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Print version ISSN 1415-4757On-line version ISSN 1678-4685
Genet. Mol. Biol. vol.23 no.4 São Paulo Dec. 2000
Patterns of heterochromatin distribution in plant chromosomes
Departamento de Botânica, CCB, Universidade Federal de Pernambuco, Cidade Universitária, 50670-420 Recife, PE, Brasil. E-mail: firstname.lastname@example.org
The C-band distribution patterns of 105 angiosperm species were compared to identify general patterns or preferential sites for heterochromatin. The base-specific fluorochrome reaction of heterochromatin for 58 of these species and the role played by the average chromosome size in band distribution were also considered. The results showed that heterochromatin was preferentially located in similar chromosome regions, regardless of the distance from the centromere. This trend results in generalized bands, with heterochromatin distribution being identical in most chromosomes of a karyotype. Such bands very often displayed the same fluorochrome reaction, suggesting possible repeat transfer between non-homologous sites. Chromosome size may also play a role in heterochromatin location, since proximal bands were much more common in small-sized chromosomes.
Heterochromatin (HC) is one of the chromosomal components that has attracted most attention from cytogeneticists, because of its still unknown function, its apparent lack of genes and the fact that it stains differently to the rest of the chromosome. Especially after the development of HC differentiation techniques in the metaphase chromosomes of a great diversity of organisms, a large number of papers were written which reported its presence, variability, molecular composition, direct and indirect effects on the karyotype, putative function, etc. (see Mizianty, 1984a,b, 1985; John, 1988; Hennig, 1999). One of the most basic, but by no means the least important aspects of HC research, is the study of its distribution in blocks or bands throughout the karyotype. Analysis of band distribution patterns can make it possible to verify whether the bands are preferentially located in certain chromosomal regions, suggesting that the HC has a functional purpose.
The analysis of HC distribution patterns in the chromosome complement of angiosperms in general is hindered by the particularly high variability of HC added to the variability of other karyotype parameters. The comparison of HC band patterns of different species should take into consideration the following aspects: 1) the HC is not homogeneous, varying qualitatively and quantitatively between species; 2) within a single species, polymorphism for the number and size of bands is frequent; 3) the amount of HC varies regardless the amount of euchromatin or the nuclear DNA content; 4) both HC and euchromatin can suffer large changes in a relatively short time; 5) different staining techniques may reveal different HC fractions; 6) very different species may present simultaneous differences in chromosome number, size and morphology, as well as in the amount, composition and distribution of HC (Greilhuber, 1982; Vosa, 1985; John, 1988; Sumner, 1990).
The functional and evolutionary meaning of HC may not be the same for all species, and a single distribution pattern for all angiosperms may not exist, only tendencies or preferential patterns for different genomes and karyotype architectures.
In spite of these difficulties in establishing general models, some authors draw attention to distribution patterns that seem to occur very frequently. Heitz (1933) was the first to observe that, in each species, the HC seemed to be distributed non-randomly in proximal, interstitial and telomeric regions. In Vicia faba, for instance, he observed that HC was preferentially located in interstitial regions, while in Allium cepa it distribution was preferentially terminal. After performing chromosome analysis of several species of plants and animals, Heitz (1957) considered that such an equilocal distribution is a universal rule.
Lima de Faria (1976a,b, 1983) considered that heterochromatin blocks observed by "cold starvation" or by C-banding, as well as chromomeres, knobs, secondary constrictions and other chromosomal markers and DNA sequences, would occupy preferential sites within the chromosome field (the chromosome region between centromere and telomere where specific DNA sequences and chromosome phenomena occur in a specific order). Greilhuber (1979), however, re-examined the available data on the distribution of "cold starved" HC and found no preferential regions.
Later, Loidl and collaborators (Loidl, 1983; Greilhuber and Loidl, 1983; Schweizer and Loidl, 1987) drew attention to the fact that in many species HC blocks tend to be distributed in the arms of different chromosomes at an equal distance starting from the centromere (see also Bennett, 1982). In such cases, the chromosomes would tend to exhibit equidistant bands (e.g., on the telomeres of similar-sized arms and in the corresponding interstitial regions of the longest arms), while, according to Heitz, the distribution of bands would be preferentially equilocal (e.g., on the telomeres of short and long arms). These tendencies would result in different karyotype patterns, with equilocalization producing a generalized banding pattern in all chromosomes of a complement (regardless of size and centromeric index variation), while equidistance would generate different banding patterns in asymmetrical karyotypes and identical patterns in symmetrical karyotypes.
