Sequence analysis and quantification of transposase cDNAs of transposon TCp3.2 in Cydia pomonella larvae

Archives of Insect Biochemistry and Physiology 63:135–145 (2006)
Sequence Analysis and Quantification of
Transposase cDNAs of Transposon
TCp3.2 in Cydia pomonella Larvae
Hugo M. Arends and Johannes A. Jehle*
The Tc1-like transposable element TCp3.2 was previously found to be horizontally transferred from the genome of Cydia
pomonella to the C÷ pomonella granulovirus (CpGV). In this study, the transcription of transposase genes of endogenous
TCp3.2 copies in the insect host genome was investigated. Cloning and sequencing of cDNAs prepared from TCp3.2 transposase
transcripts resulted in the identification of a 199-bp-long intron. Sequence heterogeneities among different cDNA clones
suggested that multiple copies of the transposase are transcribed, but that a part of these copies encode a defective transposase.
The actin gene of C÷ pomonella was cloned and sequenced, and used to standardise quantitative real time PCR on prepared
cDNA of the TCp3.2 transposase. Comparison of cDNA levels of TCp3.2 transposase prepared from mock and CpGV-infected C÷
pomonella larvae did not provide evidence that CpGV infection influenced the transcription level of TCp3.2 transposase. Arch.
Insect Biochem. Physiol. 63:135–145, 2006.
© 2006 Wiley-Liss, Inc.
KEYWORDS: Cydia pomonella; Cydia pomonella granulovirus; Tc1-mariner; TCp3.2; transposase; actin;
transcription
INTRODUCTION
(Jehle et al., 1998). Phylogenetic analysis of the
deduced amino acid sequence of the putative
In previous studies, a mutant of the Cydia
transposase gene showed that TCp3.2 belongs to
pomonella granulovirus (CpGV), designated CpGV-
the superfamily of Tc1/mariner-like transposons
MCp4, harbouring the insect transposon TCp3.2,
(Jehle et al., 1998). As is typical for these elements,
was isolated (Jehle et al., 1995, 1998). Molecular
the
characterisation of TCp3.2 demonstrated that it
TCp3.2 transposase contains a so-called D,D35E
predicted
C-terminal
part
of
the
putative
originated from the C. pomonella genome and hori-
motif and TCp3.2 inserted at a TA dinucleotide
zontally escaped into the virus genome. TCp3.2 is
(Doak et al., 1994; Radice et al., 1994; Robertson,
3,239 bp long, has 756-bp-long inverted terminal
1995). The insertion site of TCp3.2 in CpGV-MCp4
repeats (ITRs), and encodes an intron containing
is located between ORF41 (lef2) and ORF42 and
transposase gene, which is defective due to a frame
corresponds to the map position (34875–34876)
shift mutation within its open reading frame (ORF)
of the sequenced CpGV-M1 (cloned Mexican iso-
Department of Phytopathology, Laboratory for Biotechnological Crop Protection, Agricultural Service Center Palatinate (DLR Rheinpfalz), Neustadt an der
Weinstrasse, Germany
Contract grant sponsor: Deutsche Forschungsgemeinschaft; Contract grant number: JE 245/2-2.
*Correspondence to: Dr. Johannes A . Jehle, Labor für Biotechnologischen Pflanzenschutz, Abt. Phytomedizin, Dienstleistungszentrum Ländlicher Raum,
Breitenweg 71, 67435 Neustadt an der Weinstrasse, Germany. E-mail: johannes.jehle@dlr.rlp.de
Received 10 November 2005; Accepted 29 June 2006
© 2006 Wiley-Liss, Inc.
