rDNA and acrocentric chromosomes in man

Cell Differentiation 2,319-324 (1974).. © North-Holland Publishing Company

rDNA AND ACROCENTRIC

CHROMOSOMES

IN MAN

G. GUANTI and P. PETRINELLI Institute of Genetics, University o f Bari, Via Amendola 165//A, Bari, Italy Accepted 30 November 1973

DNA extracted from human subjects who are carriers of different numbers of nucleolar organizer regions (NOR) was hybridized with 3H-rRNA. The subjects with 21 and 13 trisomy, carriers of 11 NOR, show an increase of 10% in the amount of DNA complementary to rRNA; the mongol patients with 14/21 or 21/22 translocation show levels lower than normal; phenotypicaUy normal subjects, carriers of D/D or D/G translocation, show a decrease of 20% in the amount of rRNA bound per unit DNA. The experiments reported confirm that all acrocentric chromosomes carry NOR and that all rDNA is located on these chromosomes. It is probable that the number of rRNA genes, although a polymorphic character, is an inheritable attribute of given nucleolar organizer.

The genes for ribosomal RNA occur in multiple copies in the genome of all eukaryotic organisms so far studied and are located in the nucleolus organizer regions (Ritossa et al., 1965; Wallace et al., 1966). In man, cytological data (Ohno et al., 1961; Ferguson-Smith et al., 1963; Ferguson-Smith, 1964) indicated that the secondary constrictions of acrocentric chromosomes seem to be nucleolus organizers as shown by their association with nucleoli. Such indications were confirmed by other types o f investigations: a) fractions of metaphase chromosomes, enriched for smaller chromosomes, were shown to contain a proportionally increased amount of rDNA (Huberman et al., 1967); b) cytological hybridization using 3 Hlabelled ribosomal RNA on slides resulted in specific labelling of the satellite regions of acrocentric chromosomes (Henderson et al., 1972); c) clonal derivatives of a human heteroploid cell line showed different rDNA contents as a function of the relative number of D and G chromosomes (Spadari et al., 1971). Such results confirm the idea that human rRNA genes are located exclusively on the ten acrocentric chromosomes and that all these chromosomes contain a consistent amount of rRNA genes. In an attempt to determine the size and any possible difference in the number o f rRNA genes between D and G chromosomes we have examined human subjects affected by numerical and structural aberrations involving these chromosomes.

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rDNA and acrocentric chromosomes in man

MATERIAL AND METHODS Extraction o f DNA

DNA was extracted by a modification of the procedure described by Marmur (1961). Venous blood (20-25 ml) was collected using EDTA 0.11 M pH 7.5 as anticoagulant; 6 ml of gelatin (a 3% solution w/v in 0.15 M NaC1) were added and thoroughly mixed. Tile diluted blood was then left standing at a temperature of 37°C for 30 min in a siliconized tube; during this time the mixture separated into 2 visibly distinct layers. The whole of the upper layer, freed from the bulk of the red cells, was centrifuged at 1500 rpm for 10 min. The lymphocytes, sedimented as a small pellet, were lysed with the following buffer at pH 8.1: disodium EDTA (0.1 M), NaC1 (0.15 M), SDS (0.4%). NaC104 (5 M) was added to the lysate to a final concentration of 1 M. Deproteinization was obtained with 3 extractions using cldoroform-isoamyl alchool (24: 1). After precipitation with cold ethanol, DNA fibers were collected, dissolved in 1 X SSC and digested for two hours at 37°C with RNase A (bovine pancreatic from Sigma (200/ag/ml) and amylase (pancreatic from Merck (100/lg/ml)). Preincubated pronase B grade (Calbiochem, lO0/ag/ml) was then added and incubation continued for 1 hr. The solution was then made 0.4% in SDS and the enzymes removed with 3 to 4 phenol extractions followed by successive deproteinization with chloroform-isoamyl alchool. DNA was precipitated by addition of 2 vol of cold ethanol and the fibers collected and dissolved in 0.1 × SSC. The yield of DNA was a function of the number of white cells of the sample as well as of the age of the subject and ranged from 24 to 50 ~g per ml blood. Labelling and extraction o f R N A

