Comparative mapping of human alphoid sequences in great apes using fluorescence in situ hybridization

GENOMICS 2 5 , 477-484 (1995)

Comparative Mapping of Human Alphoid Sequences in Great Apes Using Fluorescence in Situ Hybridization NICOLETTA ARCHIDIACONO,* RACHELE ANTONACCI,*'t ROSALIA MARZELLA,* PALMA FINELLI,* ANGELO LONOCE,* AND MARIANO ROCCHI*'1 *lstituto di Genetica, Via Amendola 165/A, 70126 Bari, Italy, and tCattedra di Istologia ed Embriologia, L. del Pozzo 71, Modena, Italy Received August 2, 1994; revised October 10, 1994

T w e n t y - s e v e n h u m a n a l p h o i d DNA probes h a v e b e e n h y b r i d i z e d in situ to m e t a p h a s e spreads of the comm o n c h i m p a n z e e (PTR), the p i g m y c h i m p a n z e e (PPA), a n d the gorilla (GGO) to i n v e s t i g a t e the e v o l u t i o n a r y r e l a t i o n s h i p b e t w e e n the c e n t r o m e r i c r e g i o n s of the great ape c h r o m o s o m e s . The s u r p r i s i n g results s h o w e d t h a t t h e vast majority of the probes did n o t r e c o g n i z e their c o r r e s p o n d i n g h o m o l o g o u s chromosomes. A l p h o i d s e q u e n c e s b e l o n g i n g to the suprachrom o s o m a l family 1 ( c h r o m o s o m e s 1, 3, 5, 6, 7, 10, 12, 16, and 19) y i e l d e d very h e t e r o g e n e o u s results: some probes gave i n t e n s e signals, but a l w a y s o n n o n h o m o l o g o u s c h r o m o s o m e s ; o t h e r s did n o t p r o d u c e a n y hybridi z a t i o n signal. Almost all probes b e l o n g i n g to the sup r a c h r o m o s o m a l family 2 ( c h r o m o s o m e s 2, 4, 8, 9, 13, 14, 15, 18, 20, 21, and 22) r e c o g n i z e d a single chromosome: c h r o m o s o m e 11 ( p h y l o g e n e t i c IX) in PTR a n d P P A and c h r o m o s o m e 19 ( p h y l o g e n e t i c V) in GGO. Loc a l i z a t i o n of probes of s u p r a c h r o m o s o m a l family 3 ( c h r o m o s o m e s 1, 11, 17, a n d X) was f o u n d to be substantially c o n s e r v e d in PTR a n d PPA, but n o t in GGO. P r o b e pDMX1, specific for the h u m a n X c h r o m o s o m e , was the o n l y s e q u e n c e d e t e c t i n g its c o r r e s p o n d i n g c h r o m o s o m e in all three species. PPA c h r o m o s o m e s I, IIp, IIq, IV, V, VI, a n d XVIII w e r e n e v e r labeled, e v e n u n d e r l o w - s t r i n g e n c y h y b r i d i z a t i o n c o n d i t i o n s , by the 27 a l p h o i d probes u s e d in this study. These results, w i t h particular r e f e r e n c e to differences f o u n d in the t w o related species PTR a n d PPA, s u g g e s t t h a t a l p h o i d c e n t r o m e r i c s e q u e n c e s u n d e r w e n t a very rapid evolution. © 1995 Academic Press, Inc.