One of the difficulties in understanding HC distribution is the fact that bands of a single karyotype may be composed of different types of HC, i.e., unrelated families of repetitive DNA with independent distribution patterns (Flavell, 1982). Therefore, it is important to separately evaluate the distribution of each HC type by cyto-molecular techniques, as has been done for rye (Appels et al., 1978), Scilla (Deumling and Greilhuber, 1982), barley (Brandes et al., 1995), Aegilops (Badaeva et al., 1996) and a few other organisms. Working with conventional cytological techniques, only the HC associated with the nucleolar organizer region (NOR-HC) can be specifically identified, due to secondary constrictions or by silver nitrate impregnation. More reliable recognition of the NOR-HC has recently been achieved by in situ hybridization, mainly with pTa71 probe, which is able to localize rDNA 18S-5.8S-26S sites in every kind of plant (see, e.g., Galasso et al., 1996a; Hizume et al., 1992) and at least in some animals (Pendás et al., 1993). An alternative way of distinguishing some HC types is to stain the chromosomes with fluorochromes that have a preferential affinity for AT- or GC-rich DNA. This staining can differentiate the HC in only a few groups, allowing the easy observation of their distribution in the chromosomes.
HC may be revealed by different cytological techniques, with identical or very similar results (Vosa, 1976b; Lozano et al., 1990; Berg and Greilhuber, 1993). Conventional staining techniques may sometimes show heteropycnotic regions that form the so-called prophase condensation patterns (Fukui and Mukai, 1988; Ikeda, 1988; Benko-Iseppon and Morawetz, 1993). Such regions have often been confused with HC patterns which may be very different from each other (see Morawetz, 1981a; Guerra, 1988a). The HC revealed by C-banding seems to correspond to the most general conception of HC, coinciding with the sites of late replicating DNA and tandem organized satellite DNA (Baumann, 1971; Appels et al., 1978; Deumling and Greilhuber, 1982; Cortés and Escalza, 1986; Schubert and Rieger, 1991). However, the use of different C-banding techniques may result in very different banding patterns. In Citrus, for example, HC blocks were found mostly at terminal regions (Guerra, 1985, 1993a; Miranda et al., 1997; Pedrosa et al., 2000), although Ito et al. (1993), using a different technique, found exclusively proximal HC blocks. In corn, Aguiar-Perecin and Vosa (1985) reported C-bands in terminal and subterminal chromosomal regions, coinciding in number and position with the knobs. However, Molina (1981) found only proximal bands in all the chromosomes, while Chow and Larter (1981) found proximal and terminal bands. On the other hand, Carvalho and Saraiva (1993), using another technique (KHG banding), found numerous interstitial bands. In all these cases the bands were clear and unequivocal, and the differences in these results were certainly due to the procedures used, which revealed different chromatin fractions.
Besides the C-banding technique, staining with base-specific fluorochromes has been recognized as a reliable method of distinguishing some types of HC in plants (Vosa, 1970, 1976b; Schweizer, 1976). The fluorochromes quinacrine (Q), Hoechst 33258 (H) and 4'-6-diamidino-2-phenylindole (DAPI) preferentially stain AT-rich HC (Q+, H+ and DAPI+ bands, respectively) while mitramycin (MM) and chromomycin A3 (CMA) preferentially stain GC-rich (MM+ or CMA+) regions (Schweizer, 1976; Sumner, 1990). These same fluorochromes may also negatively stain AT-poor or GC-poor HC blocks, respectively, whereas double staining with two fluorochromes of different base specificity (e.g., CMA/DAPI, CMA/H or MM/DAPI) may highlight staining differences (Schweizer, 1976, 1981; Kenton, 1991). In addition, non-fluorescent DNA-ligands, like dystamycin A, may also be used to intensify the CMA or DAPI staining (Schweizer, 1983; Fuchs et al., 1998). However, not all AT-rich or GC-rich HC reacts equally to these fluorochromes (see e.g., Schwarzacher and Schweizer, 1982; Kenton, 1991; Bennett et al., 1995) and some C-banded positive regions react neutrally with fluorochromes, i.e., they fluoresce with the same brightness as euchromatin (Morawetz, 1986a,b; Röser, 1994; Galasso et al., 1996b; Cuellar et al., 1996). Other C-bands, fluoresce with the same intensity when stained with fluorochromes with different base-specificities, such as CMA and DAPI (Loidl, 1983; Guerra, 1989; Okada, 1991; Berg and Greilhuber, 1993). All these HC types were considered here as neutral bands.
