Original articleEssay on the nucleoli survey by the α- and β-satellite DNA probes of the acrocentric chromosomes in mitogen-stimulated human lymphocytes
Introduction
Nucleolar organiser regions (NORs) are loops of DNA, transcribed into ribosomal RNA (rRNA) which is processed into preribosomes in the nucleoli, and which ultimately becomes part of the mature ribosomes in the cytoplasm. Human NORs (rDNA) have been localised at the secondary constrictions of five pairs (numbers 13, 14, 15, 21 and 22) of the acrocentric chromosomes [12]. Human diploid cells contain 300–400 copies of rRNA genes [59], encoding 5.8s, 18s and 28s rRNA in block (45s RNA) and alternating with non-transcribing spacers [61]. Human acrocentric chromosomes also possess two constitutive heterochromatin blocks, α- and β-satellite DNA, near to rDNA repeats. However, little is known about the distances/junction sequences between rDNA and these neighbouring heterochromatins on the acrocentrics, even on the more studied chromosome 21 [60]. The fine structural studies of the nucleolus [9], [16], [28], [44], [53], [55], are not related to the specific position of these constitutive heterochromatins on, in or at the periphery of this organelle.
A chromosome or chromosome region may be seen as heterochromatic (in the condensed state, forming a chromocentre and therefore intensively stained) in interphase nuclei for one of three quite different reasons. 1) It is in a repressed state where, despite the fact that it includes coding DNA, that DNA is temporarily and reversibly inactivated: tissue-specific condensed euchromatin, the facultative heterochromatinisation of a given chromosome or chromosome sets. 2) It is composed of non-coding DNA and thus is permanently incapable of transcription: constitutive heterochromatin (for 1 and 2, see John [23]). 3) It is heterochromatinised in abnormal or forced condition: heterochromatin seen in some malignant cells [37], artificially heterochromatinised euchromatin due to DNA damage [8]. Most, if not all, of the constitutive heterochromatins are visualised in metaphase chromosomes by C-banding [50]. It appears to be late replicating and C-band positive (C+) on the prophase/metaphase chromosomes, to distinguish it from other chromatins. C+ heterochromatin has been classified as alpha (centromeric) or beta type [19]. Recently, it has been argued that C+ alpha (on the prophase chromosome) has tandem repeats, lacks genes and lacks chiasmata, while C+ beta lacks tandem repeats but has genes and has chiasmata [18].
Alpha-satellite DNA (C+ alpha) is a non-coding, tandemly repeated DNA family found at the centromeres (at the primary constrictions) of all human and primate chromosomes [56]. These tandem DNA repeats have been estimated as ∼3–5 % of the human genome [49].
Beta-satellite DNA (C+ beta) of five pairs of the acrocentric chromosomes is a repetitive non-coding DNA family that consists of ∼68-bp monomers, repeated in arrays of at least several hundred kilobases [13], [57].
Human lymphocytes offer a model for chromosome arrangement studies in the interphase nucleus [11], [52], [58]. They develop into large blast-like cells when stimulated by mitogens [1], [38], [45]. During this activation, the mostly single small ring-shaped nucleoli of the non-stimulated lymphocytes become double ring-shaped nucleoli, which in turn are transformed into larger nucleoli through the nucleolar association [54].
In the following, we report our findings on the acrocentric alpha-/beta-heterochromatic DNA–nucleolus overlapping in fully activated human lymphocytes at the optical level. The results have been compared with those obtained through some NOR-irrelevant heterochromatic and euchromatic probes.
Section snippets
Cell source, culture conditions and preparation of the specimens
Phytohemagglutinin-M (2.5 % v/v Difco 0528-57-5) stimulated peripheral blood lymphocytes from three healthy male volunteers (26–38 years old) were cultured in RPMI 1640 medium (Serva) supplemented with 25 % foetal calf serum (Serva), 10 mg streptomycin–10 000 U penicillin (Sigma)/100 mL at 37 °C with 5 % CO2, 95 % air for 72 h. Cells were treated with 25 μM/L colcemid (Sigma) for 2 h prior to hypotonic treatment with 0.075 M KCl for 8 min at 37 °C, and fixation was with three changes of 3:1
Results
AgNOR staining is the most reliable method of nucleolus identification in interphase nuclei [31], [46], [51] and for determining active NOR-bearing chromosome at metaphase [51]. Indeed, rDNA probes cannot discern active (transcribing)/inactive NORs in the cellular cycle [54] and inactive NORs do not form nucleoli [48]. Nevertheless, the silver-staining procedure and the FISH application on the same nucleus interfere reciprocally to some extent, following removal of the preceding stain ([54];
Discussion
Chromatin is not randomly distributed in the interphase nucleus [7], [17], [21], [35]. Chromosomes have been shown to occupy distinct and cell cycle-dependent territories [10], [11], [34] or chromosome domains [30], [41], including cell type [33] and probably tissue-specific arrangements [29]. The term ‘nucleolus’, does not explain an invariable content and structure of this organelle. Its composition and number change continuously according to the cell state in human lymphocytes [1], [54], in
Acknowledgments
This work has been supported by The Scientific and Technical Research Council of Turkey (SBAG-1604).
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