Sequence analysis and quantification of transposase cDNAs of transposon TCp3.2 in Cydia pomonella larvaeкод для вставкиСкачать
Archives of Insect Biochemistry and Physiology 63:135145 (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. 63:135145, 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 (3487534876) 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: firstname.lastname@example.org 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 cellencoded 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 ° ° 200, 35 cycles: 100 at 94 C, 100 at 54 C, 100 at ° ° In order to perform RT-PCR, total RNA was iso- 72 C; 700 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 1500, 45 cycles: 030 ° ° ° ° AGACCCGAATAAGAGCATCAGAGA-3 ) and the at 94 C, 030 at 62 C, 030 at 72 C; 700 at 72 C) amplification program (94 C for 200, 30 cycles: were used. Melting curve analyses were carried out 030 at 94 C, 030 at 62 C, 030 at 72 C; 700 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 1500, Germany). ° ° ° 45 cycles: (030 at 94 C, 030 at 54 C, 030 at 72 C; ° Cloning and Sequence Analysis of 700 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 35), 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 November 2006 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 117315, 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 (112116, 316333), 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 110129) and the intron spanning acid sequence of the actin gene of C. pomonella. The bind- primer actin-rev2 (position 311317, 425439) 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 November 2006 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) 2.46E04 (5.56E05) C(T) pgram CpGV 20 15.24 (0.25) 0.16 (0.023) 34.39 (0.29) 2.53E04 (4.30E05) 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 November 2006 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 35 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 virusinfected 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 REFERENCES been reported in Barley Stripe Mosaic Virusinfected maize plants (Peterson, 1985). In Drosophila, it was hypothesised, but never substantiated, that Arnault C, Dufournel I. 1994. Genome and stresses: reactions against aggressions, behaviour of transposable elements. Genetica 93:149160. endogenous transposable elements were involved in mutations observed after injection of the Avian Rous Sarcoma Virus (Gazaryan et al., 1984). 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