotriose was degraded
by the P hilaris cellulase, TLC detected cellobiose but not
glucose (Fig 2B) It is known that some endoglucanases
possess transglycosylation activity as the reverse reaction
[22] Oikawa et al [23] reported that the addition of acetone
to the reaction buffer increased transglycosylation activity
of the Rhodotorula glutinis cellulase Observation of
trans-glycosylation activity in the purified P hilaris cellulase was
attempted under various conditions, including the addition
of acetone to the reaction mixture Although
transglycosy-lation activity was detected, it was not enough to explain the
lack of glucose However, transglycosylation is the most
probable explanation for the missing glucose, because there
are few other potential mechanisms for eliminating it
The molecular mass of P hilaris cellulase deduced from
its DNA sequence is 36.0 kDa The apparent molecular
mass of the purified P hilaris cellulase was, however,
estimated to be 47 kDa from its mobility on SDS/PAGE
N-Terminal amino-acid sequencing analysis indicated that
the mature protein of P hilaris cellulase was a truncated
form, which lacked a signal peptide composed of the first 21 amino acids Therefore, the deduced molecular mass of mature cellulase protein is 33.8 kDa and the difference is 13.2 kDa This inconsistency may be explained by a possible post-transcriptional modification at two potential N-glyco-sylation sites, N270 and N300, in the amino-acid sequence
of P hilaris cellulase Alternatively, the mobility of the
P hilariscellulase protein on SDS/PAGE may be different from those of the standard proteins used in the experiment
A cDNA encoding a cellulase, which belongs to GHF 45, has been cloned from a gut library from the phytophagous beetle, Phaedon cochleariae [24] This GHF 45 cellulase has been confirmed to be expressed in the gut of P cochleariae
P cochleariae and P hilaris are closely related species, belonging to the same superfamily, Chrysomeloidea There-fore, a GHF 45 cellulase might have been expected from
P hilaris However, the cellulase purified from P hilaris in the current study was shown to belong to GHF 5 The activity staining indicated that there were no other CMCase activities except the GHF 5 cellulase in the larval gut of
P hilaris The sensitivity of the activity staining used is high, and CMCase activity has been detectable with 5 lL of 1000 times diluted gut juice of P hilaris larvae Therefore, if a GHF 45 enzyme is present, its level of expression would be extremely low in P hilaris larvae
Glycosyl hydrolases have been categorized into 90 families according to homologies of their amino-acid sequences, and cellulases are distributed into 14 families (http://afmb.cnrs-mrs.fr/CAZY/index.html) Most of these
14 families are composed of cellulases only, but some include other enzymes, such as xylanase and mannanase
P hilariscellulase was proposed to belong to GHF 5 and the signature sequence of GHF 5 was found in the amino-acid sequence (Fig 3) GHF 5 is the largest GHF and includes endoglycosylceramidase, b-mannanase, exo-b-1,3-glucanase, endo-1,6-b-glucosidase, b-xylanase and some other enzymes, in addition to cellulase (endo-b-1,4-gluca-nase) On the basis of sequence homology, GHF 5 enzymes can be further divided into five subfamilies [1,25] P hilaris cellulase is closely related to subfamily 2 members, which is composed only of cellulases from bacteria, fungi and nematodes It has been shown that the nematode cellulase genes contain several introns, and the positions are conserved in the deduced amino-acid sequences [26] Genomic DNA of P hilaris was amplified and sequenced
to determine whether the cellulase gene contained introns; however, none were found, despite combinations with several primers As intronless genes appear to be fairly common among insects [27,28], the lack of introns in the
P hilaris cellulase gene does not provide evidence for its recent horizontal transfer from a prokaryote A discussion
of the evolutionary origins of the cellulase enzymes in animals will be given elsewhere [29]
Acknowledgements
We thank Ms Sanae Wada for advice about rearing P hilaris larvae This work was supported by the Promotion of Basic Research Activities for Innovative Biosciences Fund from the Bio-oriented Technology Research Advancement Institution (BRAIN; Omiya, Saitama, 331-8537 Japan; www.brain.go.jp) and by the Pioneer Research Project Fund (No PRPF-0022) from the Ministry of
Trang 6Agriculture, Forestry and Fisheries of Japan N.L is supported by a
Science and Technology Agency of Japan Postdoctoral Fellowship.
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