Satellite DNA transcription in Diadromus pulchellus (Hymenoptera)

Insect Biochemistry and Molecular Biology 29 (1999) 103–111

Satellite DNA transcription in Diadromus pulchellus (Hymenoptera) Sylvaine Renault *, Florence Rouleux-Bonnin, Georges Periquet, Yves Bigot Institut de Recherche sur la Biologie de l’Insecte, UPRESA CNRS 6035, Faculte´ des Sciences, Universite´ F. Rabelais, Avenue Monge, 37200 Tours, France Received 17 April 1998; received in revised form 23 September 1998; accepted 19 October 1998

Abstract Previous studies have shown that the satellite DNAs in Hymenoptera account for 1–25% of the genome. They mainly correspond to a single family, or to several subfamilies having the same evolutionary origin. We have now showed that the satellite DNAs in the genomes of the hymenopterans Diadromus pulchellus, Diadromus collaris, Eupelmus vuilletti and Eupelmus orientalis are transcribed in both males and females. Satellite DNA transcripts could only be extracted with NP40/Urea, indicating that they are strongly associated with proteins. The satellite DNA in D. pulchellus was transcribed on the two DNA strands. The satellite DNA transcripts were single-stranded and not polyadenylated in vivo. The transcripts were found in embryos, larvae and imagos stages. The transcripts detected included one major transcript (1.9 kb) and several discrete smaller transcripts. The in vivo synthesis of these satellite DNA transcripts was explored by identifying their putative initiation sites.  1999 Elsevier Science Ltd. All rights reserved. Keywords: Insect; Satellite DNA; Transcription

1. Introduction Most eukaryotic genomes contain highly repeated tandem sequences called “satellite DNA”. These sequences are organized as large clusters in the centromeric and telomeric regions of chromosomes, and are the principal components of heterochromatin. The fraction of the genome that is satellite DNA varies widely (0–66%) between species, as does the composition of the satellite DNA and the number of evolutionarily unrelated families (for review, Beridze, 1986). The satellite DNAs in all the hymenopteran parasitoid wasps of the genus Diadromus (Ichneumonidae) belong to a single family, and satellite DNA accounts for 5– 25% of the genome. The sequences of the repeated units are all very similar, suggesting that there are no subfamilies in any species. The satellite DNAs in other parasitoids wasps (Pimpla linea, Ichneumonidae, Dinarmus basalis and Trichogramma caceociae, Chalcidoidae),

* Corresponding author. Tel.: +33-02-47-36-69-76; fax: +33-02-4736-69-66; e-mail: [email protected]

bees (Apis mellifera, Bombus terrestris and Xylocopa violacea, Apoidae) have similar features (Bigot et al., 1990; Rojas-Rousse et al., 1993; Tares et al., 1993 and unpublished results). However, the Nasonia genera (Chalcidoidae, Pteromalidae; Eickbush et al., 1992) and Diprion pini (Symphyta, Diprionidae; Rouleux-Bonnin et al., 1996) have several satellite DNA subfamilies with a common origin. Several functions have been suggested for satellite DNAs, but none has yet been demonstrated. Their possible functions have been tested by comparing strains of Drosophila melanogaster with and without satellite DNA. Deletions in the Responder locus (composed of tandemly repeated sequences) reduced the fitness of Drosophila (Wu et al., 1989). But the most significant studies to date seem to indicate that satellite DNA is involved in centromere function by association with specific such as CENP-A and CENP-B in Saccharomyces cerevisiae, the mouse and man during mitosis and meiosis (Kitagawa et al., 1995; Marshall and Clarke, 1995; Sugimoto et al., 1994). Satellites are also recovered in the scaffold attachment regions (SAR), which are implicated in a wide variety of function, including recombination and replication (Strissel et al., 1996).

