Vol. 53, no. 3-4: 213-218, 2000
CARYOLOGIA
Chromosomal characterization of three centric fusion
translocations in cattle using G-, R- and C-banding and
FISH technique
G.P. DI MEO1,L. MOLTENI2, A. PERUCATTI1, A. DE GIOVANNI2, D. INCARNATO1, G. SUCCI2,
L. SCHIBLER3:, E :P . CRIBIU3 and L. IANNUZZI1'"
1
National Research Council (CNR), IABBAM, Naples, Italy;2 Institute of Animal Science, Agricultural Faculty of Science, University of Milan, Milan, Italy;3 Research Centre, INRA, Laboratory of Genetics and Cytogenetics, Department of Animal
Genetics, Jouy-en-Josas, France.
Abstract — Five cattle, two of the Podolian breed, two Grey Alpine, and one Chianina, all of which were heterozygous carriers of three centric fusion translocations
(rob-l;29, rob-4;8, rob-25;27), underwent cytogenetic investigation. The use of G, R- and C-banding patterns, combined with the FISH technique by using both type
I molecular markers and some human chromosome painting probes, allowed the
precise identification of chromosomes involved in the chromosomal abnormalities,
compared to previous reports. While the chromosomes involved in the well known
rob (1;29) were confirmed, BTA6 (not BTA4) was involved in the second
translocation. Furthermore, BTA26 and BTA29 (not BTA25 and BTA27) were
involved in the third translocation. C-banding patterns confirmed the mono-centric
nature of rob (1;29) and revealed the dicentric nature of both rob(6;8) and rob
(26;29). The importance of these marker chromosomes in bovid chromosome
nomenclatures is also discussed.
Key words: banding, cattle, chromosome, FISH, translocations
INTRODUCTION
Cattle autosomes are all acrocentric. Standard
karyotypes at metaphase (READING CONFERENCE
1980) and prometaphase (ISCNDA89 1990)
level are available and molecular markers (and
bovine syntenic groups) have been assigned to
each cattle chromosome (TEXAS SYSTEM 1996).
However, changes and errors were made in the
position of several chromosome pairs when
comparing the different nomenclatures (4-6
among the largest chromosomes, 25- 27-28-29,
among the smallest ones, see review in
IANNUZZI 1996). Furthermore, the TEXAS SYSTEM
(1996) did not indicate to which G-
* Corresponding author: fax ++39-081-5965291; e-mail:
[email protected]
and R-banded standard chromosomes (READING
CONFERENCE 1980; ISCNDA89 1990) the bovine
syntenic groups U7, U8 and U25 map. The use
of marker chromosomes, combined with both
banding techniques and molecular markers
assignments, can easily resolve these
nomenclature discrepancies and precisely iden tify
chromosomal abnormalities.
In this study, by using G-, R- and C-banding
techniques, as well as type I markers and some
human chromosome painting probes, we definitively identify and better characterize the
chromosomes involved in three centric fusions
translocations earlier investigated: the well
known rob(l;29), widely found throughout the
world (GUSTASSON 1969; POPESCU and PECH 1990),
the rob (4;8) found in the Chianina breed (DE
GIOVANNI et al. 1988; BOUVET et al. 1989) and the
rob(25;27) found in the Grey Alpine breed (DE
GIOVANNI et al. 1979).
214
DI MEO, MOLTENI, PERUCATTI, DE GIOVANNI, INCARNATO, SUCCI, SCHIBLER, CRIBIU and lANNUZZI
Fig. 1 —Details of rob(l;29) (A-B), rob(6;8) (C-D) and rob(26;29) (E-F) treated according to GBG (left) and RBG (right) banding
techniques. All preparations were printed at the same magnification. Bar=10 µm.
MATERIALS AND METHODS
Two Podolian cows, heterozygous carriers of
rob(l;29), a cow of the Chianina breed, heterozygous
carrier of both rob(l;29) and what previously called
rob(4;8) and two Alpine Grey bulls, heterozygous
carriers of what previously designated rob(25;27),
were used for this study.
Concavalin A stimulated peripheral blood lymphocytes were cultured for three days in McCoy's
modified medium and treated for early (IANNUZZI et al.