INTRODUCTION Alpha satellite DNA is composed of tandem repeated monomers of about 170 bp, clustered in the centromeric region of primate chromosomes (Maio 1971; Manu1 To whom correspondence should be addressed at the Istituto di Genetica, Universit& di Bari, Via Amendola 165/A, 70126 Bari, Italy. Telephone: +39-80-544.33.71. Fax: +39-80-544.33.86. E-mail: [email protected].

elidis, 1978; Willard and Waye, 1987). Alphoid subsets from different chromosomes can vary in sequence homology, so that, under appropriate hybridization conditions, an alphoid clone can recognize specifically a pair of homologous chromosomes. Chromosome-specific subsets have been described for most of the human chromosomes (Choo et al., 1991). Different subsets usually have a distinct genomic organization revealed by a specific "higher-order repeat," composed of tandemly repeated n diverged monomers (Willard and Waye, 1987). Some chromosomes accommodate more than one alphoid domain; moreover, some subsets can be shared by different chromosomes (Willard and Waye, 1987; Hulsebos et al., 1988; Choo et al., 1990; D'Aiuto et al., 1993). Acrocentric chromosomes, in particular, share several subsets of alphoid sequences (Vissel and Choo, 1991). Alphoid DNA probably plays a crucial role in the centromeric function (Hadlaczky et al., 1991; TylerSmith et al., 1993; Haaf and Ward, 1994; Larin et al., 1994), but the lack of a functional assay has precluded any definitive conclusion being drawn. According to their cross-hybridization in in situ hybridization experiments under low-stringency hybridization conditions, the chromosome-specific human alpha satellite subsets have been grouped into three "suprachromosomal families" (SF) (Alexandrov et al., 1988, 1991). Suprafamily 1 (SF1) is composed of chromosomes 1, 3, 5, 6, 7, 10, 12, 16, and 19; suprafamily 2 (SF2) of chromosomes 2, 4, 8, 9, 13, 14, 15, 18, 20, 21, and 22; and suprafamily 3 (SF3) of chromosomes 1, 11, 17, and X. Chromosome 1 is the only chromosome accommodating two subsets belonging to different suprafamilies. Distinction between the three suprafamilies lies also in higher order repeat organization, revealed by restriction site periodicity: SF1 and SF2 are characterized by dimeric ancestral repeat unit organization, while pentameric ancestral repeat units characterize the SF3. Recently, Alexandrov et al. (1993) have proposed a fourth suprachromosomal family, with monomeric organization, represented by subsets located on acrocentric chromosomes and chromosome Y.

477

0888-7543/95 $6.00 Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

478

ARCHIDIACONO ET AL.

TABLE 1 A l p h o i d P r o b e s U s e d in t h e S t u d y Probe pAL1 pZ5.1 pBS4D pAE0.68 p4nl/4 pZ4.1 pEDZ6 pZ7.6B p7AL pZ8.4 pMR9A pZ10-1.3 pBRll pBR12 p14.1 pMC15 pTRA-25 pZ16A pZl7-1.6A 2XBA pBS18A pZ20 pZ21A pi90.22 p22/1:2.1 pDMX1 pZYA

Chromosome 1 1, 5, 19 2 3 4 4, 9 6 7 7 8 9 10 11 12 14, 22 15 15 16 17 18 18 20 21, 13 22 22 X Y

Locus DIZ5 DIZ7 D2Zl D3Zl D6Z1 D7Zl D7Z2 D8Z1 D9Z4 D10Z1 DllZ1 D12Z3 D14Zl D15Z3 D15Z4 D16Z2 D17Z1 D18Zl D18Z2 D20Z2 D21Zl D22Z4 D22Z2 DXZ1 DYZ3

Reference Unpublished Unpublished Rocchiet al. (1990) Baldini et al. (1989) D'Aiuto et al. (1993) D'Aiuto et al. (1993) Unpublished Unpublished Unpublished Unpublished Rocchiet al. (1991) Unpublished Unpublished Baldini et al. (1990) Unpublished Unpublished Choo et al. (1990) Unpublished Unpublished Unpublished Unpublished Baldini et al. (1992) Unpublished Rocchiet al. (1994) McDermidet al. (1986) Unpublished Unpublished

