Chromosomal characterization of three centric fusion translocations in cattle using G-, R- and C-banding and FISH technique

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1 CARYOLOGIA Vol. 53, no. 3-4: , 2000 Chromosomal characterization of three centric fusion translocations in cattle using G-, R- and C-banding and FISH technique G.P. DI MEO 1,L. MOLTENI 2, A. PERUCATTI 1, A. DE GIOVANNI 2, D. INCARNATO 1, G. SUCCI 2, L. SCHIBLER 3 :, E :P. CRIBIU 3 and L. IANNUZZI 1 '" 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 (ISCNDA ) 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, , among the smallest ones, see review in IANNUZZI 1996). Furthermore, the TEXAS SYSTEM (1996) did not indicate to which G- * Corresponding author: fax ; L.Iannuzzi@iabbam.na.cnr.it and R-banded standard chromosomes (READING CONFERENCE 1980; ISCNDA ) 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).

2 214 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

3 CENTRIC FUSIONS IN CATTLE 215 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 SYSTEM 1996), 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.

4 216 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 (rob-6;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 GIO- VANNI 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.

5 CENTRIC FUSIONS IN CATTLE 217 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 ( Bou- VET 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 q- arm 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 chromosome 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: DE GIOVANNI A., Succi G., MOLTENI L. and CASTIGLIONI M., 1979 A new autosomal translocation in "Alpine grey cattle". Ann. Genet. Sel. Anim., 11: DE GIOVANNI A., MOLTENI L., Succi G., GALLIANI C, BOSCHER J. and POPESCU C. P., 1988 A new type of Robertsonian translocation in cattle. 8 th European Colloquium on Cytogenetics of Domestic Animals, Bristol, July, p DI MEO G.P., PERUCATTI A., IANNUZZI L., RANGEL-FIGUEIREDO M.T. and FERRARA L., 1995 Constitutive heterochromatin polymorphism in river buffalo chromosomes. Caryologia, 48: 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-

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