Cytogenetic and Molecular Characterization of the MBSAT1 Satellite DNA in Holokinetic Chromosomes of the Cabbage Moth, Mamestra Brassicae (Lepidoptera)

Chromosome Research 11: 51^56, 2003. 51 # 2003 Kluwer Academic Publishers. Printed in the Netherlands

Cytogenetic and molecular characterization of the MBSAT1 satellite DNA in holokinetic chromosomes of the cabbage moth, Mamestra brassicae (Lepidoptera)

Mauro Mandrioli1, Gian Carlo Manicardi2 & Frantisek Marec3 1Dipartimento di Biologia Animale, Universita` di Modena e Reggio Emilia, Via Campi 213/D, 41100 Modena, Italy; Tel: ( þ 39) 59 2055544; Fax: ( þ 39) 59 2055548; E-mail: [email protected]; 2Dipartimento di Scienze Agrarie, Universita` degli Studi di Modena e Reggio Emilia, Viale Kennedy 17, 42100 Reggio Emila, Italy; 3Institute of EntomologyASCR, Ceske Budejovice, Czech Republic

Received 16 October 2002. Received in revised form and accepted for publication by Herbert Macgregor 10 November 2002

Keywords: heterochromatin, holocentric chromosome, Lepidoptera, Mamestra brassicae, satellite DNA

Abstract

Digestion of Mamestra brassicae DNA with DraI produced a prominent fragment of approximately 200 bp and a ladder of electrophoretic bands with molecular weights which are a multiple of 200 bp. Southern blotting revealed that this ladder is composed of DNA fragments that are multimers of the 200-bp DraI band suggesting that DraI isolated a satellite that has been called Mamestra brassicae satellite DNA 1 (MBSAT1). MBSAT1 is the ¢rst satellite DNA isolated in Lepidoptera. In-situ DraI digestion of chromosome spreads, together with £uorescent in-situ hybridization, showed that MBSAT1 sequences are clustered in heterochromatin of the sex chromosomes, Z and W. MBSAT1 was 234 bplong with an AT content of 60.7%. The curvature^propensity plot suggested a curvature in the MBSAT1 structure.