DOI: 10.1002/arch.20149
Published online in Wiley InterScience (www.interscience.wiley.com)
136
Arends and Jehle
late) genome (Jehle et al., 1997; Luque et al.,
in a cell-free system in the presence of only the
2001). Southern blot hybridisation studies sug-
transposase (Vos et al., 1996). For the transpos-
gested that the genome of C. pomonella contains
able elements Himar1 of the horn fly Haematobia
about 10 copies of TCp3.2 (Jehle et al., 1998).
irritans and Mos1 of Drosphila mauritiana, it was
The finding of TCp3.2 and the related trans-
demonstrated that purified transposase is sufficient
poson TCl4.7 in CpGV mutants was the first in
for an inter-plasmid transposition reaction (Lampe
vivo demonstration of horizontal escape of insect
et al., 1996; Tosi and Beverley, 2000). Some of
transposons into baculovirus genomes. This phe-
these Tc1/mariner-like elements showed transposi-
nomenon, however, was already previously observed
tion in heterologous hosts, thus providing some
when Autographa californica nucleopolyhedrovirus
in vivo evidence that they may not require specific
(AcMNPV) and Galleria
mellonella MNPV (Gm-
host cell–encoded factors for transposition. The Tc1
MNPV) were passaged in cultured insect cells (in
element, for example, is able to transpose in hu-
vitro). These passage experiments resulted in the
man cells (Schouten et al., 1998), the Drosophila
isolation of several AcMNPV and GmMNPV mu-
element mariner transposes in the protozoan Leish-
tants that contained insertions of different trans-
mania (Gueiros-Filho and Beverley, 1997), Tc3 is
posons (for reviews, see Fraser, 1986; Friesen, 1993;
active in the Zebrafish Danio rerio (Raz et al., 1998),
Jehle, 1996). Most of these transposons were iden-
and Himar1 efficiently transpose in bacteria (Rubin
tified when FP (few polyhedra) plaque morphol-
et al., 1999). For most of the Tc1/mariner-like ele-
ogy mutants of the virus were analysed. Due to a
ments, it is not completely clear how their activity
replication advantage under the specific conditions
is regulated. Generally, it is believed that the trans-
of in vitro replication in cell culture, the FP mu-
position activity of these elements is enhanced by
tants most probably became selected in the virus
increasing transposase concentrations in the cell
population (Volkman and Keddie, 1990; Harrison
(Tosi and Beverley, 2000; Kapetanaki et al., 2002).
and Summers, 1995).
Since it is expected that the activity of Tc1/mari-
Normally, the activity of endogenous trans-
ner-like transposons depends on the expression level
posons is strictly regulated in order to prevent ge-
of an active transposase, the levels of TCp3.2
netic disorder. Different studies, however, showed
transposase mRNA within the C. pomonella larvae
that transposon activity can be stimulated by ge-
was considered to be a good measure for TCp3.2
nomic stress factors including heat shock, expo-
activity. In this study, experiments were performed
sure to magnetic fields, or UV radiation and also
in order to analyze the possible influence of virus
virus infection (Arnault and Dufournel, 1994; Capy
infection on the transposase transcription of insect
et al., 2000; Wessler, 1996; Ratner et al., 1992;
endogenous transposons during natural infections.
Strand and McDonald, 1985; Chow and Tung,
2000; Walbot, 1992; Eichenbaum and Livneh,
MATERIALS AND METHODS
1998; Peterson, 1985). A direct determination of
the influence of CpGV infection on the transposi-
Virus Stocks and Insect Larvae
tion frequency of TCp3.2 is currently not possible
because a suitable transposition assay for this
The viruses used in this study derived from an
transposon is not available. From other well-stud-
in vivo cloned genotype of Cydia pomonella granulo-
ied Tc1/mariner-like transposons, it is known that
virus (CpGV-M) (Tanada, 1964; Luque et al., 2001).
this type of element can transpose with a minimal
The Cydia pomonella larvae derived from the insect
requirement of host functions. Experiments using
rearing at the Agricultural Service Center Palatinate,
cell-free systems have confirmed that a functional
Neustadt/Weinstr.,
transposase is the only protein required for the
reared at 26°C on a semi-synthetic diet (Ivaldi-
transposition reaction. For example, the Tc1 ele-
Sender, 1974). Larvae were inoculated by feeding
ment of Caenorhabditis elegans is able to transpose
them a small piece of medium containing a LD95
Germany.
