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Identification of Nucleolus Organizer Chromosomes in Cattle (Bos Taurus L.)

by Sequential Silver Staining + Rba Banding

Article  in  Caryologia -Firenze- · January 2014

DOI: 10.1080/00087114.1985.10797734

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Dino Di Berardino Leopoldo Iannuzzi

University of Naples Federico II Italian National Research Council
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Identification of Nucleolus
Organizer Chromosomes in
Cattle (Bos Taurus L.) by
Sequential Silver Staining + Rba
Banding
a a b
D. Di Berardino , M.B. Lioi & L. Iannuzzi
a
Animal Production Institute, Faculty of Agriculture,
University of Naples, 80055 Portici, Naples
b
CNR Institute on Adaptation of Cattle and Buffalo
to the Southern Italy Environment (I.A.B.B.A.M.), Via
Argine, Ponticelli, Naples, Italy.
Published online: 31 Jan 2014.

To cite this article: D. Di Berardino, M.B. Lioi & L. Iannuzzi (1985) Identification
of Nucleolus Organizer Chromosomes in Cattle (Bos Taurus L.) by Sequential Silver
Staining + Rba Banding, Caryologia: International Journal of Cytology, Cytosystematics
and Cytogenetics, 38:1, 95-102, DOI: 10.1080/00087114.1985.10797734

To link to this article: http://dx.doi.org/10.1080/00087114.1985.10797734

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 CARYOLOGIA Vol. 38, n. 1: 95-102, 1985

IDENTIFICATION OF NUCLEOLUS ORGANIZER

CHROMOSOMES IN CATTLE (BOS TAURUS L.)
BY SEQUENTIAL SILVER STAINING + RBA BANDING
D. DI BERARDINO, M.B. LIOI and L. IANNUZZI*
Animal Production Institute, Faculty of Agriculture, University of Naples, 80055 Portici, Naples,
* CNR Institute on Adaptation of Cattle and Buffalo to the Southern Italy Environment (I.A. B.
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B.A.M.), Via Argine, Ponticelli, Naples, Italy.

SUMMARY- A sequential «silver staining + RBA banding» procedure was applied

to cytological preparations from six individuals of the Friesian breed of Bos taurus L.
(2n = 60) for the identification of nucleolus organizer chromosomes. Chromosome
identification followed the Reading system. Of 10 sequentially stained metaphase
plates for each animal investigated, Ag-NORs were located on the telomeres of five
pairs of autosomes identified as nos. 2, 3, 6, 11 and 27, indicating that two of the five
NOR chromosomes in cattle have previously been misidentified.

INTRODUCTION

The cytological visualization of the 18S + 28S ribosomal cistrons in meta-

phase chromosomes has been demonstrated by a variety of techniques includ-
ing in situ hybridization (HENDERSON et al. 1972, 1974a and 1974b; EvANS et
al. 1974; PARDUE and Hsu 1975), N-banding (MATSUI and SASAKI 1973) and
silver staining (Ag-NORs) (GooDPASTURE and BLOOM 1975), in humans as well
as in other mammalian species, including domestic animals. In cattle (Bos taurus
L., 2n = 60) Ag-NORs have been previously located on the telomeres of five
pairs of autosomes identified, according to the Reading nomenclature (FoRD et
al. 1980) by Q-banding as nos. 2, 3, 4, 11 and 29 (DI BERARDINO et al. 1979)
and by G-banding as nos. 2, 3, 4, 11 and 28 (MAYR and CzAKER 1981; MAYR
and SCHLEGER 1982). HENDERSON and BRUERE (1979) assigned Ag-NORs to
chromosomes 2, 3, 4, 5 and 28 but without any confirmatory banding.
While these studies agree in the presence of five pairs of nucleolus
organizer chromosomes, discrepancies are evident in the identification of
individual pairs. Such differences may reflect individual variation in the

Supported by grant no. CNR CT82-02701-06 of the Research National Council, Rome, Italy.
 96 DI BERARDINO, LIOI and IANNUZZI

location of the NORs (Hsu et al. 1975) as well as limitations of the banding
techniques used.
In order to clarify the situation we decided to re-examine the identifica-
tion of the nucleolus organizer chromosomes in cattle by using the sequential
«silver staining + RBA banding» procedure, already succesfully applied to
other Bovidae (DI BERARDINO and IANNUZZI 1981; D1 BERARDINO et al. 1981).
With this technique we demonstrate that two of the five nucleolus organizer
chromosomes in cattle have been erroneously identified by earlier workers.