0965-1748/99/$ - see front matter  1999 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 ( 9 8 ) 0 0 1 1 3 - 1

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Only the structural function has been thoroughly investigated so far. However, satellite transcription has been described in vertebrates and invertebrates. It has been detected in newts, frogs, mice and arthropods. The transcripts of satellite DNA detected in lampbrush chromosomes of pigeon and chicken result from the transcription of adjacent sequences (Solovei et al., 1996; Hori et al., 1996). Some of these transcripts are produced specifically. For example, Diprion pini (Hymenoptera, Symphitae) satellite DNA is transcribed more in females than in males (Rouleux-Bonnin et al., 1996). In the crab, Gecarnicus lateralis, a G+C rich satellite DNA is transcribed in a tissue-specific and stage-specific manner (Varadaraj and Skinner, 1994). The transcription of mouse γ satellite is developmentally regulated in a cellspecific pattern and is down regulated by retinoids (Rudert et al., 1995; Sam et al., 1996). The function of these transcripts has not been determined. Satellite DNA transcripts are also tissue-specifically produced in newts. These transcripts have ribozymic activity, but no cellular function has yet been described for this ribozyme. Ribozymes can cleave themselves in some tissues (Epstein and Coats, 1991; Cremisi et al., 1992; Green et al., 1993), and the ribozyme has a hammerhead folded structure. This paper describes the transcription of the satellite DNA of the hymenopteran, Diadromus pulchellus. Discrete transcripts were detected in males and females and they resulted from the transcription of the two DNA strands. The transcription initiation site was checked by primer extension. The results obtained can explain the transcription pattern observed on Northern blots.

10 mM Tris–HCl, pH 9. The nuclei and membranes were removed by centrifugation at 15,000 g for 5 min at 4°C. The supernatant was mixed with an equal volume of 7 M urea, 350 mM NaCl, 10 mM EDTA (ethylene diamine tetraacetic acid), 1% SDS (sodium dodecyl sulfate), 10 mM Tris–HCl pH 7.2, then centrifuged for 5 min at 15,000 g, at 4°C. The supernatant was extracted with equal volumes of (i) saturated phenol, (ii) phenol/chloroform/isoamyl alcohol (12/12/0.5) and (iii) chloroform. RNAs were precipitated with absolute ethanol, washed with 70% ethanol and suspended in sterile water. Traces of DNA were eliminated by treatment with pancreatic RNAse-free deoxyribonuclease I (0.01 U/µl) in 10 mM MgCl2, 0.1 mM dithiothreitrol, 10 mM Tris– HCl pH 7.5 for 1 h at 37°C. RNAs were extracted once more with equal volumes of saturated phenol/isoamyl alcohol, phenol/isoamyl alcohol/chloroform and chloroform, precipitated with absolute ethanol, washed with 70% ethanol and suspended in sterile water. Single strand RNAs were removed from the total RNA extract by precipitation overnight with 2 M LiCl (final concentration) at 4°C and collected by centrifugation at 15,000 g for 15 min. The double strand RNAs remaining in the supernatant were precipitated with 4 M LiCl (final concentration)(Davis and Boyle, 1990). Traces of salt were removed by alcohol precipitation. The polyadenylated RNAs were purified from total RNA using the PolyATract mRNA isolation kit (Promega). RNAs were also purified by the guanidium thiocyanate and LiCl methods (Chirgwin et al., 1979; Maniatis et al., 1982), followed by treatment with RNAse-free DNAse. 2.3. Detection of satellite transcripts from D. pulchellus

2. Materials and methods 2.1. Material Diadromus pulchellus and D. collaris are endoparasitoids of the leek moth, Acrolepiopsis assectella (Lepidoptera) which infests Allium species. The parasitoids were bred in mass under standard conditions (Arnault, 1979, 1982). The ectoparasitoids E. vuilletti and E. orientalis are members of Eupelmidae and parasitizes various Bruchidae species (Coleoptera) which live on several Phasolinae species. The ectoparasitoids were bred on their host Callosobruchus maculatus (Terrasse and Rojas-Rousse, 1986). 2.2. RNA purification Total RNA was isolated from 1–5 day old imagos (about 5000–10,000 individuals=2 g). The frozen insects were crushed at ⫺80°C and the resulting powder was suspended in 6 ml 150 mM NaCl, 0.65% Nonidet P40,