Archives of Insect Biochemistry and Physiology
The
insects
November 2006
were
doi: 10.1002/arch.
TCp3.2 Transposase Transcription
(= 1,000 occlusion bodies) of CpGV. Only larvae
137
Bombyx mori (GenBank X04507), Helicoverpa armi-
that had completely ingested the virus dose within
gera (GenBank X97614), Heliothis viresens (Gen-
24 h were placed on fresh virus-free medium. Only
Bank
larvae that showed the first symptoms of infection
AF182715), Manduca sexta (GenBank L13764), and
4 days post-inoculation were included in the ex-
Mayetiola destructor (GenBank AF017427). Using C.
AF368030),
Lymantria
dispar
(GenBank
pomonella DNA or synthesised cDNA as a template,
periments.
the primers were used to amplify the actin se-
°
quence by PCR (amplification program: 94 C for
RNA Isolation and First Strand cDNA Preparation
°
°
2’00, 35 cycles: 1’00 at 94 C, 1’00 at 54 C, 1’00 at
°
°
In order to perform RT-PCR, total RNA was iso-
72 C; 7’00 at 72 C). The obtained PCR products
lated from C. pomonella larvae using TRIzol reagent.
were cloned in the pDrive cloning vector (Qiagen,
The RNA preparations were incubated with DNase
Chatsworth, CA) and sequenced (MWG Biotech
(0.1 U/
AG, Germany). The sequences were analysed us-
ml)
for 15 min at room temperature. The
first-strand cDNA was synthesised using SuperScript
ml reactions were permg
of DNase-treated total RNA and 1 ml Oligo (dT)12–
18 primer (500 mg/ml). To prevent RNA degradation, 1.0 ml RNaseOUT (40 U/ml) was added to
ing the DNASTAR (Lasergene) software.
Reverse Transcriptase. The 20-
formed as described by the supplier using 2.0
the reaction. All reagents and enzymes were purchased from Invitrogen.
Quantitative Real Time RT-PCR
Actin and transposase t32A transcripts in C.
pomonella were quantified by quantitative real time
RT-PCR. The reaction and fluorescence detection
was performed using a DNA Engine Opticon™ System (MJ Research). Transcript quantification was
done in reactions using QuantiTect SYBR green
Cloning and Sequence Analysis of
(Qiagen). In each reaction, the amount of actin
TCp3.2 Transposase cDNA
cDNA and t32a cDNA each prepared from 200 ng
total RNA (DNase treated) were quantified. For the
In order to investigate the intron region of the
TCp3.2 transposase, first-strand cDNA fragments
including the intron borders were amplified in a
¢
PCR using the primers t32a-for1 (5 -GTGCAG
¢
¢
CAATAAACGACAAAACC-3 ) and t32a-rev1 (5 -
¢
amplification of the transposase t32a transcripts,
¢
the primers t32a-for2 (5 -CGCAAAGAATCTACC
¢
¢
AGGAAC-3 ) and t32a-rev2 (5 -AGTCGTTATCTT
¢
CAAGACCTTGG-3 ) and the amplification and de-
°
tection program (94 C for 15’00, 45 cycles: 0’30
°
°
°
°
AGACCCGAATAAGAGCATCAGAGA-3 ) and the
at 94 C, 0’30 at 62 C, 0’30 at 72 C; 7’00 at 72 C)
amplification program (94 C for 2’00, 30 cycles:
were used. Melting curve analyses were carried out
0’30 at 94 C, 0’30 at 62 C, 0’30 at 72 C; 7’00 at
at 50 –95 C. For the amplification of actin tran-
72 C). The amplification products were cloned in
scripts, the primer combination actin-for2 (5 -
°
°
°
°
°
°
°
¢
¢
E. coli using the pGEM-T vector (Promega, Madi-
TGGGACAGAAGGACTCGTAC-3 ) and actin-rev2
son,
(5 -TGGGTCATCTTTTCTCTGTTG-3 ) and the am-
WI)
and
sequenced
(MWG
Biotech
AG,
¢
¢
°
plification and detection program (94 C for 15’00,
Germany).
