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Correspondence to: Laurent Héliot, Centre de Recherche de l'Hotel-Dieu, Quebec, Canada G1R-2J6.
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Summary |
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The metaphase nucleolar organizer regions (NORs) contain ribosomal genes associated with proteins such as upstream binding factor (UBF) and RNA polymerase I (RPI). These genes are clustered in 10 loci of the human acrocentric chromosomes (13, 14, 15, 21, and 22). Some NOR-associated proteins, termed AgNOR proteins, can be specifically stained by silver. In this study we took advantage of technical advances in digital imaging, image restoration techniques, and factorial correspondence analysis (FCA) to study the different AgNOR staining patterns of metaphase chromosomes in human lymphocytes. Three predominant patterns could be distinguished: pair (47%), stick-like (28%), and unstained (18%) structures. By studying the frequency of occurrence of each pattern on different chromosomes, two groups could be defined. Chromosomes 13, 14, and 21 carried predominantly pair or stick-like AgNOR structures, whereas chromosomes 15 and 22 mainly carried pair AgNOR structures or remained unstained. We suggest that the different AgNOR shapes reflect both the number of ribosomal genes carried by each chromosome and the differential recruitment of active ribosomal genes in each NOR cluster. This is the first study showing a nonrandom distribution of AgNOR shape among acrocentric chromosomes. (J Histochem Cytochem 48:1320, 2000)
Key Words: AgNOR, human acrocentric chromosomes, ribosomal gene activity, image deconvolution, multivariate analysis
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Introduction |
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AN EARLY DEFINITION of nucleolar organizer regions (NORs) described them as the morphological sites around which the nucleoli develop at the end of mitosis (
In situ studies have shown that metaphase AgNORs exhibit different morphologies independent of the cell type and fixation. Pair and stick-like AgNOR structures have been described with confocal microscopy (
In this study we addressed this question, basing our analyses on the shape of AgNORs and taking advantage of technical advances in digital imaging, image restoration, and analysis. Two groups of acrocentric chromosomes were distinguished according to the AgNOR structures that they preferentially carry. The biological significance of these results is discussed.
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Materials and Methods |
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Chromosome spreads were prepared from phytohemagglutinin (PHA)-stimulated peripheral blood by conventional cytogenetic techniques (
Preparations were observed under an epifluorescence microscope (Axiophot; Zeiss, Oberkochen, Germany) equipped with a Zeiss filter set 01 (BP 365, dichroic filter 395, LP 397) for DAPI fluorescence and by using transmitted light for AgNOR staining. The AgNOR and DAPI staining could be observed simultaneously or successively, using a x63, 1.25 NA oil-immersion objective and an intermediate magnification lens (Optovar; x1.25). Images were collected using a cooled CCD camera (C4880; Hamamatsu, Tokyo, Japan). To improve the quality of blurred images (caused by out-of-focus haze and glare), deconvolution was performed on gray level 16-bit images with image restoration software and according to procedures developed in our laboratory (
Factorial correspondence analysis (FCA) (
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Results |
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This study was carried out in 18 healthy donors (six women and 12 men) aged between 25 and 50 years. For each individual, 34 metaphases were analyzed, i.e., 68 occurrences per acrocentric chromosome (13, 14, 15, 21, and 22). Altogether, we analyzed 340 acrocentric chromosomes per donor and 1224 occurrences of each five acrocentric chromosomes. For the 18 donors, the number of AgNOR structures per metaphase ranges between 7 and 10 (mean modal value 8 ± 1) (Table 1). The number of AgNOR structures per metaphase varied within the same individual (Figure 1) and was characterized for each donor by the standard variations presented in Table 1. Figure 1 shows the distribution of AgNORs between the acrocentric chromosomes for 34 metaphases from donor P. Both chromosomes of pairs 13, 14, and 21 frequently carried an AgNOR (31 of 34). Quite often, only one homologue of pairs 22 (12 of 34) showed AgNOR staining. The observations for donor P were representative of the AgNOR distribution in the other subjects.
