John & Lewis (1960), from their observations in. Tenebrio molitor and two ladybird species, suggested that the association of X and Y is initiated by non-specific.
Chromosome Research 1993, 1, 167-174
Localization of tandemly repeated DNA sequences in beetle chromosomes by fluorescent in situ hybridization C. Juan, J. Pons & E. Petitpierre Received 17 March 1993; received in revised form 20 May 1993; Accepted for publication by J. S. Heslop-Harrison 23 May 1993 In situ hybridization to chromosomes and nuclei of Tenebrio molitor shows the massive presence of a species-specific satellite DNA in all chromosomes and six sites of rDNA in mitotic chromosomes. These sites are located in two autosomal pairs and in the X and Y chromosomes. In a related species, Misolampus goudoti, in which two different families of highly repetitive DNA have been previously characterized, one family is located in centromeric regions of all chromosomes with the exception of chromosome Y, while the other repeated DNA family is present both in centromeric and distal regions of all chromosomes, rRNA genes in this species are present in a medium-sized autosomal pair only. These results show that molecular cytogenetics can be applied to coleopteran chromosomes and open the way for a physical mapping of DNA sequences in these organisms. The results also provide insights into the type of meiotic association of the X and Y chromosomes in Coleoptera and the distribution of repeated DNAs within the genome of these insects. Key words: rDNA, satellite DNA, FISH, Coleoptera
Introduction
Fluorescent in situ hybridization (FISH) has become the most accurate technique for localizing and mapping specific nucleic acid sequences on chromosomes of different organisms (Moyzis et al. 1988, Trask 1991, Lin et al. 1991, Hamilton et al. 1992), including those with small chromosomes (Bauwens et al. 1991, Maluszynska & HeslopHarrison, 1991). The Coleoptera have, in general, 'difficult' chromosomes in the sense that they are quite small (1-5 ~m in length) and the meiotic prophases frequently show a diffuse stage until metaphase I, in which is hard to individualize the chromosomal bivalents accurately (Smith & Virkki 1978). However, the analysis of small chromosomes is made easier with fluorescent stains, which give high specificity and low background interference (Maluszynska & Heslop-Harrison 1991). We
applied FISH to beetle chromosomes in order to show the chromosomal localization of some tandemly repeated DNA sequences. This technique is more accurate and sensitive than that of silver impregnation for demonstrating the chromosomal location of nucleolar organizer regions (NORs), since the latter detects acidic ribosomal proteins and therefore ribosomal cistrons which were active in the previous interphase but not inactive cistrons (Goodpasture & Bloom 1975). A great majority of species in the Coleopteran suborder Polyphaga display a peculiar association of X and Y chromosomes in the first division of male meiosis (Smith & Virkki 1978). This was initially termed 'parachute' association by Stevens (1906) because of its shape: the Xand Y heteropycnotic chromosomes appear as a big canopy and a small load, respectively. This mode of association of sex chromosomes is also encountered in some coleopterans of the suborders Adephaga and Myxophaga, and in Megaloptera, a primitive insect order related to Coleoptera, a fact suggesting its ancient origin and a probable common ancestry for these taxa (Smith & Virkki 1978, Virkki et al. 1991). Smith (1950) symbolized this sexual bivalent as Xyp, and initially interpreted this chromosomal configuration as a terminal pairing and formation of chiasmata between the supposed homologous arms of each sex chromosome. However, no evidence of chiasmata has ever been reported between these highly condensed sex chromosomes of the Xyp bivalent (White 1973). John & Lewis (1960), from their observations in Tenebrio molitor and two ladybird species, suggested that the association of X and Y is initiated by non-specific heterochromatic joining and maintained by a persistent nucleolus developed at pachytene and lasting until anaphase I, allowing a regular orientation and segregation of the sex chromosomes. This hypothesis has been challenged by several authors because no sexual NORs have been found by standard silver staining techniques in any beetle and there is evidence for the presence of only one active autosomal NOR in several species of Coleoptera
C. Juan, (corresponding author) J. Pons & E. Petitpierre are at the Laboratori de Gen~tica, Departament de Biologia Ambiental, Universitat de les Illes Balears, E-07071 Palma de Mallorca, Spain. Tel: (+34) 71173153; Fax: (+34) 71173184.