°
°
°
45 cycles: (0’30 at 94 C, 0’30 at 54 C, 0’30 at 72 C;
°
Cloning and Sequence Analysis of
7’00 at 72 C) were used. Melting curve analyses
C. pomonella Actin
were performed at 50 –95 C.
¢
Degenerate primers actin-for1 (5 -GACAATGGM
¢
¢
TCCGGYATGTG-3 ) and actin-rev1 (5 -TYAGAA
¢
GCACTTSCKGTGDAC-3 ) were designed according
°
°
RESULTS
Characterisation of the Transposase Transcripts of
Transposon TCp3.2
to homologous DNA sequences of eight insect actin gene sequences: Spodoptera littoralis (GenBank
Z46873), Bactrocera
dorsalis (GenBank L12254),
Archives of Insect Biochemistry and Physiology
November 2006
doi: 10.1002/arch.
Based on the analysis of the DNA sequence of
TCp3.2, by Jehle et al. (1998), it was suggested that
138
Arends and Jehle
the TCp3.2 transposase gene (t32a) putatively con-
or reverse transcriptase in the reactions (Fig. 1B,
sists of two exons separated by an intron of 178
lanes 3–5), no PCR products were obtained, which
bp. In order to prove this prediction, the intron
demonstrated that the amplified fragments in lane
region and splicing borders of t32a transcripts were
1 and 2 originated from pre-mRNA and mRNA re-
identified on an RNA level. Therefore, total RNA
versely transcribed to cDNA.
was isolated from mock- and CpGV-infected lar-
The amplified fragments of t32a cDNA were
vae and first-strand cDNA was prepared by reverse
separated on an agarose gel and cloned into E. coli
transcription. A PCR using primer t32a-for1 and
using the pGEM-T vector. The inserts of twelve ran-
t32a-rev1, which are located within exon 1 and
domly picked clones were sequenced and aligned
exon 2, was applied to the prepared cDNA (Fig.
with the sequence of TCp3.2 found in CpGV-MCp4
1A). Since the PCR primers were located within
(Jehle et al., 1998). As shown in Figure 2, 10 out
the exons, fragments of spliced and unspliced (pre-
of 12 randomly picked clones represented cDNAs
mRNA) transcripts could be amplified in this RT-
(t32a-A/E) of unspliced mRNAs; two of them
PCR. As shown in Figure 1B, fragments of the
(t32a-F and -G) were RT-PCR products of spliced
expected size (371 bp) for unspliced t32a tran-
transcripts. The two sequences t32a-A were identi-
scripts were found in CpGV- and mock-infected lar-
cal to the t32a DNA sequence present in CpGV-
vae. Additionally, a 172-bp fragment that possibly
MCp4 (Jehle et al., 1998). Sequence comparison
originated from spliced transcripts was detected in
of spliced and unspliced cDNAs indicated that the
CpGV-infected larvae. In the controls without RNA
intron was 199 bp long and had typical eucaryotic
splicing borders (GT....AG). The borders corresponded to nucleotides 1,347 and 1,545 of the
TCp3.2 sequence published by Jehle et al. (1998),
suggesting an intron that is 21 bp longer than previously predicted. As shown in Figure 2, some heterogeneity among the transposase cDNAs caused
by nucleotide substitutions or deletions was observed. The sequence heterogeneity included a nonsynonymous substitution (A86G) for sequence
t32a-F resulting in a valine (GTG) codon instead
of methionine (ATG). The A114T transversion was
observed at nearly the same frequency among the
clones resulting in a codon change from CAA
(glutamic acid) to CTA (leucine). Three sequences
(t32a-E) had a deletion of an AG dinucleotide at
position 67/68 and sequence t32a-D showed a de-
Fig. 1.