In our r e c e n t p a p e r s on the cloning of alphoid sequences specific for h u m a n c h r o m o s o m e s 20 (Baldini et al., 1992) a n d 4 (D'Aiuto et al., 1993), fluorescence i n s i t u h y b r i d i z a t i o n (FISH) e x p e r i m e n t s on c h i m p a n z e e a n d gorilla c h r o m o s o m e s were also performed, w i t h unexpected results: in b o t h instances, the h u m a n sequence recognized specific c h i m p a n z e e a n d gorilla chromosomes, b u t t h e s e c h r o m o s o m e s were n e i t h e r homologous with each o t h e r nor homologous with the h u m a n c h r o m o s o m e from w h i c h the alphoid sequence w a s derived. These s u r p r i s i n g r e s u l t s p r o m p t e d us to s t a r t a s y s t e m a t i c s t u d y on the c o m p a r a t i v e m a p p i n g of alphoid sequences in g r e a t apes in a n a t t e m p t to u n d e r s t a n d t h e i r e v o l u t i o n a r y relationship. I n this p a p e r we r e p o r t the r e s u l t s for the c o m m o n c h i m p a n z e e ( P a n t r o g l o d y t e s , PTR), the p y g m y c h i m p a n z e e ( P a n p a n i s cus, PPA), a n d the gorilla ( G o r i l l a g o r i l l a , GGO). MATERIALS AND METHODS The alphoid probes used in this study are reported in Table 1. Probe pTRA25 (Choo et al., 1990) was a generous gift of Dr. A. K. H. Choo (Melbourne, Australia); probe p22/1:2.1 (McDermid et al., 1986) was kindly donated by Dr. H. E. McDermid (Edmonton, Canada). The probes reported as "unpublished" have been cloned in our laboratory, starting from monochromosomalsomatic cell hybrids, with a described strategy (Rocchi et al., 1990). The chromosomal specificity was determined by FISH experiments on normal human metaphases and, occasionally, by Southern blot analysis on a panel of somatic cell hybrids. Their genomic organization, studied by Southern blot analysis, was compared to that of one of the published

probes with the same chromosome specificity and consequently assigned to a specific locus. Metaphase spreads have been obtained from 48,XYlymphoblastoid or fibroblast cell lines of a common chimpanzee (P. troglodytes), a pygmy chimpanzee (P. paniscus), and a gorilla (G. gorilla). Chromosome preparations were hybridized in situ with clones biotinylated by nick-translation, essentially as described by Lichter et al. (1990), with minor modifications (Antonacciet al., 1994a). Briefly, 200 ng of labeled probe was used for each experiment; hybridization was performed at 37°C in 2× SSC, 50% (v/v) formamide, 10% (w/v) dextran sulfate, and 3 #g sonicated salmon sperm DNA, in a volume of 10 t~l. Posthybridization washes were at 42°C in 2x SSC-50% formamide (×3) followed by three washes in 0.1x SSC at 60°C. Biotinylated DNA was detected with FITC-conjugated avidin. Chromosome identification was obtained by simultaneous DAPI staining, which produces a Q-banding pattern. Low-stringency hybridization was performed with the following modifications of posthybridization washes: at 37°C in 50% formamide in 2× SSC (x3), followedby three additional washes in 2x SSC at 42°C. Digital images were obtained using a cooled CCD camera (Photometrics). FITC and DAPI fluorescence, detected using Pinkel No. 1 specific filter set combinations (Chroma Technology, VT), were recorded separately as gray-scale images. The filter set allows capturing of DAPI and FITC signals without any image shifting. DAPI and FITC images were occasionally pseudocolored and merged using the GeneJoin software (developed by T. Rand in the laboratory of D. C. Ward, Yale University). Ten to twenty metaphases were examined for each probe. Primers derived from conserved regions of human alphoid repeats have been described by Dunham et al. (1992) and designated alpha 27 and alpha 30. PRINS experiments have been performed according to Volpi and Baldini (1993).