In this paper the C-band distribution patterns of 105 species, belonging to 58 dicotyledons and 32 monocotyledons genera, were compared in order to identify general patterns of HC distribution or any preferential sites in which it may occur. Only the results obtained with the C-banding techniques most used in plants (Marks, 1975; Schwarzacher et al., 1980; Gill et al., 1991) will be considered. These techniques are based on chromosome hydrolysis with 45% acetic acid, or dilute hydrochloric acid, followed by denaturation in barium hydroxide, treatment with 2x SSC and Giemsa staining. Information about the reactivity to base-specific fluorochromes is included for 58 of these species. The main questions focused on are: 1) Is there a preferential distribution of C-bands in the proximal, interstitial or telomeric regions? 2) Are band distribution patterns influenced by the average chromosome size of the karyotypes? 3) Is there any relationship between GC-rich or AT-rich HC bands and their chromosomal location? 4) Is the occurrence of generalized equilocal bands influenced by their base composition, their chromosomal position, or the average chromosome size?
MATERIAL ANALYZED: CRITERIA FOR DATA SELECTION
To evaluate the variability of HC distribution patterns among angiosperms, data were compiled for 105 species belonging to 90 genera (Table I). Each species name was preceded by the first three letters of its family name to help recognize the taxa, along with diploid number, average chromosome size, band position in the chromosomes, occurrence and position of generalized bands and consulted references.
Species were selected for inclusion in this list simply because they were published or unpublished author's original data or because they were cited in papers available to the author. To avoid overrepresenting the banding patterns of more widely studied plant groups, such as Triticum, Hordeum, etc., only a single species of each genus was considered. However, in genera with large karyotype variation, either in the amount or the distribution of HC, like Capsicum and Scilla, two or more representatives were included. In these cases preference was given to the species with extreme HC patterns and to those about which data was available for both C-band and fluorochrome staining. The references used were always those with good photographic documentation which was compatible with the description in the text, the photographic data generally being more highly valued than the text description. Papers with no photographic data were not considered.
The sample was divided into three groups according to average chromosome size, based on the fact that the heteropycnotic characteristics of the chromatin of species with small, medium and large chromosomes are frequently different, resulting in different structural types of interphase nuclei (Delay, 1949; Guerra, 1987). Small chromosomes (<3.0 mm) usually have arreticulate or pro-chromosome nuclei, which present early condensed proximal areas in prophase, e.g., heterochromatic blocks. On the other hand, large chromosomes (>5.0 mm) usually have reticulate or eureticulate nuclei, with more or less homogeneous chromosome condensation. Medium-sized chromosomes (>3.0 <5.0 mm) display semi-reticulate nuclei with irregular and variable chromosome condensation (Delay, 1949; Guerra, 1987). This correlation is most evident between DNA amount and nuclear interphase type (Barlow, 1977). However, since no DNA amount estimates were available for many of the species included, nuclear interphase type was correlated with average chromosome size.
The chromosome size was preferentially based on measurements supplied by the referred authors, although in the many cases where the chromosome size was not referred to in the paper, but a scale bar was presented in the photos or idiograms, a length estimation was made with the help of the scale. In these species the calculated chromosome size are more inaccurate, since such photographs show the banding pattern, but not necessarily with a representative chromosome size. In Table I these values are indicated as circa (ca.).
Whenever possible, the chromosome size estimated from conventionally stained cells was adopted, since the procedure used for C-banding or fluorochrome staining can apparently change the chromosome size. Schwarzacher and Schweizer (1982), for instance, reported an increase in size of up to 50% for chromosomes of Cephalanthera cells after C-banding, in comparison with those stained with Feulgen, while Moscone et al. (1996) observed that Capsicum chromosomes stained with fluorochrome could be up to 1/3 larger than conventionally stained ones. On the other hand, Cyphomandra chromosomes treated with C-banding techniques were reduced to a third of the size of those stained with orcein (Pringley and Murray, 1993). In some cases, as in Arabidopsis thaliana and Arachis hypogaea, chromosome measurements made by other authors with conventional staining were used, since it was considered that any errors resulting from intraspecific polymorphism would be smaller than those arising from the measurement of a single cell. In any case, the chromosome size presented is just an approximation of the real size, used to separate the species into the three groups.