MATERIALS AND METHODS

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Peripheral blood, drawn from the jugular vein of six unrelated healthy Friesian
breed animals (2 males and 4 females), was cultured for 72 hours at 37° C in McCoy's
5A modified medium, supplemented with autologous plasma (20 per cent), antibiotic
and antimycotic Inixture, and Pokeweed Initogen. Seven hours before harvesting, 5 '-
BrdU was added to the cultures (20 ~J,g/ml, final concentration). Colceinid (0.05 ~J,g/ml,
final concentration) was added to the cultures 1.5 hours prior to harvesting. The cell
suspension was swelled in-hypotonic (0.075M KCl) for 20 minutes and fixed three
times in 3:1 methanol-acetic acid. Air dried slides were prepared soon after the fixation
which lasted 1.5 hours.

Sequential «silver staining + RBA banding>>. -The air dried cytological prepara-
tions were pretreated with Borate buffer (pH = 9.0) for 20 Ininutes, washed in
deionized water and air dried again. Three drops of filtered Ag-N0 3 solution (50%
w/v) were added to each slide and a coverglass added prior to incubating in a moist
chamber for 15-17 hours at 37° C. After the appearance of NORs the slides were
gently washed in deionized water, stained with acridine orange (1%) in Sorensen
buffer (pH = 7.0) for 20 Ininutes, washed again in deionized water, mounted in the
same Sorensen buffer and sealed with paraffin. To speed up RBA banding, the slides
were exposed to UV light for 4-6 hours.
At lest 10 metaphase plates for each animal exainined were selected and photo-
graphed following visualization of NORs with bright field optics and according to the
quality of the RBA banding pattern seen under UV light. The photoinicrographs were
taken with Kodak Microfilm Recordak 5786 and printed on Kodabroinide paper no. 3.

RESULTS AND DISCUSSION

Fig. 1 shows a metaphase plate of a bull (2n = 60, XY) sequentially stained
for NORs visualization (1A) and RBA-banding (lB). The Ag-NORs arrowed in

Fig. 1. - Metaphase plate of a bull (2n = 60, XY) sequentially stained for NORs visualization (A) and
RBA banding (B). Arrows indicate, respectively, Ag-NORs and the corresponding nucleolus organizer
chromosomes. The large arrow points to an association between two chromosomes.
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 98 DI BERARDINO, UOI and IANNUZZI