RT-PCR of t32a transposase transcripts. A: Bind-
ing sites of the PCR primers t32a-for1 and t32a-rev1 are
marked with arrows. The expected sizes of amplified
transposase fragments originating from pre-mRNA and
(spliced) mRNA as template, are indicated. B: RT-PCR
on RNA prepared from C. pomonella (Cp) larvae (third
instar) using primers t32a-for1 and t32a-rev1. M, size
standard; 1, mock infected Cp-larvae; 2, CpGV infected
Cp-larvae; 3, negative control, no RNA in cDNA synthe-
letion at position 64. These deletions cause frame
shift mutations, which lead to a translation stop
in exon 2. Since the other identified mutations are
located within the intron, they do not affect the
t32a coding region.
With the identification of the correct splicing
borders, it was possible to design a reverse primer
(t32a-rev2) that annealed to the 5¢-end of exon 2
sis reaction; 4 and 5, negative controls, RNA (without
and the 3¢-end of exon 1, thus amplifying only
reverse transcriptase) of mock and CpGV infected Cp-
cDNAs of spliced t32a mRNA but not those of pre-
larvae, respectively.
mRNA or background transposon DNA (Fig. 3).
Archives of Insect Biochemistry and Physiology
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doi: 10.1002/arch.
TCp3.2 Transposase Transcription
Fig. 2.
139
Sequence alignment of RT-PCR products of the
otide heterogeneities are indicated by A, T, G, or C; nucle-
intron and bordering regions of the transposase t32a ob-
otide identities are marked as dots; gaps in the alignment
tained from CpGV-infected larvae (compare Fig. 1, lane
are indicated by dashes. Note the putative intron from
2). t32a-A to -F represent different identified sequences
nucleotide position 117–315, which is not present in the
of twelve randomly picked clones. Numbers in brackets
sequences t32a-F and -G. The annealing site of the intron
indicate the frequency of a respective sequence. The se-
spanning primer t32a-rev2 (112–116, 316–333), which was
quence t32a-A is identical to the t32a sequence identified
used for quantitative RT-PCR, is underlined.
in CpGV mutant CpGV-MCp4 (Jehle et al., 1998). Nucle-
In control PCRs on RNase-treated genomic DNA
of C. pomonella larvae, no fragments of 313 or 172
bp were detected (results not shown), which indicated that no TCp3.2 copies without intron are
present within the genome of C. pomonella and that
the products in the RT-PCRs originate from spliced
transcripts. These results showed that the primer
pair (t32a-for2/t32a-rev2) was suitable for the
quantification of t32a transcription.
Characterisation of the Actin Transcripts in
C. pomonella
For the quantification of the t32a transcription
in virus- and mock-infected larvae, the transcripFig. 3.
A: RT-PCR on t32a transposase transcripts. The
binding sites of the PCR primers t32a-for2 and t32a-rev2
are marked with arrows. The expected size of the amplified transposase fragments originating from pre-mRNA
and (spliced) mRNA as template are indicated. B: RT-PCR
on RNA prepared from CpGV-infected C. pomonella (Cp)
tion of the actin gene as a housekeeping gene was
chosen as an internal standard. In order to obtain
sequence information on the unknown DNA sequence of the C. pomonella actin gene, degenerate
primers based on actin sequences of other insects
larvae (third instar) using primer combination t32a-for2/
were designed in such a way that nearly the com-
t32a-rev2. M, size standard; 1, complete reaction; 2, nega-
plete coding region of the gene could be ampli-
tive control (no reverse transcriptase).
fied (Fig. 4). Application of these primers on C.
Archives of Insect Biochemistry and Physiology
November 2006
doi: 10.1002/arch.