Total RNAs (15 µg) were linearized by the dimethylsulfoxide/glyoxal method and separated through denaturing gels containing formaldehyde (Maniatis et al., 1982). Northern blots were performed on nitrocellulose membranes (C+ membrane) as described by the manufacturer (Amersham). The fragments used as probes were purified from the satellite monomer 2 clone for D. pulchellus (length: 320 bp; Bigot et al., 1990). Monomers of satellite DNA from D. collaris, E. vuiletti and E. orientalis were purified as described in Bigot et al., 1990. The 28S gene probe used as control for RNA quantity was a subclone of pY12 (Terracol and Prud’homme, 1986). It contains an internal 600 bp fragment of the gene encoding the 28S RNA of Drosophila melanogaster. This probe is well conserved with the 28S gene of hymenopteran species (Bigot et al., 1992). The D. pulchellus actin probe was obtained by reverse transcription of 15 µg total RNA with 50 pmole oligoT and 16 U avian myeloblastoid virus (AMV) reverse transcriptase for 1 h at 42°C under the conditions specified by the supplier (Promega). 5 µg aliquots of reverse tran-

S. Renault et al. / Insect Biochemistry and Molecular Biology 29 (1999) 103–111

scription reaction product plus 100 pmoles of Act1(5⬘TGA TCA CCA TTG GCA ACG AG-3⬘) and Act2(5⬘ACA TCT GCT GCAA GGT GGA C-3⬘) primers in 200 µM dACGT, 10 mM Tris pH 9.0, 25 mM MgCl2, 50 mM KCl and 0.5 U Taq polymerase (Appligene) were amplified by Polymerase chain reaction (PCR). The PCR program used was 1 min at 95°C, 30 s at 50°C, 1 min at 72°C for 30 cycles. The internal fragment encoding the actin gene was cloned and sequenced (Accession number X919510). The probes were labelled with [α P32dATP] (ICN) by random priming using the Klenow fragment (Boerhinger Mannheim). Five oligonucleotides from conserved regions of the two satellite DNA strands, chosen in conserved regions were used to detect satellite transcripts (5⬘-CGG GAT CGC TG-3⬘; 5⬘-CGA TCC CGA GCT-3⬘; 5⬘-CTC TCT TCA TTT G-3⬘; 5⬘-CAG TTT GAC TAA-3⬘; 5⬘-CAG GAT TTG AC-3⬘). 50 pmol of each oligonucleotide were end-labelled with [γ P32dATP] (ICN) using 10 U polynucleotide kinase (Promega). Hybridization was performed in 0.5 M NaP, pH 7, 7% SDS overnight at 65°C for satellite monomer, 28S rDNA and actin probes and at 42°C for oligonucleotides. The final washing was done with 2XSSC or 0.2XSSC (20XSSC: 3 M NaCl, 0.3 M Na3citrate, pH 7.0), 0.1% SDS at 65°C. Hybridized filters were autoradiographed on FUJI RX films at ⫺80°C. 2.4. Cloning of satellite RNA 2.4.1. Reverse transcription (RT) and PCR A 15 µg sample of total RNA from D. pulchellus females was reverse transcribed with 20 pmole of each of the following primers (RT1 or RT2) and 16 U of AMV reverse transcriptase for 1 h at 42°C under the conditions specified by the supplier (Promega). The oligonucleotides used (RT1 and RT2) were chosen from conserved regions of satellite monomers: RT1: 5⬘GAATTCCGTTTAGCCAGAAAAGTG-3⬘ and RT2: 5⬘-AAGGTACCGGCTAAACGAAAGTAGC-3⬘. PCR were performed in the conditions previously described. The PCR program used was 1 min at 95°C, 30 s at 42°C, 1 min at 72°C for 30 cycles. The RT1 and RT2 primers amplified a complete 320 bp monomer of satellite DNA. 2.4.2. Cloning and sequencing of RT–PCR products PCR fragments were separated on 1.5% agarose GTG Nusieve gels (FMC products), eluted and purified by two phenol extractions, followed by a chloroform extraction. Purified fragments were cloned in dT-tailed M13mp18 at the SmaI site. Positive clones were sequenced by the Sanger dideoxynucleotide method, using the Sequenase II kit (USB). These clones were numbered 1–4 (EMBL accession number X94969 to X94972). The sequences were analysed using Infobiogen facilities (Dessen et al., 1990).