Introduction usually small and numerous, lack distinct primary constrictions (centromeres) and sister chromatids Most studies concerning satellite DNAs have been separate by parallel disjunction at mitotic meta- focused on organisms with monocentric chro- phase (Murakami & Imai 1974). Some ¢ndings mosomes, whereas organisms possessing holo- suggest that lepidopteran chromosomes are not kinetic (holocentric) chromosomes have been truly holokinetic but exhibit a localized kine- almost neglected. Only nematodes (Roth 1979, tochore that, in contrast to that in the typical Naclerio et al. 1992, Grenier et al. 1997, Cas- monokinetic chromosomes, covers a relatively tagnone-Sereno et al. 1998a, 1998b), aphids large portion of the chromosomal surface. These (Bizzaro et al. 1996, Spence et al. 1998, Mandrioli ¢ndings favour a chromosome type intermediate et al. 1999a, 1999b, 1999c), a bug (Lagowsky et al. between the traditional holokinetic and mono- 1973) and a plant (Collet & Westerman 1987) have kinetic chromosomes (for a review, see Wolf 1996). been studied in this regard. In accordance with the absence of distinct cen- Moths and butter£ies, the Lepidoptera, have tromeres, lepidopteran chromosomes lack peri- holokinetic chromosomes. Their chromosomes are centric heterochromatin that is common in 52 M. Mandrioli et al. monocentric chromosomes. In most species slides were stained with 40-60-diamidino-2-phe- examined, heterochromatin was found only in sex nylindole (DAPI) as described by Donlon & chromosomes and, with a few exceptions, only in Magenis (1983). the heterogametic female sex. The Lepidoptera Fresh chromosome spreads were digested at possess a WZ/ZZ (female/male) sex-chromosome 37C with 0.6 U/mlofDraIandApaIinthe system or its numerical variations (Traut & Marec appropriate bu¡er solution, for time periods 1997, Sharma & Sobti 2002). Like many Y ranging from 10 min to 2 h. Nick translation was chromosomes in the XY/XX system, the W carried out for 20 min according to Mandrioli et al. chromosome often consists partly or largely of (1999c). heterochromatin (Traut & Marec 1997). In addi- DNA probes were labelled by random priming tion, most species display one or more hetero- using the ‘DIG high prime’ (Roche Molecular chromatin bodies in female somatic interphase Biochemicals, Mannheim, Germany) following nuclei but not in male nuclei. This female-speci¢c the manufacturer’s protocol. FISH was carried heterochromatin (so-called ‘W-chromatin’ or ‘sex- out as described by Mandrioli et al. (1999c) chromatin’) is derived from the W chromosome making the following stringency washes after (Traut & Marec 1996). Data on the DNA com- hybridization: twice in 0.1 Â SSC for 10 min at position of W heterochromatin and repetitive room temperature and twice in 0.1 Â SSC at 45C DNAs in Lepidoptera are scarce and limited to a for 10 min. few studies reporting microsatellites (e.g. Reddy DNA extraction from cultured CRL-8003 cells et al. 1999) and transposable elements (e.g., Abe was performed using a standard protocol given in et al. 1998, Ohbayashi et al. 1998) in Bombyx mori. Mandrioli (2002). Southern hybridization, dot To date, no published information about satellite blotting and restriction enzyme digestion were DNAs is distributed in any lepidopteran species. carried out as described in Mandrioli et al. (1999c). Here we present data on structure and chromo- RNA extraction and RT-PCR were performed somal distribution of a satellite DNA identi¢ed with the SV Total RNA Isolation System (Pro- in the cabbage moth, Mamestra brassicae (Lepido- mega Corporation, Madison, USA) and with the ptera: Noctuidae), a serious pest of agricultural Access RT-PCR System (Promega), respectively, crops. according to the supplier’s suggestions. MBSAT1 fragments were cloned using the ‘pGEM-T-easy’ cloning kit (Promega Corpora- Material and methods tion, Madison, USA) according to manufacturer’s protocol. We used the IZD-MB-0503 cell line of Mamestra Sequence alignments and search for open reading brassicae (ATCC number: CRL-8003). The cells frame and internal repeats were performed using were cultured in Ex-Cell 405 medium (JRH the GCG software (GCG Computer Group, Biosciences, KS, USA) at 26C. Madison, USA). The curvature^propensity plot CRL-8003 cells were spread following the was calculated with DNase I parameters of the method described by Mandrioli (2002). Brie£y, the bend.it server (http://www2.icgeb.trieste.it/*dna/ cells were kept in a hypotonic solution (0.8% bend it.html) according to Gabrielian et al. sodium citrate) for about 45 min, then