Fig. lA are carried, in this particular cell, by the six nucleolar chromosomes
indicated in Fig. lB. From this metaphase plate, the RBA banded karyotype
shown in Fig. 2A has been prepared on the basis of the RBA banded karyotype
previously proposed by the authors (DI BERARDINO and IANNUZZI 1982), which
was arranged according to the G-banded «standard» karyotype of the Reading
Conference. Cut out nucleolus organizer chromosomes from the same karyo-
type have been arranged, side by side, in Fig. 2B, and identified as follows:
both homologues of pair no. 2, one chromosome no. 3, one chromosome no. 6,
and both homologues of pair no. 27 in association. Nucleolar chromosome 11,
shown in Fig. 2C, is taken from another cell of the same animal.
Figs. 3A-B show a nucleolar association between three chromosomes
identified by RBA banding as nos. 3,11 and 27; other associations are shown in
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Figs. 3C-D-E to occur between chromosomes 6-11, 2-6 and 6-27, respectively.
The present results confirm the presence of telomeric NORs on chromo-
somes 2,3 and 11, but demonstrate that NORs ate carried by chromosome 6
instead of 4, and by chromosome 27 instead of either 28 or 29. The possibility
of individual variation in the location of the NORs can be ruled out since in all
six animals examined in this study the Ag-NORs were consistently located on
chromosomes 2, 3, 6, 11 and 27.
The misclassification between chromosome 4 and 6, as well as between
chromosomes 27 and 28 or 29 is, in our opinion, mainly due to the banding
techniques previously used. Cattle cytogeneticists, in fact, know that quite
exceptionally Q or G banding in this species can provide an unambigous
banding pattern, especially· when contracted chromosome preparations are
used, whereas the RBA technique, which involves acridine orange staining
after late BrdU incorporation (DuTRILLAUX et al. 1973) has been foud to give
improved resolution and reliability. It is, therefore, to be preferred for the
detailed description and definite identification of individual metaphase chro-
mosomes of domestic animals (PoPEscu 1975; GusTAVSSON and HAGELTORN
1976; D1 BERARDINO et al. 1981; D1 BERARDINO ~tnd IANNUZZI 1982). Since the
Q or G banding pattern of chromosome 4 is very similar to that of chromosome
6 it is possible, in less than perfect preparations, to be misled in their
identification. By using RBA banding this ambiguity can be avoided: in fact,
while chromosome 6 shows a uniform banding pattern, chromosome 4 shows
three brilliant regions, one proximal, one central and one distal; furthermore,
when Q or G banded, chromosome 27 can also be confused with chromosome

Fig. 2. - (A) RBA banded karyotype prepared from the same metaphase shown in Fig. lB according
to the Reading system. - (B) identification of the nucleolus organizer chromosomes nos. 2 (both
homologues), 3,6 and 27 (in association) from the same karyotype. Notice the large block of silver
material on the telomeres of chromosome 6. - (C) nucleolar chromosome 11 taken from another cell of
the same animal.
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 100 DI BERARDINO, UOI and IANNUZZI
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Fig. 3.- Nucleolar ~ssociations between chromosomes 3, 11 and 27 (A-B), 6-11 (C), 2-6 (D) and 6-27
(E).

28 or 29, especially when contracted preparations are used. The unique RBA
banding pattern of chromosome 27 (four positive bands equally distributed)
makes this chromosome easily recognizable among the others.
Further details of individual RBA banded chromosomes in cattle are
reported in DI BERARDINO and IANNUZZI 1982).
The fact that Ag-NORs in cattle are located on chromosome 6 rather than
on 4, and on chromosome 27 instead of either 28 or 29, necessitates a
reconsideration of the cytotaxonomic relationships between cattle and buffalo
(Bubalus bubalis L.) previously reported (DI BERARDINO and IANNUZZI 1981; DI
BERARDINO et al. 1981). By using the same sequential «silver staining + RBA
banding» procedure, telomeric NORs have been located in river buffalo
(2n =50) on chromosomes 3p, 4p, 8, 21, 23 and 24, and in the swamp buffalo
(2n = 48) on chromosomes 4p, 8, 20, 22 and 23 (six and five pairs of NORs
respectively). These chromosome pairs are homologous in banding pattern to
cattle chromosomes 19, 28, 6, 23, 26, 27 and 19, 6, 23, 26, 27, respectively, as
defined by the Reading system and by the RBA banded karyotype previously
 NUCLEOLUS ORGANIZER CHROMOSOMES IN CATTLE 101

proposed by the authors (DI BERARDINO and IANNUZZI 1982). Since nucleolar
chromosomes 8 and 24 in the river, as well as chromosomes 8 and 23 in the
swamp buffalo, correspond to chromosomes 6 and 27 in cattle, we may
conclude that during the divergence of the genus Bubalus from the cattle
ancestor, two nucleolus organizer chromosome pairs have been conserved.
The adoption of a common system of nomenclature, such as the Reading
system, and the use of the RBA technique for identification and detailed
description of individual chromosomes in domestic animals are highly recom-
mended in order to avoid misclassifications of chromosomes involved in
numerical or structural rearrangements, as well as misinterpretations when
evolutionary relationships among breeds or species have to be traced.
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 102 DI BERARDINO, UOI and IANNUZZI

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Received 5 April 1984; revision accepted 24 July 1984

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