In this paper the position of the bands was classified as proximal when located in the proximal region or immediately after the primary constriction, as telomeric when located in the terminal regions of the chromosome, and as interstitial or intercalar when they occupied neither of the chromosome arm extremities. When the band occupied the whole chromosome arm, it was classified as telomeric or proximal depending on the dominant band pattern in the karyotype.
Concerning HC reactivity in relation to base-specific fluorochromes, the bands were classified as AT, GC or N (for neutral), depending on how they reacted with fluorochromes that preferentially stain DNA sequences rich in AT or GC. Species analyzed with the fluorochromes DAPI, quinacrine, Hoechst 33258 or CMA are indicated in Table I by the numbers 1, 2, 3 or 4, respectively, after the species name. The other species with an indication of main base composition but without a number were stained with CMA plus DAPI.
The bands associated with the NOR can involve the secondary constriction and/or the chromatin adjacent to this region, and for this kind of band the description in the papers was more important than photographs, since secondary constrictions have very variable expression and may not be expressed in that cell (Sato et al., 1980).
RESULTS AND DISCUSSION
In spite of the great variability known in HC distribution patterns, drastic and discontinuous changes within a related group of species are not common. In most of the genera in which data were available for at least five species, the number of bands and the HC amount varied but the general pattern was relatively well conserved, as in Anacyclus (Schweizer and Ehrendorfer, 1976), Secale (Bennett et al., 1977), Vigna (Zheng et al., 1991; Galasso et al., 1996b) and Citrus (Guerra, 1993a; Miranda et al., 1997). The diversity was much higher in genera with karyotypes exhibiting numerous interstitial bands, like Anemone (Marks and Schweizer, 1974), Scilla (Greilhuber, 1982), and Tulipa (Blakey and Vosa, 1982). Data from very different, non-angiosperm taxa, like some gymnosperm or bryophyte genera, also show similar tendencies (see Tanaka and Hizume, 1980; MacPherson and Filion, 1981; Newton, 1986a,b; Davies et al., 1997).
Considering Table I as a representative sample of angiosperms, the genera and species most frequently analyzed with C-banding techniques are those with large chromosomes, like Triticum, Gibasis, Allium, Scilla, etc. Most of these genera are monocotyledons, whereas those with small chromosomes and only a few studied species are mainly dicotyledons. The diploid number of the sampled species is quite representative of angiosperms, although very high numbers are not represented. No relationship between chromosome number and any HC characteristic was found.
Frequency of telomeric, interstitial, and proximal C-bands
Table II presents the frequency of species with telomeric, interstitial or proximal C-bands, in relation to chromosome size. In general, C-bands were slightly more frequent in the proximal region than in other regions. Considering only the karyotypes with HC restricted to a single chromosome region one can observe that it was found in 33 of the 105 species, 23 of which had proximal bands, although these numbers may contain some bias. The frequency of telomeric bands may be overestimated due to the common occurrence of NOR-HC in this region, which is not always identified with this technique and may be wrongly counted as common C-bands. On the other hand, the very reduced frequency of interstitial HC blocks in small chromosomes may be partly due to technical difficulties in localizing these bands on the arms of very small chromosomes.
The distribution of C-bands seems to depend at least partly on chromosome size. Proximal bands, in general, were more common in karyotypes with small chromosomes, decreasing in frequency with increasing chromosome size (Table II). Furthermore, karyotypes with only proximal bands were found in 11 of the 34 species with small chromosomes but in only 12 of the 71 species with large and medium-sized chromosomes, suggesting that this region develops or conserves C-bands more frequently.
The predominance of proximal bands in karyotypes with small chromosomes is more evident in the distribution of generalized equilocal bands, considered here as those bands occurring in the same region in at least 80% of the chromosomes of a karyotype. In general, such bands were less common in species with small chromosomes (Table III). However, among 24 species with small chromosomes and generalized equilocal bands, 21 showed generalization in the proximal region, three in the telomeric region and only one in the interstitial region. Such contrast is far less evident in species with medium or large chromosomes (Table III). Very often generalized proximal bands are largely conserved throughout a genus, as in Crepis (Ikeda, 1988) or Sesbania (Forni-Martins and Guerra, 1999). The frequencies of generalized bands in Table III are overestimated due to the a priori selection of species with the maximum number of bands in those genera with a single representative, although this bias was the same for every chromosome size class.