transferred (1996). to minitubes and centrifuged at 350 g for 3 min. A ¢xative solution (methanol^acetic acid, 3:1) was successively added to the pellet, which was made to Results £ow upand down for 1 min through a needle of a 1-ml hypodermic syringe. Finally, the pellet was In order to study M. brassicae heterochromatin at resuspended in 200 ml of fresh ¢xative, and 20 mlof a molecular level, total genomic DNA was cellular suspension was dropped onto clean slides digested with di¡erent restriction endonucleases and air dried. (ApaI, AluI, DdeI, MspI, HpaII, DraI) and C-banding was performed according to the separated in a 1.2% agarose gel by electrophoresis. technique of Sumner (1972). After the treatment, Digestion with AluI, DdeI, MspI, and HpaII, Satellite DNA in Mamestra (Lepidoptera) 53 respectively, resulted in a di¡use smear on the gel. that MBSAT1 is not methylated since there was no A faint electrophoretic band corresponding to a di¡erence between the two restriction patterns DNA fragment of 1800 bpwas observed after (Figure 1b, lanes 5 and 6). digestion with ApaI, whereas DraI produced a By means of densitometric scanning of dot blots, prominent band of approximately 200 bp and a obtained by hybridizing the MBSAT1 probe to the ladder of electrophoretic bands that appeared as M. brassicae genomic DNA, we estimated that multiples of the 200-bp fragment (Figure 1a). MBSAT1 accounts for 1.9 Æ 0.3% of the genome. As the ladder observed is typical for a satellite In-situ digestion with restriction endonucleases DNA, the DraI 200-bpfragment was isolated from (RE) followed by nick translation (NT) showed the gel, cloned, and used as a probe for hybridi- that DraI restriction sites are clustered in C- zation experiments. Southern blotting after DraI positive segments of two chromosomes (Figure 2a) digestion (Figure 1b, lane 1) revealed a regular previously identi¢ed as the sex chromosomes, Z ladder of bands composed of basic-length multi- and W (Mandrioli 2002). ApaI targets were mers.AladderofbandswasalsopresentinDdeIand localized in the NOR-bearing telomeric segments AluI (Figure 1b, lanes 3 and 4) digested DNAs but, of Z and W chromosomes (Figure 2b, c). GC- compared with the pattern obtained after DraI richness of the ApaI restriction target together digestion, DdeI lacked the ¢rst two multimers, with the results of silver staining after ApaI/NT whereas AluI showed only three multimers. Based indicated that the 1800-bp ApaIband(Figure1a) onitsrepetitivenature,theDraIfragmentwascalled contained rDNA genes. M. brassicae satellite DNA 1 (MBSAT1). Com- FISH experiments, carried out on M. brassicae parison of hybridization patterns of MBSAT1 after mitotic chromosomes using an MBSAT1 probe, MspIandHpaII digestion, respectively, showed con¢rmed the results of in-situ DraI/NT. It was concluded that MBSAT1sequences are clustered on Z and W chromosomes (Figure 2d) in chromosomal regions that are composed of heterochromatin as shown by C-banding and DAPI staining (Figure 2e). Cloning and sequencing revealed that MBSAT1 length is 234 bpwith an AT content of 60.7% (MBSAT1 Genbank accession number: AY- 136944). A search for homology of MBSAT1 with other DNA sequences in GenBank and EMBL databases yielded negative results. Apart from short poly-A tracts, no signi¢cant direct or inverted repeats or open reading frames were revealed by sequence analysis. The curvature^propensity plot that was calculated with DNase I parameters of the bend.it server revealed an MBSAT1 region, located between nucleotide 108 and 182, with a high value of curvature propensity (13.6). Its magnitude Figure 1. Genomic DNA of Mamestra brassicae digested with roughly corresponds to the value calculated for a DraI (lane 1), ApaI(lane2),AluI (lane 3), DdeI (lane 4), MspI highly curved motif described in Columba risoria (lane 5), and HpaII (lane 6), respectively. (a) Electrophoresis DNA satellite (CRBENSAT) (Figure 3) indicating in a 1.2% agarose gel stained with ethidium bromide; (b) that MBSAT1 is curved. Southern hybridization with the MBSAT1 probe. Note a posi- Transcriptional activity of MBSAT1 was tive band of about 1800 bp(indicated by asterisk) corresponding to the ApaI digested DNA and a ladder of bands after examined by Northern blotting and RT-PCR digestion with DraI. The ladder of bands is still evident in DraI without producing any positive results. Thus, it lane after hybridization with the MBSAT1 probe, whereas sev- was concluded that MBSAT1 is not transcribed in eral bands are present in AluIandDdeI lanes. M. brassicae cells. 54 M. Mandrioli et al.