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A morphological analysis of AgNOR staining distinguished three patterns: stick-like, pair, and singlet structures (Figure 2), which were described by 3D electron microscopy in our previous work (
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Partial karyotyping was performed using DAPI banding and image deconvolution to study the relationship between the AgNOR shape and the identity of the labeled chromosomes. The distribution of acrocentric chromosomes into two groups according to their AgNOR structure was clearly emphasized by a multivariate analysis. The FCA (Figure 4) summarizes the distribution of the 6120 chromosomes in this study. The symbols correspond to the 68 observations (34 metaphases) of individual acrocentric chromosomes of each donor. For example, arrows indicate the chromosomes of a single donor (donor P, Table 1). The chromosomes are distributed between three major attractors corresponding to their AgNOR structures (i.e., stick-like or pair patterns, or unstained). The first factorial plane encompasses 93% of stress information (Figure 4; see Materials and Methods). The third factorial axis bears only 5% of stress information (corresponding to the atypical class) and consequently was not plotted. The FCA graph shows two groups (Figure 4). The first group, containing chromosomes 13, 14, and 21, spreads along a line between stick-like and pair patterns (Figure 4). The second group, composed of chromosomes 15 and 22, spreads along a line between unstained cases and pair patterns. Pair patterns are evenly distributed between acrocentric chromosomes (Figure 4). Table 2 summarizes data extracted from the multivariate analysis exposed as the distribution of AgNOR shapes on acrocentric chromosomes. The number of pair structures per chromosome ranges between 41 and 54 (average 47 ± 11%). In contrast, 96 ± 4% of the stick-like structures are carried by chromosomes 13 (53 ± 10%), 14 (32 ± 8%), and 21 (48 ± 13%). This structure is only found at 4 ± 3% on chromosome 15 and 1 ± 1% on chromosome 22. Most of the unlabeled chromosomes (93 ± 7%) are chromosomes 15 (46 ± 11%) and 22 (39 ± 15%). These data are summarized in Table 2. AgNOR patterns are dependent neither on the size of chromosomes (i.e., cytogenetic groups D and G) nor on the sex of the donor (Table 1).
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Discussion |
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Here we describe a new aspect of human AgNORs, the nonrandom location of differently shaped structures on the five NOR-carrying chromosomes. Eighteen healthy donors were included in this study and 6120 chromosomes were analyzed. The mean modal number of AgNOR structures per individual was 8, ranging from 7 to 10 with a very low SD (between 0.61 and 2.37) for each donor (Table 1). Our data are in agreement with the results of six previously published studies (Table 3) except for those of
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Two chromosome groups, independent of cytogenetic classes D and G and of donor sex, could be distinguished by multivariate analysis (Figure 4; Table 2). Chromosomes 13, 14, and 21, constituting the first group, preferentially carry stick-like and pair structures; on average, 96% of stick-like structures are borne by these chromosomes. The second group, containing chromosomes 15 and 22, preferentially exhibits pair structures or is unstained; this group contains 93% of the unstained chromosomes. Pair structures are most numerous (47%) and can be found on all acrocentric chromosomes, whereas stick-like structures and unstained cases are mutually exclusive (Figure 4; Table 3). Homologous chromosomes may carry AgNORs of different shapes, and the occurrence of singlet structures shows that sister chromatids do not always carry a similar AgNOR. These differences may reflect either rDNA deletion or variations in expression, a question that requires further studies of singlet structures.
During interphase, the size of AgNORs and the number of fibrillar components are correlated with the level of ribosomal gene activity after the stimulation of lymphocytes with PHA or cell fusion (
The transcriptional activity of ribosomal cistrons requires the binding of transcription factors to their promoters, and AgNOR staining could also be influenced by the number of initiated transcription complexes. Models to explain the differences between NOR shapes on homologous and nonhomologous chromosomes must take into account how, when, and to what extend the binding of transcription factors takes place. Each transcription factor (UBF and others) may bind randomly to ribosomal cistrons in all clusters, or once the first factors are bound the others may bind in a cooperative manner (
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Acknowledgments |
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We thank Drs R. Hancock and M. Thiry for their thoughtful discussions and suggestions, and Drs M. J. O'Donohue, D. Morel, and S. Stephan for revising the English style of the manuscript. We thank the reviewers for their suggestions, which significantly improved the quality of this manuscript.
Received for publication March 16, 1999; accepted August 26, 1999.
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Appendix |
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Factorial correspondence analysis (FCA) is basically a graphic representation of large contingency tables. A contingency table K is an array of p rows and n columns where an element kij (with 1<i<p and 1<j<n) contains the score of individuals of type i and bearing the character j. For example, it could be a table with 4 rows corresponding to the hair color of boys in a classroom (blond, black, brown, and red) and with 4 columns corresponding to the eye color (blue, brown, green, and black). An element of this array corresponds to the number of boys sharing the same hair color and the same eye color.
In a first stage, the K table is converted into a frequency table F by applying the following transform:
Let us consider that pn. In this case, the FCA consists in solving the following equation:
where t denotes transposition, I is the identity matrix, Ul are the latent roots or eigenvalues of the matrix, and Ul are the ancillary eigenvectors.
The projection of the row vectors in the factorial space defined by Ul and Ul are obtained as follows:
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