HeLa cells were grown in Eagle's medium, supplemented with 10% serum, in the presence of 3H-uridine ( 2 0 - 3 0 C/mM, Amersham) for 48 hr at 37°C and then chased for 20 hr by adding 10-s M cold uridine. The RNA was extracted from the supernatant of homogenized cells, by repeated treatments with phenol in the presence of 0.4% SDS. The final product was precipitated by addition of 2 vol of a mixture of ethanol-0.02 M Na acetate pH 5.9 (9 : 1). Total RNA was dissolved in a buffer (0.4 M NaC1; 0.05 M Tris pH 6.8) and fractioned on a methylated albuminKieselghur (MAK) column as described by Mandell et al. (1960). 18 S and 28 S rRNA elute in a single peak at 0.7 M NaC1. The fractions containing rRNA were pooled and dialyzed against 100 vol of 2 X SSC. Hybridization

The rRNA-DNA hybridization was made with DNA immobilized on nitrocellulose filters according to the technique of Gillespie and Spiegelman (1965). The nitrocellulose filters, first soaked in 6.6 X SSC, were loaded with about 50/ag of DNA per filter. Hybridizations were made in 2 X SSC at 60°C for 12 hr. Each incubation mixture contained also 2 pg of cold mRNA (gift of Dr. Spadari, Pavia).

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The mRNA, extracted from EUE cells and purified by absorption to and elution from a benzoylated DEAE cellulose column (Sedat et al., 1967, 1969), allowed us to obtain flat plateaux even at high 3 H-rRNA/DNA ratios (Spadari et al., 1972). At the end of the hybridization period, the filters were washed several times with 2 × SSC and treated with RNase (20/ag/ml) in 2 × SSC with gentle shaking for 1 hr at 30°C.

Source of DNA Different groups of human subjects were used in the present study to prepare DNA containing various numbers of nucleolus organizer regions (NOR). In the case of normal subjects and 21 trisomic patients, particular attention was devoted to avoiding the presence of.polymorphisms (p+; p - ; s+; s-) of the short arm of acrocentric chromosomes. The groups analyzed were the following: 1) normal subjects (46,XY or 46,XX), who have ten chromosomes with NOR (6 D + 4 G); 2) mongoloid subjects (47,XY,21+ or 47,XX,21+), who have 11 chromosomes with NOR (6 D + 5 G); 3) subjects with Patau's syndrome (47, XX, 13+), who have 11 chromosomes with NOR (7 D + 4 G); 4) mongoloid subjects with D/G translocation (46,XX,t(14q21q)+, 14-), who have 9 NOR (5 D + 4 G); 5) mongoloid subjects with G/G translocation (46,XY,t(21q22q)+, 22-), who have 9 NOR (6 D + 3 G); 6) phenotypically normal subjects, carriers of D/D translocation (45,XY,t(13q 14q)+, 13-, 14-), who have eight chromosomes with NOR (4 D + 4 G); 7) phenotypically normal subjects, carriers of D/G translocation (45,XX,t(14q2 lq)+, 14-, 21-), who have 8 chromosomes with NOR (5 D + 3 G). The mechanisms involved in the chromosomal aberrations observed in the subjects of groups 4, 5, 6 and 7 has generally been regarded as a centric fusion of the entire long arms of two acrocentric chromosomes preceded by breakage on opposite sides of the centromeres (Robertson, 1916). The rearrangement results in the loss of one centromere and of the two short arms with their nucleolar organizer regions. More recently, with the current banding methods and the special centromeric staining technique, it has been possible to demonstrate that a centric fusion-like translocation might be the result of a reciprocal translocation (Niebuhr, 1972; Hsu et al., 1973). The presence of dicentrics in several cases of robertsonian translocations seems to indicate, according to Niebuhr (1972), that the breaks in the short arms of both acrocentric chromosomes are more frequent in man than has been previously recognized. No cytological evidence, which clearly indicates the persistence of secondary constrictions, are reported.

RESULTS AND DISCUSSION The saturation curves of fig. 1 show that the amount of DNA complementary to

322

rDl~M a~td acrocentric chromosomes h~ man

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~Ug of 3H-RNA Fig. 1. Saturation curves of hybridization experiments between DNA of different groups of subjects and 3H-rRNA. About 50/~g of DNA were loaded on nitrocellulose filters and incubated with increasing quantities of 3H-rRNA (1-6/~g/ml). r R N A is different in the different groups o f subjects examined. The results are summarized in table 1. In normal subjects, 0.029% o f D N A hybridized to r R N A . This value is in reasonable agreement with those reported by H u b e r m a n and Attardi (1967) in H e L a cells (0.036%) and Bross et al. (1972, 1973) in different h u m a n Table 1 Karyotype