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2.5. Analysis of the 5⬘ end of satellite DNA transcripts Two oligonucleotides (153: 5⬘-CGA TCC CGA GCT3⬘; 196: 5⬘-CTC TCT TCA TTT G-3⬘) were chosen using the sequence of satellite monomer 1 (Fig. 4), and used for 5⬘ end analysis. Total RNA (20 µg) extracted by the NP40/Urea method, was annealed for 30 min at 65°C, followed by 30 min at room temperature with 0.8 pmole of each end-labelled oligonucleotide, in a buffer containing 150 mM KCl, 10 mM Tris–HCl pH 8.3, 1 mM EDTA. The RNA was precipitated with ethanol, the pellet was resuspended in a buffer containing 25 mM Tris–HCl pH 8.3, 25 mM KCl, 5 mM MgCl2, 5 mM DTT, 0.25 mM spermidine, 0.22 mM of each dNTP, 10 U RNAse inhibitor (Promega) and 12.5 U of AMV reverse transcriptase (Promega) incubated for 1 h at 42°C; 5 µl RNAse (10 mg/ml) was added for the last 15 min. The reverse transcripts were extracted with phenol/chloroform and precipitated. The pellet was resuspended in 10 µl water and 4 µl formamide dye and boiled for 1 min. The reaction products were analysed on a sequencing gel.

3. Results 3.1. Satellite DNA transcription in different hymenopteran species D. pulchellus satellite DNA is composed of about 100,000 repeats of 320 bp monomers which differ from each other by about 10%. RNAs were extracted from D. pulchellus by the guanidium isothiocyanate or NP40/urea methods. They were spotted as dots onto membranes and hybridized with previously cloned satellite monomer of D. pulchellus (Bigot et al., 1990). Hybridization signals were detected only when RNAs were extracted with NP40/urea (Fig. 1(A)). However, the same dot blot hybridized with the 28S rDNA probe gave signals of similar intensity after both extraction methods. The LiCl method was also tested and did not extract satellite transcripts (data not shown). These results suggested the satellite transcripts were associated with specific proteins which prevented the purification of these RNAs by the usual methods (guanidium isothiocyanate or LiCl). We confirmed that the signals observed on dot blots resulted from hybridization with RNAs by treating the NP40/urea extracts with either RNAseA-free DNAseI, or DNAseI-free RNAseA one more time. RNAs were dotted and hybridized with satellite DNA. Signals were recovered only when RNAs were treated with DNAseI and were absent when treated with RNAseA (Fig. 1(B)). This result demonstrates that the dot blot signals resulted from hybridization with RNAs. Northern blot hybridization studies with oligonucleot-

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Fig. 1. Satellite DNA transcription. Northern blots and dot blots were hybridized with satellite oligonucleotides (SAT), actin, and 28S (28S) probes. (A) Dot blots of RNA extracted from Diadromus pulchellus females with NP40/urea or guanidium isothiocyanate. (B) Dot blots of RNA extracted with NP40/urea. These RNAs were treated with RNAseA-free DNAseI or with DNAseI-free RNAseA. (C) Northern blots of total RNAs (15 µg) extracted from females and males were hybridized with satellite oligonucleotides, actin and 28S rDNA probes. The same hybridization is shown after exposure for 24 h (left) and for 7 days (right). Arrows and stars show the positions of discrete bands.

ides based on the satellite sequence detected two main discrete bands of 1.9 kb and 0.62 kb with 24 h exposure (Fig. 1(C), left, stars). Longer exposure (7 days) revealed four discrete bands of 0.32, 0.96, 1.2 and 1.5 kb and a weak smear (Fig. 1(C), right, arrows). As the satellite DNA monomers are 320 bp long, these bands may correspond to transcripts containing 1–6 of the satellite DNA units resulting from multiple transcription initiations and terminations in satellite DNA. They may also result from specific cleavages of the 1.9 kb transcript. The hybridization of the same blot with a complete monomer of satellite DNA showed the same discrete bands but the smear was more intense (data not shown). RNA degradation, which could cause the observed smear, was checked by hybridizing the same blot with the actin and 28S probes (Fig. 1(C)). The discrete bands observed with these probes indicate that the RNAs were not degraded. Its also indicates that the amounts of satellite DNA transcripts in male and female imagos were similar (Fig. 1(C)), even though the smear observed in males was slightly greater than that of females. The NP40/urea extraction method was used to check satellite DNA transcripts from three hymenopteran spec-