140
Fig. 4.
Arends and Jehle
Alignment of different insect actin sequences. The
otide identities are marked by dots; dashes indicate se-
alignment was used for the design of degenerate primers
quence gaps. The sequences of the degenerate primers ac-
for the amplification of the C. pomonella actin gene. Nucle-
tin-for1 and actin-rev1 are indicated at the bottom of the
otide heterogeneities are indicated by A, T, G, or C. Nucle-
alignment.
pomonella DNA and cDNA resulted in the amplifi-
ably represents an intron within the actin gene
cation of fragments of 1.2 and 1.1 kb, respectively
since this sequence is flanked by the typical splic-
(data not shown). Sequence analyses of the cloned
ing borders (GT....AG) and conceptual deletion of
PCR fragments revealed that the fragment that
it results in a continuous open reading frame (Fig.
originated from the DNA template was 1,205 bp
5). A Blast search revealed that the obtained nucle-
long and thus 107 bp longer than the smaller frag-
otide sequence of the partial C. pomonella actin
ment (1,098 bp) that was obtained from the cDNA
gene was most similar to the sequence of the actin
template. The difference of the 107 bp most prob-
A3b gene of H. zea (data not shown). In order to
Fig. 5.
Partial nucleotide sequence and deduced amino
tin-for2 (position 110–129) and the intron spanning
acid sequence of the actin gene of C. pomonella. The bind-
primer actin-rev2 (position 311–317, 425–439) that were
ing sites of the actin-for1 and actin-rev1 primers that were
used for the quantitative RT-PCR are underlined. The
used for PCR amplification are in bold. The splicing bor-
GenBank accession number for this sequence is AY524976.
ders (gt...ag) are highlighted. The sequence of primer ac-
Archives of Insect Biochemistry and Physiology
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doi: 10.1002/arch.
TCp3.2 Transposase Transcription
141
develop a PCR that could specifically amplify
(data not shown). For the quantification of actin
spliced actin cDNA, the identified intron border-
and t32a, standard curves were generated by apply-
ing sequence was used to design the intron span-
ing six different concentrations of target DNA to the
ning primer actin-rev2 (Fig. 5). PCR using primer
PCR reaction. The obtained linear standard curves
pair (actin-for2/actin-rev2) on C. pomonella DNA
for actin and t32a had correlation coefficients of
and cDNA as template confirmed its specificity for
0.985 and 0.998, respectively. The results of the
spliced transcripts (Fig. 6). Fragments of the ex-
quantification of the experimental actin and t32a
pected size for spliced transcripts were amplified
cDNA samples are depicted in Table 1. For actin, it
from cDNAs prepared from mock- and CpGV-in-
was found that the amount of cDNAs in the mock-
fected larvae. PCR on DNase-treated RNA did not
and CpGV-infected larvae was 0.23 and 0.16 pg, re-
result in an amplification product. This demon-
spectively. On the other hand, the amounts of t32a
strated that the DNase treatment of the isolated
cDNAs were very similar in mock (2.46E-4 pg) and
RNA was very efficient and that the amplification
CpGV infected (2.53E-4 pg) larvae (Table 1). In or-
products in the RT-PCR originated from first-strand
der to avoid that the variability of transcription be-
cDNA and not from DNA. These results showed that
tween the larvae would superpose the variation
the primer pair (actin-for2/actin-rev2) was suitable
within the larvae, the individual t32a:actin ratio for
for the quantification of actin transcription.
each larvae was quantified (Fig. 7). The t32a:actin
ratio in CpGV-infected larvae was 2.20E-03 com-
Quantification of Actin and Transposase Transcription
pared to 1.25E-03 in mock infected larvae, suggesting a 1.8-fold increase of the t32a transcription rate.