1989) and late-BrdU incorporation to obtain G-and
R-banding patterns, respectively. Slides were treated
for GBG- and KBG-banding techniques (IANNUZZI
1996), while only R-banded preparations were used for
FISH-technique. CBA-banding was
215
CENTRIC FUSIONS IN CATTLE
achieved by using SUMNER'S (1972) protocol and
acridine orange staining (DI MEO et al. 1996).
As probes, goat BAC-clones mapping the following
type I markers (SCHIBLER et al. 1998) were used: CP
(BTA1, bovine U10); COX8 (BTA29, bovine U7),
GNRHR (BTA6, bovine U15), CTSLL (BTA8,
bovine U18),ACTA (BTA26, bovine U26). As painting
probes, HSAl0 and HSA11 (painting kit from
Cambio, England), which paint BTA26 and BTA29,
respectively (TEXAS SYSTEM1996), were also used.
Chromosome identification followed cattle G- an
R-banded karyotypes (IANNUZZI 1996) and the TEXAS
SYSTEM(1996).
RESULTS AND DISCUSSION
Chromosome Identification
Fig. 2 — Details of rob(l;29) (a-b), rob(6;8) (c-d) and
rob(26;29) (e-f) treated for simultaneous visualization of RBA banding and FISH-mapping with CP (BTA1), COX8 (BTA29),
GNRHR (BTA6), CTSLL (BTA8) and ACT A (BTA26). The
arrows indicate the sites of hybridization signals. Notice the
FITC-signals with COX8 on both telomeric regions of p-arms of
rob(l;29) (b) and rob(26;29 (f). Bar=10 m.
Figure 1 shows details of G-, R-banded
preparations of the three centric fusion translocations. The analysis showed that BTA 1 and
BTA29 (rob 1;29), BTA6 and BTA8 (rob 6;8),
BTA26 and BTA29 (rob 26;29) were involved.
The use of molecular markers confirmed our
chromosome identification (Figure 2). Indeed,
CP and COX8 map on q- (BTAl) and p-(BTA29)
arms of rob(l;29), respectively (Figure
Fig. 3 — Details of rob(26;29) sequentially treated for RBH- (left) and RBA- (middle) banding and FISH-technique (right) with
HSAlO (c) and HSAll (f) painting probes mapping on BTA26 and BTA29, respectively. Bar=10 µm.
216
DI MEO, MOLTENI, PERUCATTI, DE GIOVANNI, INCARNATO, SUCCI, SCHIBLER, CRIBIU and IANNUZZI
Fig. 4 —Details of rob(l;29) (a-d), rob (6;8) (b-e) and rob (26;29) (c-f) treated according to CBA- (top) and sequential GBG/CBAbanding (bottom) techniques. Notice the large C-band present in q-arms of rob(l;29) (a) and the two HC-blocks in both rob(6;8) (b)
and rob(26;29) (c). The application of sequential GBG/CBA-banding techniques reveal the loss of chromosomal material from the
proximal p-arm region of rob(l;29) and the presence of a larger chromosomal region in proximal q-arm region of rob(l;29) when
compared with normal chromosomes 29 and 1, respectively (d). While both rob(6;8) (e) and rob(26;29) (f) reveal their dicen-tric nature
when comparing the HC-blocks of the two translocations with those found in normal chromosomes 6/8 and 26/29, respectively.
Arrows indicate primary constrictions in all centric fusions. Bar=10 m.
2A-B); GNRHR and CTSLL map on q-(BTA6)
and p- (BTA8) arms of rob(6;8), respectively
(Figure 2C-D); ACTA and COX8 map on q(BTA26) and p- (BTA29) arms of rob(26;29),
respectively (Figure 2E-F). A FISH-mapping
control with the markers HBA (BTA/CHI25,
bovine U8) and ANK (BTA/CHI27, bovine
U25) gave negative signals in both arms of
rob(26;29). Furthermore, the use of HSA10 and
HSA11 painting probes showed clear positive
signals on rob(26;29) q- and p-arms, respectively
(Figure 3), in agreement with the TEXAS SYSTEM
(1996).