Generalized proximal HC has also been observed in several species only when using modified C-banding methods, as in onion (Fiskesjo, 1974; Cortés and Escalza, 1986), or after in situ hybridization, as in Vigna unguiculata (Guerra et al., 1996), or after sequential CMA/DAPI staining and C-banding, as in Cardiospermum grandiflorum (Hemmer and Morawetz, 1990). In some other species proximal C-bands have been observed only occasionally. Figure 1 illustrates the occurrence of occasional C-bands in Santolina (Asteraceae-Anthemidae). In two diploid species, S. rosimarinifolia and S. chaemacyparissus, only telomeric bands were found, whereas in a tetraploid, Santolina sp., telomeric and proximal bands were always observed (Figure 1a-c). However, in a single slide of S. rosimarinifolia, proximal bands were found in all chromosomes (Figure 1d). Such occasional results are certainly not rare, but usually go unreported. These data suggest that the occurrence of any detectable kind of proximal HC is much higher than that observed here, and it may be a common feature of all angiosperm chromosomes (see also Vosa, 1985).
Another interesting aspect is that the occurrence of generalized bands in the interstitial region was not only much lower but almost always accompanied by generalization in another region, since 15 of 20 species with generalized insterstitial bands also presented generalization in the telomeric or proximal region.
NOR associated heterochromatin
Secondary constrictions were not observed or not mentioned in only 14 of the 105 species of Table I. They are usually more difficult to visualize when they are very small or terminally located in the chromosomes (Guerra et al., 1997; Pedrosa et al., 2000). In general, the secondary constrictions were located in the short arm, close to the telomere, as observed by Lima de Faria (1976a), and the NOR-HC was located distally, occupying one or both sides of the constriction.
The NOR-HC is clearly differentiated from other HC types because it contains rDNA genes, although it may also contain repeated sequences not related to the NOR, such as the 120-bp repeat unit of rye (Cuadrado and Jouve, 1994). Analysis of rDNA site distribution by in situ hybridization shows that they are always present in the NOR and often in other regions where secondary constrictions have not been located. In Vigna unguiculata, for instance, only one or two chromosomes with secondary constrictions were observed, whereas 10 rDNA sites were revealed by FISH (Guerra et al., 1996), eight of them terminally located. NOR-HC often stains differently from other HC types (Sato et al., 1979; Guerra, 1988a) and in extreme cases it does not react positively after C-banding or fluorochrome staining, as in Tulbaghia pulchella (Vosa, 1970), Buglossoides purpurocaerulea (D'Amato et al., 1981), Cyprepedium segawai (Kondo et al., 1994; Hoshi et al., 1995), Lathyrus aphaca, L. tingitanus (Ünal et al., 1995) and Orchis coriophora (D'Emerico et al., 1996).
Bands observed with fluorochromes
The most commonly used fluorochromes were CMA and DAPI, and among the 58 species analyzed with fluorochromes in Table I, 44 were stained with this fluorochrome combination, almost always using the double-staining technique proposed by Schweizer (1976). In some cases, besides CMA and DAPI, other fluorochromes were also used as primary stains (Hoshi et al., 1995; Sato and Yoshioka, 1984) or as counterstains (Deumling and Greilhuber, 1982; Jamilena et al., 1990; Cuellar et al., 1996). Quinacrine was the third most frequently used fluorochrome (10 species), followed by Hoechst 33258.
Bands observed with fluorochromes were also positively stained after C-banding, although not all C-bands were differentiated by fluorochromes. In some species, however, a small number of fluorescent bands were found at sites where C-bands were not detected, as in Aconitum sanyoense (Okada, 1991) and Cestrum fasciculatum (Berg and Greilhuber, 1993). This fact, together with the apparent lack of NOR associated C-bands in some species, indicates once again that not all heterochromatin is detectable with the most usual C-banding techniques (see, Vosa, 1985).