The amounts of actin and t32a transcripts were
This difference in actin and t32a transcription in
quantified in single larvae, which had been mock-
mock- and CpGV-infected larvae, however, was sta-
or CpGV-infected 4 days prior to RNA isolation.
tistically not significant (t-test,
a = 0.05).
Quantitative real time PCR was applied on the prepared cDNA fractions using primer pair (actin-for2/
DISCUSSION
actin-rev2) for actin and (t32a-for2/t32a-rev2) for
t32a. Melting curve analyses of the actin and t32a
The transcription of the endogenous transposon
PCR products showed single peaks at melting tem-
TCp3.2 transposase (t32a) in larvae of C. pomonella
peratures of 84° and 79°C, respectively, indicating
was investigated by sequencing and quantification
that no unspecific PCR products were amplified
of cDNAs prepared from total RNA of larvae. Sequence analyses of 12 randomly isolated cDNA
clones demonstrated the presence of an intron in
t32a. The nucleotide positions of the splicing borders differed from the borders predicted earlier on
the basis of DNA sequence (Jehle et al., 1998). The
intron in t32a was 199 bp in length and is considerably longer than those found in other Tc1/mari-
Fig. 6.
RT-PCR of the actin gene of C. pomonella (Cp)
Table 1. The Actin and Transposase t32a Transcription Level in Mock and
CpGV-Infected C. pomonella Larvae*
larvae (third instar) using the primers actin-for2 and acActin
tin-rev2 on RNA samples isolated from mock- and CpGVinfected Cp-larvae. M: size standard; 1, RNA from mockinfected Cp-larvae; 2, negative control, RNA from mock-
Treatment
Transposase t32a
n
C(T)
pgram
Mock
25
14.58 (0.20)
0.23 (0.027)
35.13 (0.46) 2.46E–04 (5.56E–05)
C(T)
pgram
CpGV
20
15.24 (0.25)
0.16 (0.023)
34.39 (0.29) 2.53E–04 (4.30E–05)
infected Cp-larvae (no reverse transcriptase); 3, RNA from
CpGV-infected Cp-larvae; 4, negative control, RNA from
CpGV-infected Cp-larvae (no reverse transcriptase).
Archives of Insect Biochemistry and Physiology
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doi: 10.1002/arch.
*In each reaction, the amount of actin or t32a cDNA prepared from 200 ng DNasetreated total RNA was determined using quantitative RT-PCR. Numbers in parentheses indicate the standard error.
142
Arends and Jehle
due to a suggested frame shift mutation in the
transposase gene (Jehle et al., 1998). Since TCp3.2
horizontally escaped into the genome of CpGV, at
least one of the copies in the host genome must
have enough coding capacity to trans-activate transposition of defective copies.
In order to quantify the transcription level of
t32a in C. pomonella larvae, the transcription of the
Fig. 7.
The t32a:actin transcription ratio in 25 mock- and
20 CpGV-infected C. pomonella larvae. The t32a:actin ratio was calculated from the independent measurements
in single larvae: 1/n
S(pgram t32a:pgram actin). The stan-
dard error is indicated.
actin gene was intended to be used as an internal
reference. Actin is generally considered as a housekeeping gene, which is assumed to be expressed
on a similar level under different cellular conditions (Thellin et al., 1999). Degenerate PCR primers for the actin gene were designed from eight
ner-like transposons that are generally between 40–
actin sequences that originated from species be-
60 bp. Similar to other Tc1/ mariner -like trans-
longing to different insect families. The partial C.
posases, the intron of t32a is located near the
pomonella actin gene was amplified by PCR and se-
middle of the open reading frame and upstream
quenced. The identified nucleotide sequence of the
of the coding region of the D,D(35)E catalytic do-
C. pomonella actin gene was most similar to the ac-
main (Rosenzweig, 1983; van Luenen et al., 1993;
tin A3b gene of H. zea and also showed a very high
Franz et al., 1994). Our analyses also revealed some
sequence similarity to cytoplasmic actins of H.
sequence heterogeneity among the cDNA clones.