According to the present data, while the
identification of chromosome 1 and 29 was
confirmed in rob(l;29), BTA6 (not BTA4)was
involved in the second translocation (rob6;8).
The chromosomes involved in the small
translocation of Grey Alpine cattle are 26 and
29, not 25 and 27 as previously reported (DE
GIOVANNI et al. 1979). This different result can
in part be explained by the poor banding
techniques available at that time (DE GIOVANNI et al. 1979). Since rob (26;29) has been
found in several animals of Alpine Grey breed
(DE GIOVANNI et al. 1979), it is interesting to
note that the same small chromosome is involved in both rob(l;29) and rob(26;29)p-arms.
CENTRIC FUSIONS IN CATTLE
C-banding
While rob(l;29) was monocentric (Figure
4a), as demonstrated in other studies (!ANNUZZI
etal. 1987, 1992), both rob(6;8) and rob(26;29)
were dicentric (Figure 4b-c). This accounts for
an ancient origin of rob(l;29) and recent origin of
both rob(6;8) and rob (26;29). Indeed, dicentric
translocations are unstable until one of two
centromeres is lost or inactivated. The dicentric
nature of rob(6;8) agrees with the complete synapsis of trivalent figures found at meiosis ( BouVET et al. 1989). Sequential GBG/CBA-band- ing
techniques (Figure 4d-f) revealed that: (a)
chromosomal material (mostly heterochromatin) was lost from the proximal p-arm region of
rob(l;29) when compared with normal chromosome 29, and larger chromosomal material
was observed in the proximal q-arm region of
rob(l;29), when compared with normal chro
mosome 1 (Figure 4d); (b) HC was retained in
both arms of both rob(4;8) and rob(26;29) when
compared with normal chromosomes 6/8 and
26/29 (Figure 4e-f), confirming the dicentric
nature of these two translocations, although
rob(26;29) was reported to be monocentric by
DE GIOVANNI et al. (1979). According to these data,
while the HC was conserved in the centromere of
BTA29 from rob(26;29) p-arms, that of BTA29
from rob(l;29) was apparently lost (Fig ure 4), as
also reported in previous studies (IAN-NUZZI et al.
1987, 1992), although a pericentric inversion
seems to have occurred during or af ter the centric
fusion event (EGGEN et al. 1994). Indeed, a
cosmid (INRA143) has been clearly FISHmapped to both pericentromeric regions of the
free BTA29 and q-arms of rob (1;29) (EGGEN et
al. 1994). The larger chromosome region found in
the proximal q-arm region of rob(l;29) (Figure
4) is in agreement with this finding. Therefore,
it is possible that part of BTA29 chromosomal
material apparently lost from p-arm of rob(1;29)
(Figure 4), has been retained in the proximal qarm region of rob(l;29) by the hypothesized
pericentric inversion (EGGEN et al. 1994).
Further studies on more animals from several
breeds are necessary to better understand this
phenomenon occurring in the most important
abnormality of cat-tie.
Since cattle chromosomes 4, 6, 25, 27, 28
and 29 (and goat and sheep homologues) have
been miss-class-classified during the chromo-
217
some standard nomenclatures (see review in
IANNUZZI 1996), the G- and R-banding patterns of
these three centric fusion translocations,
combined with their physical assignments of
type I markers, will be very useful for a defini tive
and clear construction of G- and R-banded
standard karyotypes for domestic bovids. Furthermore, chromosome painting probes produced from these chromosome arms will be very
useful in further investigations even when using
contracted chromosome preparations. When
cattle chromosome painting probes are
commercially available, as for human chromosomes, the clinical cytogenetics of domestic animals will increase in importance: breeding stock
will be selected more effectively and animals
with reproductive disorders will closely examined. Indeed, several chromosomal abnormalities, such as reciprocal translocations and inversions, can easily elude cytogenetic investigation
with normal banding techniques, especially
when contracted chromosome preparations are
employed.
Acknowledgements — This study was in part
supported by "Consiglio Nazionale delle Ricerche,
Progetto Strutturale FESR, Valorizzazione Prodotti
Tipici" and "Progetto Speciale Biodiversita".