© 1993 Rapid Communications of Oxford
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C. Juan, J. Pons & E. Petitpierre showing the Xyp sex chromosome system (Virkki 1983, Virkki & Denton 1987, Virkki et al. 1990; Postiglioni & Brum-Zorrilla 1988, Postiglioni et al. 1991). On the other hand, Drets et al. (1983) claimed that heterochromatic nonhomologous associations between the sex chromosomes were responsible for the formation of the Xyp in Epilachna paenulata (Coccinellidae). Fluorescent in situ hybridization may be of critical value for a deeper study of NORs in different beetle species, thereby throwing light on this debate about the chromosomal locations of rDNA genes and the mode of association of sex chromosomes in these insects. On the other hand, some species-specific abundant satellite DNAs have recently been described in several Coleoptera species belonging to the family Tenebrionidae (Juan et al. 1991, 1993, Ugarkovi4 et al. 1992, Plohl et al. 1993, Pons et al. 1993). The mealworm beetle Tenebrio molitor has a unique tandemly repeated unit of 142 bp highly conserved in sequence (Petitpierre et al. 1988, Davis & Wyatt 1989, Ugarkovi4 et al. 1989, Plohl et al. 1992). The related species Misolampus goudoti has about 58% of its chromosome length formed by C-banded heterochromatin and shows two different tandemly repeated satDNAs. One, characterized by the digestion of total DNA with EcoRI, is composed of units of 196 bp (short tandem repeat, STR) and the other, isolated by digestion with PstI, of about 1.2 kb (long tandem repeat, LTR) in length; they show no similarity in sequence either between each other or with the T. molitor satellite DNA sequence (Pons et al. submitted). In this paper we describe the detection of X and Yspecific rDNA sequences in the chromosomes of the mealworm beetle Tenebrio molitor but not in the related species Misolampus goudoti despite a positive Ag-NOR reaction. These results are discussed in relation to the Xyp mode of association. The localization of the STR and LTR satellite DNAs of M. goudoti was also investigated on these chromosomes in relation to their distribution within the genome.
Material and methods Chromosome preparations Chromosome spreads were obtained from adult or larval male gonads. The gonadal tissue was fixed in ethanolacetic acid (3:1) for 1 h and stored at -20°C. After squashing in 50% acetic acid, the coverslips were removed after freezing the preparations in liquid nitrogen. The slides were monitored for the presence of chromosome divisions by phase contrast microscopy. Conventional staining was performed in 4% Giemsa in phosphate buffer pH 6.8 for 15 min, and C-banding was performed according to Sumner (1972). In situ hybridization
Chromosome spreads were pretreated with RNAase A in 2 × SSC for I h at 37°C, 0.005% pepsin in 10 mM HCI for
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10 min, and dehydrated in an ethanol series. Probes were labelled with biotin-16-dUTP by nick translation. The hybridization mixture contained 4 n g / ~ l of labelled probe, 0.1~g/~l denatured salmon sperm DNA, 0.1 ~g/~1 yeast RNA and 60% formamide (satellite DNA probes) or 50% formamide (ribosomal probe). A 5 ~1 aliquot of this mixture was placed under a 18 × 18 m m coverslip. Denaturation of probe and chromosomes were performed at the same time at 80°C for 3 rain and hybridization was performed at 37°C overnight in a humid chamber. After hybridization, three stringent washings were performed in 50% formamide, 2 × SSC at 37°C, each for 5 min. Immunological detection was performed using the avidin-FITC/anti-avidin-biotin system with one (satellite DNA probes) or two (ribosomal probe in some cases) rounds of amplification (Pinkel et al. 1986). The chromosomal DNA was counterstained using propidium iodide. A Zeiss Axiophot photomicroscope with the appropriate set of filters for fluorescence was used for micrography.
DNA probes The ribosomal probe used was pDm238, consisting of 28, 18 and 5.8 S ribosomal genes plus interspacer regions of Drosophila melanogaster cloned in pBR322 (Roiha et al. 1981). Satellite DNA probes were as follows: pTm700 consisted of purified pUC-cloned pentamer of Tenebrio molitor satellite DNA, pMg200 was replicative form (RF) of a M13 clone of the monomeric unit of the light satellite DNA of Misolampus goudoti and pMg1200 comprised the monomer unit of the heavy satellite DNA from the same species cloned in a pUC vector.