ies belonging to the Apocrita suborder (D.collaris, E. vuiletti, E. orientalis). Northern blots of RNAs extracted from males and females of D. collaris, E. vuiletti and E. orientalis were hybridized with their respective satellite DNA (Fig. 2). The satellite DNA was transcribed in each species as a smear in E. vuiletti and E. orientalis (Figs. 2(B,C)) or discrete bands of RNAs in D. collaris (arrows, Fig. 2(A)) in both males and females, except in females of E. vuiletti, where no signal was detected (Fig. 2(B), lane 1). The amount of satellite DNA transcripts in males and females differed as the same blots hybridized with a 28S probe gave discrete bands of similar intensity in males and females of each species. Hence, transcription of satellite DNA seems to be a general phenomenon in Hymenoptera. D. pulchellus satellite DNA transcripts were further characterized using RNAs extracted from females by the NP40/urea method. Single strand RNA was separated from double strand RNA by differential precipitation with LiCl (2 M–4 M)(Fig. 3(A)). PolyA+ RNA were also prepared from female single strand RNA. They were blotted and hybridized with D. pulchellus satellite DNA. A signal was detected only in the single strand non-poly-

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Fig. 2. Satellite transcription in several hymenopteran species. Northern blots of 15 µg RNA extracted with NP40/urea from males and females of Diadromus collaris (A), Eupelmus vuiletti (B) and Eupelmus orientalis (C) were hybridized with probes from their respective satellite DNA and the 28S rDNA probe. Arrows show the positions of discrete bands.

adenylated fraction (Fig. 3(A)). Hybridization of the same samples with an actin probe showed that actin was present in the total and polyadenylated RNA. This indicates that satellite DNA transcripts are present in the single-stranded non-polyadenylated fraction. However, the absence of any open reading frame from the sequence of the basic monomer and the fact that no polyadenylated RNA was detected suggest that they are not translated into proteins. The hybridization with the satellite DNA probe on Northern blot could be due to hybridization with the two RNA strands or to hybridization with only one strand. We checked this by hybridizing replicated dots of RNA with each strand of satellite DNA. Both strands gave the same intensity showing that both satellite DNA strands were transcribed in vivo (data not shown). RNAs purified from different developmental instars of D. pulchellus were also checked for the presence of satellite DNA transcripts (Fig. 3(B)). Satellite DNA transcripts were detected in the embryonic instar, 4-day old larvae and imagos. The signal intensities with satellite DNA probe were similar in all three stages. As the signal obtained with the actin probe was more intense in imagos than in embryos than in larvae, satellite DNA seems to be differentially transcribed during the development.

3.2. Satellite transcript sequences Transcripts of D. pulchellus satellite DNA were reverse transcribed and amplified by PCR with primers chosen from regions highly conserved in all the eight of the published satellite DNA monomers (Bigot et al., 1990). The amplification produced a complete 320 bp monomer. Four were cloned and sequenced (Table 1: sequences 1–4, Accession number X94969–X94972). Their sequences were compared to those of D. pulchellus satellite monomers isolated from genomic DNA (Table 1: sequences 5–12, Accession number M31306). The Infobiogen facilities (Dessen et al., 1990) showed that the four sequences obtained from satellite DNA transcripts were more similar to each other (94–97%) than to the eight sequences of satellite DNA repeats isolated from genomic DNA (75 to 90%) (Table 1). This suggests that a sub-family or a small part of D. pulchellus satellite DNA is transcribed or is better suited for transcription than any random satellite monomer. 3.3. Determination of the initiation site A search for conserved nucleic motifs of eucaryotic promoters transcribed by RNA pol II (TATA and CAAT boxes) and by RNA pol III (DSE sequence) were first

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Fig. 3. D. pulchellus RNAs were hybridized with a satellite DNA (SAT) and an actin probe (ACTIN). (A) Northern blot of total (lane 1) and polyadenylated single-stranded (lane 2) or double stranded (lane 3) RNA. (B) Northern blot of 15 µg RNA extracted from embryos (lane 1), larvae (lane 2) and females (lane 3).