Figure 2. Mitotic metaphase chromosomes prepared from the Mamestra brassicae CRL-8003 cells. (a^c) In-situ RT/NT shows that DraI cutting sites (a) are clustered in heterochromatic segments of both sex chromosomes, whereas ApaI cutting sites (b) are restricted to one telomeric end of each sex chromosome containing rDNA genes as shown by NOR silver staining (c); (d, e) FISH with the MBSAT1 probe (d) demonstrates that the Z and W sex chromosomes contain each several copies of MBSAT1 located in heterochromatic regions as shown by C-banding followed by DAPI staining (e). Arrows indicate sex chromosomes. Bar represents 10 mm.

Discussion that the same satellite DNA was located in both sex chromosomes but absent in all autosomes is of Digestion of M. brassicae genomic DNA with particular interest. Taken together with the DraI showed a new DNA satellite, called absence of recombination in lepidopteran females MBSAT1. The satellite sequences are arrayed in (for a review, see Marec 1996), it implies that the tandem with some subsets containing more than MBSAT1 was pre-existing in the chromosomes ¢ve units, and represent about 1.9% of the genome. before they di¡erentiated as sex chromosomes. By means of in-situ RE/NT and FISH, we The presence of NOR on both the sex chromo- showed here that the MBSAT1 repeats are strictly somes strongly suggests that M. brassicae possess concentrated in heterochromatin of both sex neo-W and neo-Z chromosomes that have evolved chromosomes, Z and W. In addition, the MBSAT1 by fusion of the original WZ pair with the hybridization sites corresponded to DAPI-positive autosome pair bearing the NOR. Similar neo-W/ heterochromatic segments of Z and W, indicating neo-Z sex chromosomes were reported in Orgyia a high AT content in MBSAT1. This was subse- antiqua and O. thyellina (Traut & Marec 1997). quently con¢rmed by sequence analysis. The fact The karyotype of M. brassicae favours this Satellite DNA in Mamestra (Lepidoptera) 55

The MBSAT1 repeat, isolated from M. brassicae, is a short DNA satellite with a consensus sequence of 234 bp. It exhibits a high AT content (60.7%) which is a general feature of satellite DNA (Singer 1982). The MBSAT1 is the ¢rst satellite DNA identi¢ed in Lepidoptera.

Acknowledgements

This work was supported by grants from The University of Modena and Reggio Emilia and from the Ministero dell’Istruzione, dell’Universita` e della Ricerca (MIUR) of Italy. F.M. was supported by the Grant Agency of the Czech Republic Figure 3. Curvature^propensity plot of MBSAT1 satellite (grant no. 206/00/0750) and from the Entomology DNA showing the presence of a strong curvature propensity Institute projects Z5007907 and K5052113. in the region between nucleotides 108 and 182. References hypothesis. It shows a low number of chromo- Abe H, Ohbayashi F, Shimada T, Sugasaki T, Kawai S, Oshiki somes (n ¼ 11) that di¡er considerably in their T (1998) A complete full-length non-LTR retrotransposon, sizes (Mandrioli 2002), thus indicating multiple BMC1, on the W chromosome of the silkworm, Bombyx mori. Genes Genet Syst 73: 353^358. chromosome rearrangements in the genome. This Bizzaro D, Manicardi GC, Bianchi U (1996) Chromosomal might have happened recently as a population- localization of a highly repeated EcoRI DNA fragment in speci¢c event, since the early study of Saitoh Megoura viciae (Homoptera, Aphididae) by nick translation (1959), performed in a Japanese population, and FISH. Chromosome Res 4: 392^396. reported a completely di¡erent karyotype of M. Castagnone-Sereno P, Semblat JP, Leroy F, Abad P (1998a) A new AluI satellite DNA in the root-knot nematode Melo- brassicae consisting of 31 small and uniform ydogyne fallax: relationshipwith satellites from the chromosome pairs. Thus, it appears that M. sympatric species M. hapla and M. chitwoodi. Mol Biol Evol brassicae could be an interesting model, not only 15: 1115^1122. for the study of sex chromosome di¡erentiation, Castagnone-Sereno P, Leroy H, Semblat JP, Leroy F, Abad P, but also for karyotype evolution in Lepidoptera. Zijlstra C (1998b) Unusual and strongly structured sequence variation in a complex satellite DNA family from the nema- The present study clearly shows that the satellite tode M. chitwoodi. J Mol Evol 46: 225^233. DNA in M. brassicae is localized in heterochro- Collet C, Westerman M (1987) Interspecies comparison of the matic chromosome segments and, in addition, highly repeated DNA of Australian Luzula (Juncaceae). spreads throughout the entire length of the seg- Genetica 74: 95^103. ments. This suggests that the constitutive het- Donlon TA, Magenis RE (1983) Methyl green is a substitute for distamycin A in the formation of distamycin A/DAPI erochromatin of holokinetic chromosomes is C-bands. Hum Genet 65: 144^146. principally made of the satellite DNAs as in Gabrielian G, Simoncsits A, Pongor S (1996) Distribution of monocentric chromosomes (John & Miklos 1979, bending propensity in DNA sequences. FEBS Lett 393: John 1988). However, the distribution of hetero- 124^130. chromatin is not equilocal in holokinetic chro- Grenier E, Castagnone-Sereno P, Abad P (1997) Satellite DNA sequences as taxonomic markers in nematodes of agronomic mosomes. In other words, heterochromatin is not interest. Parasitol Today 13: 398^401. located at speci¢c sites such as, for example, John B (1988) The biology of heterochromatin. In: Verna RS, centromeres in monocentric chromosomes. Our ed. Heterochromatin: Molecular and Structural Aspects. ¢ndings in M. brassicae thus support a hypothesis Cambridge: Cambridge University Press, pp 1^147. of Schweizer & Loidl (1987), who stated that the John B, Miklos GLG (1979) Functional aspects of satellite DNA and heterochromatin. Int Rev Cytol 58: 1^114. theory of ‘equilocal distribution of hetero- Lagowsky JM, Yu MY, Forrest HS, Laird CD (1973) Disper- chromatin’ is not valid for holokinetic chromo- sion of repeat DNA sequences in Oncopeltus fasciatus,an somes. organism with diffuse centromere. Chromosoma 43: 349^373. 56 M. Mandrioli et al.