REFERENCES
BOUVET A., POPESCU C.P., DE GIOVANNI-MACCHI A.,
COLOMBO G. and MOLTENI L., 1989 —Synaptonemal
complex analysis in a bull carrying a 4;8
robertsonian translocation. Ann. Genet., 32: 193199.
DE GIOVANNI A., Succi G., MOLTENI L. and CASTIGLIONI
M., 1979 —A new autosomal translocation in
"Alpine grey cattle". Ann. Genet. Sel. Anim., 11:
115-120.
DE GIOVANNI A., MOLTENI L., Succi G., GALLIANI C,
BOSCHER J. and POPESCU C. P., 1988 — A new type
of Robertsonian translocation in cattle. 8th
European Colloquium on Cytogenetics of
Domestic Animals, Bristol, 19-22 July, p. 53-59.
DI MEO G.P., PERUCATTIA., IANNUZZIL., RANGEL-FIGUEIREDO
M.T. and FERRARA L., 1995 — Constitutive
heterochromatin polymorphism in river buffalo
chromosomes. Caryologia, 48: 137-145.
EGGEN A., OUSTRY A., VAIMAN D., FERRETTI L., FRIES R.
and CRIBIU E.P., 1994 — bovine syntenic group
U7, previously assigned to G-banded chromosome
25 in the ISCNDA nomenclature, as-
218
DI MEO, MOLTENI, PERUCATTI, DE GIOVANNI, INCARNATO, SUCCI, SCHIBLER, CRIBIU and IANNUZZI
signs to R-banded chromosome 29. Hereditas,
121:295-300.
GUSTAVSSON I., 1969 — Cytogenetics, distribution and
phenotypic effects of a translocation in Swedish
cattle. Hereditas, 63: 68-169.
IANNUZZI L., 1996 — G- and R-banded prometaphase
karyotypes in cattle (Bos taurus L.). Chromos.
Res., 4: 448-456.
IANNUZZI L., DI BERARDINO D., GUSTAVSSON L, FERRARA L.
and DI MEO G.P., 1987 — Centromeric loss in
translocations of centric fusion type in cattle and
water buffalo. Hereditas, 106: 73-81.
IANNUZZI L., GUSTAVSSON L, DI MEO G.P. and FERRARA L.,
1989 — High resolution studies on late replicating
segments
(G+C-bands)
in
mammalian
chromosomes. Hereditas, 110: 43-50.
IANNUZZI L., RANGEL-FIGUEIREDO T., DI MEO G.P. and
FERRARA L., 1992 — A new Robertsonian
translocation in cattle, rob (15;25). Cytogenet.
Cell Genet, 59: 280-283.
ISCNDA89, 1990 — International System for Cytogenetic Nomenclature of Domestic Animals. Di
Berardino D., Hayes H., Fries R., Long S. ( eds).
Cytogenet. Cell Genet., 53: 65-79.
POPESCU C.P.
and PECH A., 1991 — Une bibliographie
sur la translocation 1/29 de bovins dans le monde
(1964-1990). Ann. Zootech., 40: 271-305.
READING CONFERENCE, 1980 — Proceedings of the first
international conference for the standardisation of
banded karyotypes of domestic animals. Ford
C.E., Pollock D.L., Gustavsson L, (eds).
Hereditas, 92: 145-162.
SCHIBLER L., VAIMAN D., OUSTRY A., GIRAUD-DELVILLE C.,
CRIBIU E.P., 1998 — Comparative gene mapping: a
fine-scale survey of chromosome rearrangements
between ruminants and humans. Genome Res., 8:
901-915.
SUMNER A.T., 1972 —A simple technique for demonstrating centromeric heterochromatin. Exp. Cell
Res., 75: 304-306.
TEXAS SYSTEM, 1996 — Standardization of cattle
karyotype nomenclature: report of the committee
for the standardization of the cattle karyotype.
Popescu C.P., Long S., Riggs P., Womack J.,
SCHMUTZ S., FRIES R., GALLAGHER D., (eds). Cytogenet.
Cell Genet., 74: 259-261.
Received 14 July 2000; accepted 20 September 2000