Table 1 Homology between satellite monomers. Monomer sequences 1–4 were obtained by reverse transcription of satellite transcripts. Monomer sequences 5–12 are random cloned genomic satellite DNA. Sequences 1 to 4 have been deposited in the EMBL nucleotide sequence database under accession numbers X94969 to X94972. The % homology was determined for each monomer against each other monomer. The numbers in bold are the % of homology between reverse transcribed monomers Satellite monomer

1 2 3 4 5 6 7 8 9 10 11

Satellite monomer

2

3

4

5

6

7

8

9

10

11

12

95

97 96

98 94 97

88 88 89 88

82 82 83 82 83

88 88 89 88 88 77

84 84 85 85 76 79 79

90 89 91 91 84 79 83 81

85 85 85 85 79 79 80 85 82

90 90 89 89 85 79 85 80 85 81

75 75 76 76 74 73 69 72 73 72 73

% homology

performed. Six putative TATA boxes, four CAAT boxes and one DSE were located (Fig. 4). Transcription initiation sites were checked by primer extension analysis.

Extracts of total RNA extracts from males and females were annealed with two different oligonucleotides chosen in the sequence of transcribed satellite DNA monomer (oligonucleotides 153 and 196)(Fig. 4). The

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Fig. 4. Nucleotide sequence of the reverse transcribed satellite monomer 1 (s1). The positions of oligonucleotides 153 and 196 are given in italics, in the lowest lane. Initiation site is in bold. The CAAT boxes are in gray boxes, TATA boxes are hatched and the DSE sequence is in a white box. Star indicates the putative initiation site detected by primer extension analysis.

satellite DNA transcripts–oligonucleotide hybrids were reverse transcribed and the products were analysed on sequencing gel (Fig. 5). These assays were repeated twice on males and gave the same results. The same experiments were done on total RNA extracted from females. The same initiation sites were identified (data not shown), and the signal intensities in males and females were the same. The primer extension results obtained with the 196 oligonucleotide revealed a major band of 394 nucleotides (Fig. 5, lane 1) indicating that a major transcription initiation site might be present at position 130 (Fig. 4). Under this hypothesis, a 74 nucleotide band was expected if each satellite monomer contains a functional transcription initiation site. As the major 394 bp band was larger than a satellite monomer (320 bp), this suggests that the sequence complementary to the 196 oligonucleotide was divergent in the satellite DNA monomer containing the initiation site. This would explain the absence of a 74 nucleotide band in the primer extension products. Taken together, these results suggest that the transcribed satellite DNA region has a dimeric substructure, that we did not detect with the cloning strategy used in this work. The primer extension performed with the 153 oligonucleotide revealed bands of 20–28 nucleotides (Fig. 5, lane 2). This is in agreement with the presence of an initiation site at position 130, and confirms that the transcription initiation site is inside satellite DNA monomers. Thus, the primer extension experiments revealed one main putative transcription initiation site at position 130. This result was obtained with two different oligonucleot-

ides. The sequences upstream of the initiation sites had potential CAAT and TATA boxes, but they were in nonclassical positions (Fig. 4). Unfortunately, the tandemly repeated structure of the satellite DNA and the intermonomer divergence made it impossible to confirm these results by nuclease S1 mapping.

4. Discussion The satellite DNA of the hymenopteran species, D. pulchellus, is transcribed in the imagos and during development. Satellite DNA transcripts were also found in other hymenopteran species, three belonging to the Apocrita suborder (D. collaris, E. vuiletti, E. orientalis) and one to the Symphyta suborder (D. pini; RouleuxBonnin et al., 1996). Thus the transcription of satellite DNA is quite common in Hymenoptera. Satellite DNA transcription has also been detected in a wide range of animals including D. melanogaster, newts, mice, crabs, birds and frogs, suggesting that the transcription of satellite is a general phenomenon even though its function remains unknown. Only one method (NP40/urea) successfully isolated satellite transcripts. Other classical methods, like guanidium isothiocyanate and LiCl, did not do so in D. pulchellus. This suggests that satellite transcripts are strongly associated with proteins, as reported for Het-A transposable element transcripts (Danilevskaia et al., 1994). It has also recently been demonstrated that the newt ribozyme is a part of a ribonucleotide complex in vivo (Luzi et al., 1997). Transcripts of discrete sizes are detected. The 1.9 kb transcript corresponds to 6 monomers. It might result from