No. of NOR

% DNA hybridized mean value

Relative hybridized value

No. of subjects examined*

Range of values

46, 46, 47, 47, 47, 46, 46, 46,

10 10 11 11 11 9 9 9 8 8

0.0290 0.0291 0.0317 0.0316 0.0320 0.0260 0.0259 0.0261 0.0229 0.0233

100 100 109.3 108.9 110.5 89.6 89.3 90 78.9 80.3

10 10 10 10 2 3 5 1 3 9

0.0284-0.0299 0.0285-0.0298 0.0310-0.0326 0.0308-0.0322 0.0316 0.0328 0.0252--0.0266 0.0254 0.0262 0.0260-0.0262 0.0228-0.0232 0.0230-0.0239

XX XX XY, 21+ XX, 21+ XX, 13+ XY, t(14q21q)+, 14XX, t(14q21q)+, 14XY, t(2 lq22q)+, 2 2 45, XY, t(13q14q)+, 13-, 14 45, XX, t(14q21q)+, 14-, 2 1 -

* For each sample 1-7 experiments were made.

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tissues (0.030%; 0.026%). The trisomic subjects with 11 NOR carry an amount of DNA complementary to rRNA which is higher than normal. The DNA from those patients with 21 trisomy show saturation levels (0.0317%) which are only slightly lower than those observed with subjects affected by 13 trisomy (0.0320%). These saturation levels are corrected for DNA content; the corrections are made assuming that the presence of a supernumerary G chromosome and a supernumerary D chromosome gives an increase in the amount of nuclear DNA of, respectively, 0.8 and 1.8% (DuPraw, 1970). There are two kinds of carriers of translocation in phenotypically normal subjects: the carriers of D/D translocation show a decrease of 21% in the amount of rRNA bound per unit DNA (0.0229%) and the carriers of D/G translocation contain slightly more rDNA (0.0233%) than the D/D. These data confirm those previously reported (Guanti et al., 1973) which were obtained using rRNA of Cricetus hamster for the hybridization experiments. The mongol patients with 14/21 or 21/22 translocation, who are carriers of 9 NOR, show similar (0.0260%; 0.0261%) saturation levels which are always lower than normal. The experiments reported here are consistent with previous reports which indicate that all acrocentric chromosomes carry NOR and that all the DNA complementary to rRNA is located on these chromosomes. In fact, the presence of one supernumerary D or G chromosome is paralleled by an increase of about 10% of rDNA while, in the absence of one or two acrocentric chromosomes, one observes a decrease of, respectively, 10% and 20% of the content of rRNA genes. In order to verify the validity of this assertion, we made saturation curves using equal amounts of DNA from mongoloid subjects with 21 trisomy and 21/22 translocation. The saturation curves in this case overlapped those of normal subjects. The number of genes for rRNA, in each of the ten acrocentric chromosomes, seem quite similar, though in some cases the components of the D group seem to exhibit slightly higher values. Minor differences are also observed in the various DNA obtained from normal (46,XX or 46,XY) donors, while in our experience the carriers of any translocation within the same family show identical saturation plateaux. Bross et al. (1973) analyzed 4 D/G translocation carriers. Two of these showed saturation plateaux significantly lower than normal; the other two showed almost normal values. The authors consider the possibility that the divergence observed might be correlated with the mono- or dicentric status of the respective translocation. The number of NOR lost in the case of dicentric translocation could vary between zero and two with the possibility of partial loss. In our cases there was no cytological evidence of the presence of dicentric translocations, and the molecular data seem to be in accordance with the cytological observations. Bross et al. (1973) reported also that three of six subjects with 21 trisomy presented an increase of 10% in the amount of rDNA, two showed an increase that

324

rDNA and acrocentric chromosomes in man

exceeds the expected values and one had a normal saturation value. We did not find so wide a range of variations, perhaps because the group of patients examined was homogeneous regarding morphological characteristics of the short arm of satellited chromosomes. We think, however, that it is possible to find appreciable differences in the content of rDNA genes within normal subjects too, as a function of the polymorphism of the short arm of acrocentric chromosomes. This might be due to the often reported occurrence of unequal exchanges within rRNA genes (Ritossa et al., 1966; Schalet, 1969). Accordingly, when observed values are different from expected, they are significant only if compared with those of the parents. This is because the number of rRNA genes, although a polymorphic character, is expected to be an inheritable attribute of a given nucleolar organiser.

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