RESULTS The results of F I S H e x p e r i m e n t s on PTR, PPA, a n d GGO chromosomes, u s i n g 27 distinct h u m a n alphoid probes, are s u m m a r i z e d in Tables 2, 3, a n d 4, respectively. I n the left-most c o l u m n of each table, the alphoid probes, along with the h u m a n chromosome(s) t h a t t h e y recognize, are reported. Most of the probes are specific for a single h u m a n chromosome. For each probe F I S H e x p e r i m e n t s h a v e been p e r f o r m e d u n d e r low (L) a n d h i g h (H) s t r i n g e n c y h y b r i d i z a t i o n conditions. The hum a n alphoid probes h a v e been r e p o r t e d according to t h e i r s u p r a c h r o m o s o m a l family g r o u p i n g s (Alexandrov et al., 1988, 1991). G r e a t ape c h r o m o s o m e s h a v e been r e p o r t e d according to t h e i r phylogenetic n o m e n c l a t u r e a n d h a v e been sorted a s s u m i n g , arbitrarily, the existence of a h u m a n - l i k e s u p r a c h r o m o s o m a l family organization. GGO c h r o m o s o m e 19 is derived from a t r a n s l o c a t i o n b e t w e e n a n c e s t r a l c h r o m o s o m e s homologous to h u m a n c h r o m o s o m e s 17 (17pl2-~pter) a n d 5 ( 5 p t e r ~ q l 3 ) ( S t a n y o n et al., 1992). The l a t t e r region includes the c e n t r o m e r i c region, a n d for this r e a s o n signals on GGO c h r o m o s o m e 19 h a v e been r e p o r t e d in Table 3 as being on phylogenetic V, while those on GGO c h r o m o s o m e 4 h a v e been r e p o r t e d as being on phylogenetic XVII. The signal i n t e n s i t y h a s been visually e v a l u a t e d as strong, m e d i u m , faint, a n d very faint (see notes to the tables). An e x a m p l e of F I S H experim e n t s is r e p o r t e d in Fig. 1.

COMPARATIVE MAPPING OF ALPHA SATELLITE SEQUENCES IN GREAT APES

479

TABLE 2

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480

ARCHIDIACONO ET AL,. TABLE 3

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COMPARATIVE MAPPING OF ALPHA SATELLITE SEQUENCES IN GREAT APES

481

TABLE 4

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Note. Left column: alphoid probes (below), along with the human chromosome(s) that they recognize (above). For each probe FISH experiments under low (L) and high (H) stringency hybridization conditions are reported. Human alphoid probes have been grouped according to suprachromosomal family organization. Great ape chromosomes have been reported according to their phylogenetic nomenclature, and arranged, arbitrarily, assuming the existence of analogous suprachromosomal family organization in great apes. The signal intensity has been visually evaluated as strong (m), medium (m), faint (.), and very faint (-).

482

ARCHIDIACONO ET AL.

I

G FIG. 1. DAPI-banding(a) and FITC signals (b), detected with the CCD camera, from a male PPA metaphase hybridizedin situ with biotinylated pRBll probe, specific for human chromosome 11, under high-stringencyhybridization conditions. The pseudocoloringand merging of the images were not performed to allow the fluorescence signals to be seen as they appear on the microscope. Signals on chromosomes 9 (phylogeneticXI, large arrowheads) and X (small arrowhead) were evaluated as strong, while those on chromosome 19 (phylogeneticXVII, dash) were faint.

Suprachromosomal Family 1 SF1 probes gave the most heterogeneous results. At high stringency, strong cross-hybridization signals were found in PTR chromosomes IV, VIII, XII, XIV, XVI, XVIII, XIX, and XXII and in GGO chromosomes I, X, XI, XII, XVII, XIX, and X, but they were never found on chromosomes homologous to the one from which the h u m a n alphoid probe was derived. In PPA only faint signals were recorded, and in GGO crosshybridization signals were concentrated on GGO SF1 and SF3.