Results Conventional staining and C-bands The chromosome complement of Tenebrio molitor is composed of 18 meta- or submetacentric autosomes of gradually decreasing sizes, a metacentric X chromosome of similar size plus a minute Y chromosome which is apparently metacentric (Juan et al. 1991). The presence of large blocks of heterochromatin in all chromosomes is well documented (Weith 1985, Juan et al. 1990, 1991). This typical tenebrionid karyotype is also present in the related species Misolampus goudoti (Juan & Petitpierre 1989). Figure 1 shows conventionally stained chromosomes of this species at different stages of meiosis and mitosis. In pachytene all autosomic bivalents show procentric heteropycnotic blocks, while the sex-bivalent Xyp appears as a highly condensed heteropycnotic mass (Figure la). As prophase I progresses, and during metaphase I, the Xyp is visible with the typical parachute morphology, with a non-stained lumen between X and Y (Figure lb,c). Figure ld shows a C-banded mitotic metaphase of M. goudoti-the autosomic pairs and the X chromosome have large median and faint sub-telomeric blocks of heterochro-
Localization of tandem D N A repeats in Coleoptera
t
b
C
d
Figure 1. Chromosomes of Misolampusgoudotiatdifferent cellular stages after conventional Giemsa staining. Pachytene nucleus (a), note nine autosomic bivalents showing procentric heteropycnotic blocks plus the highly condensed heteropycnotic sex bivalent (marked with an arrowhead). Diakinesis (b) and Metaphase I (c), the Xyp sex bivalent is arrowheaded in both plates. (d), C-banded mitotic spermatogonial chromosomes, note faint sub-telomeric bands in many chromosomes in addition to the large and dark pericentromeric ones; the minute Y chromosome is marked by a small arrowhead. Bar represents 5 p.m. matin, while the minute Y chromosome is apparently completely heterochromatic.
Ribosomal genes In Tenebrio molitor, in situ hybridization with the pDm238 probe (Roiha et al. 1981), showed a signal in six mitotic chromosomes at metaphase (Figure 2a). Four of these were in the sub-telomeric regions of two pairs of medium size autosomes, and the remaining two were in the subtelomere of the X chromosome and in the Y chromosome. This figure is confirmed in the meiotic chromosomes: pachytene cells showed a signal attached to two autosomal bivalents plus one or, in some cases, two signals in the sex bivalent (Figure 2b). The hybridization signal in the Xy~ is in one side of the bivalent, apparently in the region of contact between X and Y (see inset of Figure 2b). This suggests that the two sexual NORs are separated in early meiotic phophase, but they fuse when prophase I progresses and the Xyp forms. However, FISH with the same probe in Misolampus goudoti chromosomes gave a hybridization signal in one pair of autosomes of medium size only (Figure 2d). Accordingl~ only one signal was seen in pachytene nuclei attached to an autosomal bivalent (Fisure 2e).
Silver staining showed the presence of only one nucleolar precipitate in the sexual bivalent of T. molitor which persists until metaphase I (Figure 3a), while in M. goudoti there were two, one in the Xyp sex bivalent and another in an autosomal bivalent (Figure 3b). In metaphase I only the silver precipitate corresponding to the sex bivalent was visible (Figure 3c).
Highly repetitive DNAs Fluorescent in situ hybridization of the pTm700 probe showed the massive presence of these DNA sequences in large pericentromeric areas and arms of all T. molitor chromosomes, including the minute Y chromosome (Figure 2c), as suggested previously by isotopic or colorimetric in situ hybridizations (Davis & Wyatt 1989, Juan et al. 1991). In fact, in this species most of the length of mitotic chromosomes is occupied by the 142 bp tandem repeat, which makes up more than 70% of the total length of the chromosome complement. Hybridization of the pMg200 probe, corresponding to the STR family of M. goudoti, was detected at the centromeric regions of all chromosomes of this species, except the small Y chromosome (Figure 2f). In pachytene nuclei, the pericentromeric heterochromatic blocks con-
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C. Juan, J. Pons & E. Petitpierre
a
d
e