amigera and B. mori. Cytoplasmic actin genes in
Though the observed differences were only small
these lepidopteran species comprise a pair of
and no major deletions were identified, the detec-
tandemly duplicated paralogous genes, which both
tion of putatively non-functional transposase tran-
contain a highly conserved intron interrupting the
scripts indicated that a substantial amount of the
actin coding sequence at codon 117 (Mangé and
TCp3.2 transposons within the C. pomonella ge-
Prudhomme, 1999; Li et al., 2002). This intron po-
nome encode a defective transposase gene. This is
sition appears to be lepidopteran specific and was
typical for many transposons found in Eucaryotes,
also observed for the actin gene sequence of C.
where most of the elements are not intact due to
pomonella.
small deletions, nonsense, and frame shift muta-
The determined cDNA amounts of the actin
tions (Robertson, 1995). Beside Tc1, Tc3, Mos1, and
transcripts were used as an internal standard to
Minos, only a very limited amount of active trans-
quantify t32a transposase transcription. The results
posons have been identified in genomes (Emmons
showed a lower concentration of actin transcripts
et al., 1983; Collins et al., 1989; Medhora et al.,
in virus-infected than in mock-infected larvae. This
1988; Franz et al., 1994). The Tc1/ mariner-like
was not unexpected since the RNA preparations
Sleeping Beauty, Himar1 , and Frog
from CpGV-infected larvae contained not only in-
Prince showed a high activity but they were recon-
sect RNA, but also viral RNA. Synthesis of virus
structed from bits of consensus sequences of inac-
RNA is initiated in midgut cells within the first
tive copies in their salmon, horn fly, and frog host
few hours of infection. Virus infection spreads to
genomes (Ivics et al., 1997; Robertson and Lampe,
fat body, tracheal matrix, epidermis, and Mal-
1995; Lampe et al., 1996; Miskey et al., 2003). An
pighian tubules within 3–5 days, resulting in a con-
intact copy of these transposons in their host ge-
siderable amount of viral transcripts (Hess and
nomes has not been identified. The completely se-
Falcon, 1987; reviewed by Crook, 1991). Therefore,
quenced
CpGV
the relative amount of actin mRNA used in the
mutant CpGV-MCp4 is most probably defective
cDNA synthesis reaction could have been less than
transposons
copy
of
TCp3.2
found
in
the
Archives of Insect Biochemistry and Physiology
November 2006
doi: 10.1002/arch.
TCp3.2 Transposase Transcription
143
in the samples of mock-infected larvae. The cDNA
tion of the four nucleotides causing this frame shift
concentrations in mock- and CpGV virus–infected
and a correct splicing of the intron identified in
larvae was nearly the same. In order to ensure that
this work, a putative 358 amino acid (aa) long
the variability of transcription between the larvae
transposase could be encoded by this gene. This
would not superpose the change within the lar-
size is similar to other identified active trans-
vae, the individual t32a:actin ratio for each larvae
posases. Tc1, Tc3, and mariner encode transposases
was determined and the average ratio per experi-
of 343, 329, and 345 aa (Rosenzweig et al., 1983,
mental group was calculated. The t32a:actin ratio
Collins et al., 1989; Jacobson et al., 1986). With
was nearly 1.8-times higher in CpGV-infected lar-
the sequence information presented in this report,
vae than in mock-infected larvae, but this differ-
complemented with sequence information of other
ence was statistically not significant. The induction
regions of TCp3.2 copies in the genome of C.
of transposon activity by stress factors including
pomonella, it might be possible to reconstruct an
viral infection has been studied in several organ-
active TCp3.2 element in a similar way as was suc-
isms. However, it has been demonstrated for only
cessfully achieved for Sleeping beauty, Himar1, and
a few elements (Arnault and Dufournel, 1994;
Frog Prince.
Capy et al., 2000). For example, the mobilization
and insertion of transposable Element Uq have
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