Suprachromosomal Family 2 The cross-hybridization pattern revealed by SF2 probes was peculiar: almost all probes gave a strong signal on chromosome IX in both PTR and PPA and on chromosome V in GGO. The only exception was found in GGO, where probe p21A (13 and 21) gave a more intense signal on chromosome XXII t h a n on chromosome V. Chromosome 9 in HSA is a member of SF2, while chromosome 5 belongs to SF1.

Suprachromosomal Family 3 Localization of alphoid SF3 sequences appeared to be conserved in PTR and PPA, with negligible crosshybridizations on chromosomes not belonging to PTR or PPA SF3. In GGO, on the contrary, the pDMX1 probe (X) was the only SF3 sequence t h a t recognized its homologous chromosome. PPA chromosomes XI and X behaved similarly when hybridized with probe p B R l l (11) and pDMX1 (X). No signal with any probe was detected on PPA chro-

mosomes I, IIp, IIq, IV, V, VI, and XVIII. To investigate these findings closely, the alpha 27 and alpha 30 primers were used in PRINS experiments on PPA chromosomes, but, again, the above reported chromosomes did not show any hybridization signal (data not shown). Alpha 27 and alpha 30 primers are derived from conserved regions of the h u m a n alphoid repeats consensus sequence (Dunham et al., 1992; see Materials and Methods). PRINS experiments on h u m a n metaphases using alpha 27 and alpha 30 primers highlight intensely the centromeric regions of all chromosomes (data not shown). DISCUSSION

We have presented data on comparative mapping of 27 h u m a n alphoid sequences on PTR, PPA, and GGO chromosomes. The most relevant findings can be summarized as follows: (i) The majority of the probes do not recognize their homologs; the probe specific for chromosome X is the only sequence showing localization consistency in all three species. (ii) Each suprachromosomal family exhibits a distinct and peculiar evolutionary history. (iii) Almost all members of SF2 recognize a single chromosome: chromosome IX in PTR and PPA and chromosome V in GGO. (iv) PPA chromosomes I, IIp, IIq, IV, V, VI, and XVIII never showed cross-hybridization signals. In evaluating these results one must consider t h a t the homology between h u m a n and great ape chromosomes, suggested by the striking banding pattern similarities (Yunis and Prakash, 1982; ISCN, 1985), has been recently investigated and confirmed at the level of DNA sequence using whole-chromosome painting libraries (Jauch et al., 1992).

COMPARATIVE MAPPING OF ALPHA SATELLITE SEQUENCES IN GREAT APES

The suprachromosomal family grouping of great ape chromosomes has been done arbitrarily. The assessment of the suprachromosomal family organization in PTR, PPA, and GGO would require the use of a large set of species-specific alphoid probes, representative of the entire chromosome complement. Unfortunately, only a few data on the in situ hybridization of great ape alphoid probes are available (Baldini et al., 1991a,b). Probes belonging to the SF1 gave the most heterogeneous results. Some sequences (probe pZ7AL, No. 7, in gorilla, for example) did not show any cross-hybridization signal; others gave intense signals, but on nonhomologous chromosomes. This suggests that SF1 represents recently evolved sequences. Their rapid evolution is also supported by differences found between PTR and PPA, which are supposed to have diverged much more recently than the other species. The fact that all of the h u m a n alphoid sequences belonging to SF2 recognize a single chromosome in PTR and PPA (chromosome IX) and a single, but different, chromosome in GGO (chromosome V) is intriguing. FISH experiments using a fully representative panel of chimpanzee and gorilla alphoid probes, when available, will probably help, as stated, in the understanding of these results. The size of the heterochromatic region of PTR and PPA chromosome IX and GGO chromosome V did not appear larger than that of the remaining chromosomes. Therefore, the most likely interpretation is that a single subset present in these two chromosomes strongly cross-hybridizes with all h u m a n SF2 sequences. In chimpanzees (both P. troglodytes and P. p a n i s c u s ), SF3 is conserved, while in the gorilla the only SF3 alphoid sequence that recognizes its homologous chromosome is the one specific for chromosome X. We stress again that this sequence is the only one showing localization consistency in all three species examined. Sequence and genomic organization comparisons of chromosome X-specific alpha satellite probes of the human, the gorilla, and the chimpanzee have been reported (Durfy and Willard, 1990; Laursen et al., 1992). These studies have shown substantial organization similarities, suggesting the existence of an ancestral alpha satellite component, identical or similar to the 12-monomer unit of the h u m a n X chromosome. On the basis of present results, the conclusions reported by the authors should be restricted to chromosome X. The peculiarity of the sex chromosome probably plays a crucial role in the homology maintenance of this alpha satellite subset. In contrast, PPA chromosomes I, IIp, IIq, IV, V, VI, and XVIII did not show any hybridization signal, even under low-stringency hybridization conditions, suggesting that some subsets have substantially diverged in their evolutionary history. Some h u m a n chromosomes accommodate two or more alphoid subsets. Different approaches have been used to study the organization of subsets present on the same chromosome: Southern blot analysis of partial genomic DNA digestions (Waye et al., 1987), PFGE