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Fig. 5. Determination of the initiation sites (+1) on Diadromus pulchellus satellite DNA transcripts. Oligonucleotides 196 and 153 (see precise locations in Fig. 4) were used to initiate reverse transcription of 20 µg RNA extracted from males of D. pulchellus. The primer extension products were obtained with primer 196 (lane 1), with primer 153 (lane 2). The size of the extension product was determined by comparison with the satellite DNA sequence ladder prepared with oligonucleotide 153 on the s1 monomer (lane 4).

transcription of 6 monomers. It is more likely that initiation was outside the satellite sequences, since transposable elements are embedded in heterochromatin; this could drive the transcription in adjacent satellite sequences. The 0.62 kb band corresponds to the transcription of two monomers, which is in agreement with the detection of an initiation site, only every two monomers. They could be obtained by specific initiation and termination, or from specific cleavage of longer transcripts. The smear could be due to multiple initiation and/or termination sites along the 100,000 copies of satellite DNA in D. pulchellus. The amounts of satellite DNA transcripts in females and males of D. pulchellus are similar. As the pro-

portions of satellite DNA in the two sexes are quite similar, the transcription detected is probably due to constitutive transcription in imagos. A different result was obtained in D. collaris, E. vuiletti, E. collaris and D. pini, where the expression in males and females is different. Hence, there may be different rates of satellite DNA transcription in Hymenoptera, depending on the presence of specific transcription factors in males and females. This suggests that the satellite DNA transcription in Hymenoptera is not regulated by cis-elements located in satellite DNA monomers. The most important regulatory factors seem to act in trans. Tissue-specific transcription of satellite DNA occurs in amphibians, mice, drosophila and crabs, but no specific regulatory elements have yet been found for these RNAs. Satellite DNA is also differentially expressed between embryos, larvae and imagos stages. Some developmentally regulated transcription factors might regulate satellite DNA transcription during development. The satellite DNA monomers obtained by reverse transcription seem to be more closely related to each other than to genomic sequences. This suggests that only a fraction of the monomers of satellite DNA is transcribed. The RNAs detected by hybridization with satellite DNA are transcripts of the two DNA strands. They are transcribed at similar rates in males and females, which is not the case in Diprion pini, where the two strands are differently transcribed (Rouleux-Bonnin et al., 1996). Their presence raises the question of how satellite DNA transcription is regulated. Transcription may be initiated from genes or transposable elements interspersed in satellite DNA, as in lampbrush chromosomes (Hori et al., 1996; Solovei et al., 1996). In the work presented here, transcription initiation and the promoter elements were analyzed to determine how satellite DNA is transcribed. Sequence analysis of one of the two strands of satellite DNA shows potential transcription regulatory elements for RNA pol II and RNA pol III (Murphy et al., 1989). Primer extension analyses revealed that there is at least one potential initiation site for transcription. This result cannot be confirmed by S1 mapping experiments due to the repeated structure of satellite DNA transcripts. And the sequences analysis reveals that the putative regulatory elements for RNApol II and RNApol III are not in classical positions. In this study, the uncertainty of the localisation of transcription initiation sites may originate from two phenomena. First, the initiation site could be outside the satellite DNA, in adjacent sequences like genes or transposable elements. If this were so, primer extension would not detect the specific initiation sites. However, the result of primer extension suggest that this error source is weakly probable. The second source of uncertainty may come from the fact that the extension products could result from reverse transcriptase arrest due

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to the secondary structure of the satellite transcripts. Sequences analyses indicate that these sequences can form intrastrand dyads (Bigot et al., 1990). A combination of these two phenomena, together with multiple termination sites, could explain the different sizes of satellite DNA transcripts. However, these features cannot explain the occurrence of discrete bands on Northern blots. They may result from specific self-cleavage of longer transcripts, as in amphibian satellite RNA, which has ribozyme activity (Green et al., 1993). Preliminary experiment to test the potential ribozymic activity of the D. pulchellus satellite transcripts detected no such activity (data not shown). However, this could be due to a lack of essential proteins in the in vitro detection method. The ribozyme activity of the ovarian transcripts from the newts is detected only in the presence of ovary proteins (Luzi et al., 1997).

Acknowledgements This work was supported by grants from the CNRS (URA 1298), M.E.S.R., ASTRE and BIOTECHNOCENTRE. The English text was edited by Owen Parkes.

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