Figure 2. Localization of rRNA genes and satellite DNAs in Tenebrio molitor and Misolampus goudoti by fluorescent in situ hybridization. The hybridized sites are visualized by FITC (yellow) and the chromosomal DNA by staining with propidium iodide (red-orange). Six mitotic chromosomes show sites for rRNA genes (pDm238 probe) in T. molitor(a). The minute Y chromosome is arrowed. Pachytene nucleus of the same species with two hybridization rDNA sites in autosomic bivalents plus one in the sex bivalent Xyp (arrowed) (b). The inset in b shows a magnified Xyp sex bivalent showing the signal in the region of contact between the X and Y chromosomes. Hybridization of pTm700 satellite DNA probe with T. molitormitotic chromosomes (c). Mitotic chromosomes (d) and pachytene nuclei (e) of M. goudotihybridized with pDm328 probe showing rRNA genes in an autosomic medium sized chromosome pair. Hybridization of pMg200 satellite DNA probe with mitotic chromosomes (f) and a pachytene nucleus (g) of M. goudoti. The signal is present at pericentromeric positions of all chromosomes except in the Y chromosome (arrowed in the sex bivalent of g). In situ hybridization of pMg1200 satellite DNA probe with mitotic chromosomes (h) and pachytene nuclei (i) of M. goudoti. The signal is present in both pericentromeric and distal regions of all chromosomes. Bar in (i) represents 5 i~m. centrated the signal, but in the Xyp parachute the part corresponding to the Y chromosome lacked any hybridization signal (Figure 2g). On the other hand, the pMg1200 probe, consisting of a cloned monomer of the LTR family of M. goudoti, gave hybridization signals in both telomeric and centromeric regions of all chromosomes (Figure 2h-i). In this case, the Y chromosome showed the presence of the heavy satellite DNA sequences. This pattern of hybridization is coincident with the C-bands of M. goudoti chromosomes (compare with Figure ld), composed by large pericentromeric C-positive 170 ChromosomeResearch Vol 1 1993
heterochromatin and thin telomeric C-bands only visible when a weak C-banding treatment is performed (Juan & Petitpierre 1989).
Discussion In situ hybridization in Coleopteran chromosomes In a previous paper (Juan et al. 1990) we showed that in situ digestion with restriction enzymes can be a useful technique for chromosome banding of beetle chromosomes, in which these techniques have been poorly
Localization of tandem D N A repeats in Coleoptera
Figure 3. Silver staining in Tenebrio molitor and Misolampus goudotL Pachytene nucleus of T. rnolitor (a), one active nucleolus is associated to the Xyp sex bivalent
(marked by an arrowhead). Pachytene nucleus of M. goudoti (b), two silver precipitates are present, one being in the sex bivalent Xyp (marked by an arrowhead). Metaphase I of the same species, showing that in this stage only the sex chromosome associated nucleolus is active
(marked by an arrowhead). Bar represents 5 #m. developed. This technique can even provide information about chromosome rearrangements which are cryptic by C-banding. The results on in situ hybridization demonstrate that physical mapping of DNA sequences is possible in small chromosomes like those of beetles. Although the hybridization signals obtained are high, gentle denaturation and hybridization procedures are essential to preserve the chromosome morphology in such small chromosomes. Similar conclusions have been reached in previous work using FISH in either mitotic chromosomes or interphase nuclei of Arabidopsis thaliana (Maluszynska and Heslop-Harrison 1991, Bauwens et al. 1991), a plant species which also has very small chromosomes. rRNA genes in Coleoptera and the mode of association
of the Xyp Much discussion has taken place about the mode of meiotic association of sex chromosomes in the Xyp bivalent of beetles. The three main opinions are: subterminal pairing and chiasmata formation (Smith 1950); terminal heterochromatic non-specific association (Drets et al. 1983); and nucleolar association by a sex- or autosomalorganized nucleolus (John & Lewis 1960). The first seems unlikely to some authors since there is a strong condensation of X and Y chromosomes in very early stages of
meiosis and usually there is a great difference in size between the two chromosomes (White 1973). However, some chiasmate XY systems, like some found in mammals, also show both large size differences and strong condensation of X and Y chromosomes in early meiosis (Solari 1974, Jones 1987, John 1990). The use of surfacespreading and electron microscopy techniques should determine whether or not synapsis occurs in the Xyp bivalents. Postiglioni et al. (1991) have found, using such microspreading techniques, that there is only one NOR in the fifth bivalent of the chrysomelid Chelymorpha variabilis, and that the sex chromosomal axes are always unpaired, supporting a non-chiasmatic association of the Xyp in this species. The very small Y chromosome is often considered dispensable in species with Xyp because of its heterochromatic nature and presumably little genetic information (Smith & Virkki, 1978). This may explain why the Y chromosome in species with ancestral Xyp systems has frequently been lost in the karyological evolution of beetles, resulting in the acquisition of an XO sex-chromosomal system or other derived systems such as neo XY (Smith & Virkki 1978). Drets et al. (1983) proposed a heterochromatin association as the cause of maintenance of Xyp in the coccinellid Epilachna paenulata. They interpreted the polar appearance of the parachute as a non-chiasmatic association of heterochromatin of X and Y chromosomes (canopy) and a terminal