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(Wevrick and Willard, 1991; Trowell et al., 1993), and FISH experiments on elongated chromosomes or chromatin (Antonacci et al., 1994b; H a a f and Ward, 1994). All of the data suggest that they are organized in distinct domains. In the present study two distinct probes for h u m a n chromosomes 1, 4, 7, 9, 15, and 18 and three probes for chromosome 22 have been used. The results obtained showed that, in general, subsets localized on the same chromosome had a similar evolutionary history, with the exception of pZ5.1 and pAL1, localized on chromosome 1, and pZ7.6 and pZ7AL, both specific for h u m a n chromosome 7. Repetitive DNA undergoes a very rapid evolution compared to unique DNA sequences with coding or regulatory functions, which are under strict selective pressure. As an example, the subterminal satellite DNA located adjacent to the telomeres in the chimpanzee and the gorilla, and organized in large arrays, has no counterpart in other great apes or in humans (Marks, 1993; Royle et al., 1994). Alphoid sequences are probably involved in centromeric function and may be subject to evolutionary constraint. However, if there is a selective pressure, we do not know at which level (sequence or secondary structure) a selection occurs. Chromosomal variations can play an important role in species evolution. The data reported here should be taken into consideration in this respect (Laursen et al., 1992). At present, however, it would be difficult to support the hypothesis that variation in centromeric sequences is the cause and not the consequence of speciation. As stressed before, a full understanding of the data reported here would require the availability of a panel of PTR, PPA, and GGO alphoid probes comparable to the h u m a n one used in this study. Efforts in this direction are in progress in our laboratory. ACKNOWLEDGMENTS This study was supported in part by grants from AIRC and Telethon. REFERENCES Alexandrov, I. A., Mitkevich, S. P., and Yurov, Y. B. (1988). The phylogeny of human chromosome specific alpha satellites. Chrom o s o m a 96: 443-453. Alexandrov, I. A., Mashkova, T. D., Akopian, T. A., Medvedev, L. I., Kisselev, L. L., Mitkevich, S. P., and Yurov, Y. B. (1991). Chromosome-specific alpha satellites: Two distinct families on human chromosome 18. Genomics 11- 15-23. Alexandrov, I. A., Medvedev, L. I., Mashkova, T. D., Kisselev, L. L., Romanova, L. Y., and Yurov, Y. B. (1993). Definition of a new alpha satellite suprachromosomal family characterized by monomeric organization. Nucleic A c i d s Res. 21: 2209-2215. Antonacci, R., Colombo, I., Archidiacono, N., Volta, M., DiDonato, S., Finocchiaro, G., and Rocchi, M. (1994a). Assignment of the gene encoding the B-subunit of the electron-transfer flavoprotein (ETFfl) to human chromosome 19q13.3. Genomics 19: 177-179. Antonacci, R., Rocchi, M., Archidiacono, N., and Baldini, A. (1994b).

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