The relationship between the intensity of the brightness of a fluorochrome stained chromatin and its base composition was originally established for cells not treated with C-banding or any other treatment promoting DNA denaturation-renaturation or preferential extraction of the euchromatin (Casperson et al., 1969; Schweizer, 1976; Sumner, 1990). However, some authors have used fluorochrome staining after the C-banding technique, obtaining bright bands which may not be an indication of AT- or GC-richness (Silva and Guerra, 1998). Bennett et al. (1995), for instance, after chromosome staining with quinacrine or DAPI of Zingeria biebersteiniana (n = 2), observed fluorescent bands only in the proximal region of one chromosome pair. Using C-banding plus the same fluorochromes, they observed proximal DAPI+ bands in both chromosome pairs and a terminal band in one of them, coinciding with Giemsa stained C-bands. These same DAPI+ bands were also observed after in situ hibridization. Vosa (1976a,b) observed that the NOR-HC of some species was positive for Hoechst and quinacrine after C-banding but negative for the same fluorochrome without pretreatment. Such fluorescent bands obtained only after C-banding were considered as neutral in Table I.
The most characteristic HC detected with fluorochromes was the NOR-HC, almost always CMA+ and DAPI-. The reaction intensity of CMA with this HC was variable. Most frequently it was more intense than with other GC-rich bands, but in Muscari comosum and Psygmorchis pusilla it was faintly stained or neutral. Interestingly, in Arabidopsis thaliana the NOR associated HC was apparently positive after staining with DAPI (Maluszynska and Heslop-Harrison, 1991; Ross et al., 1996), although its reaction with CMA is unknown. In this case, staining with CMA or mithramycin is important to verify whether or not a small CMA+ region exists in the secondary constriction itself or very close to it. Positive bands for DAPI or Hoechst, very close to the NOR, have been reported in several other species (Kenton, 1991; Cremonini et al., 1994).
Distribution of fluorochrome bands
Most species have at least one pair of CMA+ bands, since all of them have at least one pair of NORs (Moravetz, 1986a; Röser, 1994; Guerra et al., in press). In some species there are one or two pairs of CMA+ blocks at a very similar chromosome position to that of the NOR, although no secondary constriction is observed associated with them. This occurs in Hedera helix (König et al., 1987), Cicer arietinum (Galasso et al., 1996a) and Hypochoeris chillensis (Cerbah et al., 1995). However, after in situ hybridization with rDNA probes, these CMA bands are also often labelled, suggesting that it is the same type of HC (Galasso et al., 1996a; Cerbah et al., 1998).
In Table I, excluding the NOR bands, AT-rich blocks were found in 29 species, GC-rich in 19 species and neutral blocks in 23 species. Note that the total number of AT- or GC-rich sites per karyotype is not under consideration, but rather the number of species exhibiting at least one such site per chromosomal region. AT-rich sites were more frequently found at the interstitial region in species with medium and large chromosomes size (Table IV). Particularly in the case of bimodal karyotypes with long acrocentrics and small metacentrics or acrocentrics, interstitial bands were almost always AT-rich, as in some Brimeura species (Vosa, 1979), Fortunatia arida, Muscari comosum, Ornithogalum tenuifolium, Eleutherine bulbosa and Cephalanthera longifolia (Table I). A noteworthy exception was Nothoscordum fragrans, with interstitial bands that were positive for mithramycin and chromomycin A3 and negative for quinacrine (Sato and Yoshioka, 1984). Other karyotypes basically composed of long acrocentrics, like Anemone blanda and Vicia faba (Table I), were also characterized by interstitial AT-rich bands.
Among GC-rich bands, some were certainly non-identified rDNA sites, suggesting that the proportion of species with AT-rich HC is even higher. Nevertheless, in some taxa, GC-rich bands are very diversified and highly dominant or even exclusive, as in the genus Capsicum (Moscone et al., 1996), in the subfamily Aurantioideae (Guerra et al., in press) or in the family Lemnaceae (Geber, 1989).
Equilocal bands almost always stain in the same way with fluorochromes, suggesting that most of these bands are made up of the same or very similar repeats. Analyses in several organisms with in situ hybridization of specific satellite DNA fractions have shown that such repeats usually have equilocal distribution (Appels et al., 1978; Badaeva et al., 1996; Brandes et al., 1995). In Aegilops, for instance, a specific satellite sequence was observed in the terminal and subterminal regions of all species investigated, as well as in other chromosomal regions (Badaeva et al., 1996). In some species, two or more unrelated satellite DNAs have been observed occupying the same equilocal sites (Brandes et al., 1995), which may explain those sites that stain positively with different base specific fluorochromes.