association of the long arms from both chromosomes (load). In this species, as in several other Coleoptera, no sexual NORs have been detected by the most commonly used silver staining technique (Goodpasture & Bloom 1975). The third hypothesis implies the existence of a nucleolus formed early during prophase I between X and Y, allowing the joining of both chromosomes until anaphase I (John & Lewis 1960). These authors found nucleolar material by histochemical methods in the Xyp lumen, beginning at pachytene and lasting until anaphase I. However, they indicated that 'the sex-chromosomeassociated nucleolus may not be a true nucleolus in the sense that both sex chromosomes carry specific organisers responsible for organising nucleoli in physiologically active somatic cells'. In fact as mentioned above, several authors failed to find sex-specific NORs species of Coleoptera. The present in situ hybridization data of T. molitor chromosomes with a rDNA probe are consistent with the John & Lewis hypothesis, although the two previous hypotheses cannot be excluded. There are sex-specific rDNAs in this species in addition to the two autosomal ones. At zygotene, two close hybridization signals corresponding to the X and Y NORs are seen in the early parachute, fusing into one at pachytene. They could organize two nucleoli which in turn fuse into one sex nucleolus or sex vesicle which maintains both chromosomes in contact until segregation. Silver staining indicates that the meiocytes of T. molitor have only one active NOR attached to the sex bivalent lasting in metaphase I,
Chromosome Research Vol 1 1993 1 7 1
C. Juan, J. Pons & E. Petitpierre while the remaining two autosomal NORs seem to be inactive at this cellular stage. However, this situation is not confirmed in the case of Misolampus goudoti, another tenebrionid species with Xyp and in species of the distant family Chrysomelidae (unpublished observations), in which only one hybridization signal is present in an autosomal pair but not in the sexual one. Silver staining highlights the presence of two active NORs (or at least acidic NOR-like proteins), one in an autosomal pair and another, surprisingly, in the parachute Xyp. The apparently contradictory results obtained with FISH and AgNOR staining techniques in M. goudoti could be explained if the rRNA genes in the sex bivalent are present in an insufficient copy number to yield a FISH signal. This seems unlikely because two or even one round of signal amplification should detect genes at low copy number by FISH, and on the other hand, the size of the Xyv-associated silver precipitate indicates a significant synthesis of nucleolar material. One possibility is that the sex nucleolus is not organized by the NORs present in the sex chromosomes but by the autosomal NOR, as was previously suggested (John & Lewis 1960, Virkki et al. 1991). This would imply that nucleolar material produced in the active autosomal rDNA be imported to the sex bivalent, but there is no evidence favouring this hypothetical situation. Another possibility is that neither the presence of acidic argyrofilous proteins in the Xyp nor the existence of sex-specific nucleolar organizing regions are directly related to the mode of assocation of X and Y in coleopterans. The association of tandemly repeated DNA sequences, either ribosomal genes or satellite DNA present in the sexual heterochromatin, could account for the meiotic association and normal segregation of X and Y in this case. The absence of synapsis between X and Y chromosomes has been shown in many marsupial and some eutherian mammalian species (Jones 1987, John 1990). In some of these species a sex chromatin body seems to be produced by non-specific terminal associations between the sex chromosomes, as in the case of Microtus agrestis. In other species, the euchromatic long arm of the X chromosome has a terminal segment which associates with the totally C-positive Y as in Mus dunni (Pathak & Hsu 1976). The precise nature of such specific or non-specific terminal associations of sex chromosomes in meiosis remains unclear (John 1990). In beetles, in spite of the apparent conservation of the Xyp, its structure and mode of association of sex chromosomes may be diverse and has probably changed several times in the course of evolution of coleopteran chromosomes, as suggested by several authors (Smith & Virkki 1978, Virkki et al. 1991). Localization of satellite DNAs and organization within the genome The two species studied here also show differences in the chromosome localization of satellite DNAs. While T. molitor is a striking case of a very abundant satellite DNA
