Essay on the nucleoli survey by the α- and β-satellite DNA probes of the acrocentric chromosomes in mitogen-stimulated human lymphocytes

Abstract

The two constitutive heterochromatin (α- and β-satellite DNA) probes of human acrocentric chromosomes were essayed separately to label the nucleoli in the phytohemagglutinin (PHA)-stimulated human lymphocytes. Fluorescent in situ hybridisation (FISH) results have shown that: a) whole (100 %) signal–nucleoli overlapping was obtained with both heterochromatin probes in maximally activated nuclei (MANs); b) partial overlapping was observed in non-activated or slightly activated nuclei; c) random signal–nucleolus overlapping (background level) was found to be ∼6 % by the NOR-irrelevant euchromatic probe (D5S23); d) Yq–nucleolus association in the MANs was found to be ∼97 % without the subtraction of the background level. We concluded that: a) acrocentric α- or β-satellite DNA probes may be used as nucleolar markers only in the MANs and not in slightly activated or non-activated nuclei; b) the distances between rDNA loci and α-/β-satellite DNA on human acrocentrics are short enough to permit their observation on the same nucleolus.

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.