Mandrioli M (2002) Cytogenetic characterization of telomeres Roth GE (1979) Satellite DNA properties of the germ line in the holocentric chromosomes of the lepidopteran limited DNA and the organization of somatic genomes in Mamestra brassicae. Chromosome Res 9: 279^286. the nematode Ascaris suum and Parascaris equorum. Mandrioli M, Manicardi GC, Bizzaro D, Bianchi U (1999a) Chromosoma 74: 355^371. NORs heteromorphism within a parthenogenetic lineage Saitoh K (1959) The chromosomes of some species of moths. of the aphid Megoura viciae. Chromosome Res 7: 157^162. Jpn J Genet 34: 84^87. Mandrioli M, Bizzaro D, Giusti M, Manicardi GC, Bianchi U Schweizer D, Loidl J (1987) A model for heterochromatin dis- (1999b) The role of rDNA genes in X chromosomes associ- persion and the evolution of C-band patterns. Chromosomes ation in the aphid Acyrthosiphon pisum. Genome 42: Today 9: 61^74. 381^386. Sharma VL, Sobti RC (2002) Cytogenetics of lepidopteran Mandrioli M, Bizzaro D, Giusti M, Gionghi, D, Manicardi insects. In: Sobti RC, ed. Some Aspects of Chromosome GC, Bianchi U (1999c) Cytogenetic and molecular char- Structure and Functions. New Delhi, India: Narosa acterization of a highly repeated DNA sequence in the peach Publishing House, pp. 192^206. potato aphid Myzus persicae. Chromosoma 108: 436^442. Singer ML (1982) Highly repeated sequences in mammalian Marec F (1996) Synaptonemal complexes in insects. Int J Insect genomes. Int Rev Cytol 76: 67^112. Morphol Embryol 25: 205^233. Spence JM, Blackman RL, Testa JM, Ready PD (1998) A Murakami A, Imai HT (1974) Cytological evidence for 169 bptandem repeat DNA marker for subtelomeric holocentric chromosomes of the silkworms, Bombyx mori heterochromatin and chromosomal re-arrangement in and B. mandarina (Bombycidae, Lepidoptera). Chromosoma aphids of the Myzus persicae group. Chromosome Res 6: 47: 167^178. 167^175. Naclerio G, Cangiano G, Coulson A, Levitt A, Ruvolo V, La Sumner AT (1972) A simple technique for demons- Volpe A (1992) Molecular and genomic organization of trating centromeric heterochromatin. Exp Cell Res 75: clusters of repetitive DNA sequences in Caenorhabditis 304^306. elegans. J Mol Biol 226: 159^168. Traut W, Marec F (1996) Sex chromatin in Lepidoptera. QRev Ohbayashi F, Shimada T, Sugasaki T et al. (1998) Molecular Biol 71: 239^256. structure of the copia-like retrotransposable element Traut W, Marec F (1997) Sex chromosome differentiation in Yokozuna on the W chromosome of the silkworm, Bombyx some species of Lepidoptera (Insecta). Chromosome Res 5: mori. Genes Genet Syst 73: 345^352. 283^291. Reddy KD, Abraham EG, Nagaraju J (1999) Microsatellites in Wolf KW (1996) The structure of condensed chromosomes in the silkworm, Bombyx mori: abundance, polymorphism, and mitosis and meiosis of insects. Int J Insect Morphol Embryol strain characterization. Genome 42: 1057^1065. 25: 37^62.