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equilocated in large pericentromeric heterochromatic areas, M. goudoti chromosomes show the presence of two different families of satellite DNA in heterochromatic pericentromeric regions. One of these satellite DNA families also localizes in distal heterochromatic C-bands. The STR satellite DNA family has a monomer unit of 196 bp, AT-rich (64.3%), with a minimum of 120 000 copies per haploid genome (Pons et al. 1993). Its localization in pericentromeric areas is in accordance with the results of in situ digestion with EcoRI, which produces unstained gaps in those regions (Pons et al. submitted). This satellite is absent from the minute Y chromosome, but is present in the X chromosome. Moreover, the LTR family of satellite DNA is made of 1.2 Kb units whose DNA sequence is only partially known, accounting for some 70000 copies per haploid genome (Pons et al. submitted). It is localized in the telomeric heterochromatin of all chromosomes, including the sex chromosomes and with a lower signal intensity in pericentromeric areas. The procentric presence of both STR and LTR satellite DNAs demonstrate a heterogeneity of the centromeric heterochromatin indicated by C-banding in this species. In partial DNA digestions of total DNA with PstI restriction enzyme the maximum number of tandem repeats of the LTR family was found in form of tetramers of about 4.8 Kb, suggesting that this satellite DNA is probably interspersed among larger clusters of satellite DNA from the STR family. This would be in accordance with the lower and heterogenous signal found in centromeric areas by in situ hybridization using the pMg1200 probe compared with the signal in distal areas or with that produced by pMg200 in pericentromeric regions. Two general patterns of heterochromatin distribution can be distinguished in animals and plants (john et al. 1986): i) proximal C-bands constituted by either a unique or more than one highly repetitive DNA family and ii) proximal and distal C-bands. It is well known that the heterochromatin tends to accumulate in the same regions of the non-homologous chromosomes, usually surrounding centromeres or/and in telomeres, a tendency called equilocal distribution of the heterochromatin (Verma 1988). The satellite DNA present in large pericentromeric regions of Tenebrio molitor, Tribolium freemani or Tribolium confusum are examples of centromeric heterochromatin (Juan et al. 1991, 1993; Plohl et al. 1993), as is the mouse centromeric heterochromafin composed of major and minor satellite DNAs (Pietras et al. 1983), and that of Drosophila melanogaster, where several families of highly repetitive sequences are found in the pericentromeric heterochromatin (Peacock 1977). Both proximal and distal C-bands are present in other species, composed of either particular different satellite DNAs in each region, as in whales (Arnason et al. 1978), or only one satellite family differentially distributed within the genome, as in Scilla siberica (Deumling and Greilhuber 1982), Allium cepa (Barnes et al. 1985) or Apodemus sylvaticus (Hirning et al. 1989). In this last species the telomeric heterochromatin seems to have appeared in the evolution and separation of the two Apodemus species, A. flavicollis and A. sylvaticus
Localization of tandem D N A repeats in Coleoptera (Hirning et al. 1989). Usually, when there are both proximal and distal heterochromatin in the same species, they differ to some extent in composition, in line with their location (John et al. 1986, Verma 1988). The distribution of the heterochromatic DNA in the genome of Misolampus goudoti has the characteristics of both distribution patterns since there are two different families of satellite DNAs in the proximal C-bands, and one of these families (or certain related sequences) are present in the distal telomeric C-bands. The origin of the distal C-bands in this species is unclear, but several authors have suggested that C-band polymorphisms and the presence of a similar highly repetitive DNA sequence in proximal, distal a n d / o r intercalar regions of the chromosomes could be due to spreading of the satellite DNA from particular chromosomal sites and subsequent amplification in other areas (John et al. 1986, Hirning et al. 1989). The physical polarization of chromosomes in the interphase diploid nuclei (Rabl polarization) and the bouquet stage of meiotic nuclei provide proximity of chromosomal sites and opportunity for DNA sequence mobility among them, producing an equilocal dispersion of specific DNA sequences within the genome (Schweizer & Loidl 1987, Verma 1988). A detailed sequence analysis of a significant n u m b e r of cloned LTR repeats should clarify the possible evolutionary patterns of the satellite DNA in Misolampus goudoti.
Acknowledgements Drs G. M. Hewitt and G. H. Jones kindly made suggestions that substantially improved the manuscript. We acknowledge Drs D. Ugarkovi4 and M. Plohl for providing a cloned satellite DNA pentamer from Tenebrio molitor and Professor M. Ashburner for a gift of the pDm238 probe. This work has been supported by project DGICYT n u m b e r PB90/0357 (Spain).
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