The analysis of C-band patterns in a sample of over one hundred angiosperm karyotypes with different chromosome sizes showed that HC is not randomly distributed but rather that it is preferentially located. The NOR-HC is a notorious example, with a very marked distribution at subtelomeric sites. This HC type is very frequently, but not always, positively stained by C-banding techniques and by fluorochromes with high affinity for GC-rich chromatin. The remaining HC is mainly AT-rich and, regardless of its DNA base composition, it has a generalized equilocal distribution. In small chromosomes the bands are conspicuously preferentially located in proximal regions. Such generalized proximal bands may be related to the high frequency of prochromosomal nuclei in species with small chromosomes and low DNA content (Delay, 1949; Barlow, 1977; Guerra, 1987).
The bands seen here showed a tendency to be preferentially distributed in some specific chromosome regions, regardless of distance from the centromere. Thus, generalized telomeric C-bands were found in similar- and dissimilar-sized arms of a single karyotype, as in Paris tetraphyla (Miyamoto and Kurita, 1990), Aconitum sanyoense (Okada, 1991) and Fortunatia arida (Fernández and Daviña, 1991). Likewise, NOR-HC was terminally located in karyotypes with similar-sized arms, as in Allium subvillosum (Jamilena et al., 1990), or different-sized arms, as in Paeonia tenuifolia (Schwarzacher-Robinson, 1986). Generalized interstitial C-bands can be also equidistant, as in Anemone blanda (Marks and Schweizer, 1974) and Lathyrus tingitanus (Ünal et al., 1995), or, more frequently, occupy different positions in interstitial regions, as in several species of Lilium (Smyth et al., 1989) and Cyphomandra (Pringle and Murray, 1993).
One of the important assumptions in the heterochromatin dispersion model proposed by Schweizer and Loidl (1987) is that interstitial bands in long arms are equidistant to the terminal bands of short arms. Such symmetrical distribution was attributed to HC amplification and transposition occurring during mitotic interphase between non-homologous chromosomes showing Rabl orientation. In the present sample, such interstitial bands corresponding to non-homologous terminal HC were sometimes observed, although often along with several non-equidistant interstitial bands, as in Gibasis karwinskyana (Kenton, 1978, 1991) and Secale cereale (Mukai et al., 1992). Chromosomes of Allium subvillosum, for instance, exhibited telomeric bands in all short arms and interstitial bands in all long arms, but, according to the idiogram of Jamilena et al. (1990), the interstitial bands were located at distances almost always larger than the longest of the short arms. Actually, an exact equidistancing is not expected. Since different chromosome arms may have different condensation/decondensation patterns (Okada, 1975; Fukui and Mukai, 1988), the distance between one band and the centromere in non-homologous arms may be quite different in metaphase and in interphase.
These data suggest that generalized bands are not just an accident due to chromatin proximity of more or less equidistant non-homologous regions but most probably are due to the functional or structural similarity of these regions. The equilocal or equidistant chromatin "contamination" by the same type of repetitive DNA sequence may be facilitated, but not determined, by Rabl orientation. This assumption is supported by HC distribution in species with holocentric chromosomes, which are free from the influence of Rabl orientation. Karyotypes of Drosera (Sheik and Kondo, 1995, 1996) and Rhynchospora (Vanzela et al., in press), genera with holocentric chromosomes and belonging to widely different angiosperm families, exhibit HC distribution patterns similar to those with monocentric chromosomes. In both genera, generalized equilocal C-bands were found, which also gave identical fluorochrome reactions. In several species of Rhynchospora, Vanzela et al. (1998) observed the occurrence of multiple rDNA sites always distributed in the terminal regions. Similarly, satellite DNA sequences with generalized equilocal distribution were observed in the holocentric chromosomes of the peach potato aphid (Spence et al., 1998; Mandrioli et al., 1999). Therefore, HC equidistribution may not depend fundamentally on Rabl orientation and band-centromere distance, but rather on some structural or functional similarity of each chromosomal region that makes them equally receptive to the installation and/or amplification of the same or similar DNA sequence (Fry and Salser, 1977; Flavell, 1982).
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