The existence of an apomorphic reciprocal chromosomal translocation in the gorilla lineage has been asserted or denied by various cytogeneticists. We employed a new molecular cytogenetic strategy (chromosomal in situ suppression hybridization) combined with high-resolution banding, replication sequence analysis, and fluorochrome staining to demonstrate that a reciprocal translocation between ancestral chromosomes homologous to human chromosome 5 and 17 has indeed occurred

Bigoni, F.

Cremer, Thomas

Jauch, Anna

Romagno, D.

Stanyon, Roscoe

Wienberg, Johannes

English

Open Access LMU ( Ludwig-Maximilians-Univ. München)

AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 88:245-250 (1992) Molecular and Classical Cytogenetic Analyses Demonstrate an Apomorphic Reciprocal Chromosomal Translocation in Gorilla gorilla R. STANYON, J. WIENBERG, D. ROMAGNO, F. BIGONI, A. JAUCH, AND T .  CREMER Istituto di Antropologia fisica, University of Genoa, 16126 Genoa, Italy (R.S., F.B.); Institut fur Humangenetik und Anthropologie, University of Heidelberg, D-6900 Heidelberg, Germany (J.W., A.J., T.C.); Istituto di Antropologia, University of Florence, Florence, Italy (D.R.) KEY WORDS logeny, primates In situ hybridization, chromosome banding, phy- ABSTRACT The existence of an apomorphic reciprocal chromosomal translocation in the gorilla lineage has been asserted or denied by various cytogeneticists. We employed a new molecular cytogenetic strategy (chromo- somal in situ suppression hybridization) combined with high-resolution band- ing, replication sequence analysis, and fluorochrome staining to demonstrate that a reciprocal translocation between ancestral chromosomes homologous to human chromosome 5 and 17 has indeed occurred. o 1992 Wiley-Liss, Inc. Comparative studies of banded chromo- somes in primates have demonstrated that particular chromosomal mutations, or rear- rangements, are often characteristic of dif- ferent clades. For example, while fusions characterize the prosimians, fissions are common in the evolution of guenons, and inversions (especially pericentric inver- sions) are thought to account for the major- ity of chromosomal changes in the homi- noids. However, Dutrillaux (1973,1975) and later Yunis and Prakash (1982) pointed out the possibility of a reciprocal translocation in the gorilla lineage between chromosomes homologous to human chromosomes 5 and 17. This rearrangement was not mentioned by other cytogeneticists interested in hu- man and ape evolution (Seuanez, 1979; Stanyon and Chiarelli, 1982). Marks (1982) has suggested that these conflicting reports are due to problems of precision of resolution in cytogenetic studies. Seuanez (1987:72), however, provided a more definitive state- ment “Though the pattern of chromosome band- ing might give partial support to this pre- sumptive rearrangement, comparative gene assignment of the thymidine kinase locus (TK) has ruled out the possibility that such rearrangement has ever occurred.” In making this statement Seuanez was following the normal procedure because gene mapping is usually given priority in establishing homologies (Ruddle, 1981). However, gene mapping is not always a fool- proof indication of homology. For example, genes mapped to human 2p and 2q are found on the “wrong” great ape arm homologs (Seuanez, 1987). A conflict also exists for the inosine-triphosphatase (ITPA) locus. This gene is mapped to human 20, but it is not on the seemingly identical great ape chromo- some (O’Brien and Graves, 1990). Perhaps, then, TK1 gene assignment has not defini- tively ruled out the existence of an apomor- phic 5/17 translocation in the gorilla lineage and this possibility could be profitably reex- amined. We decided to study this region systemat- ically using high resolution banding tech- niques (Yunis, 1981), fluorochrome staining (Schmid et al., 1986; Wienberg and Stanyon, Received May 14,1991; accepted October 30,1991 0 1992 WILEY-LISS, INC.  246 R. STANYON ET AL. 1987, 1988), replication banding (Weber e t  al., 1986, Stanyon et al., 1990) and in situ suppression (CISS) hybridization (Lichter et al., 1988; Pinkel et al., 1988) of human DNA libraries to gorilla chromosomes and to the chromosomes of other primate species. These data amply demonstrate that a 5/17 translocation occurred in the gorilla lineage. MATERIAL AND METHODS Chromosome preparations were made from both phytohemagglutinin stimulated whole blood and fibroblast cultures of hu- man, chimpanzee, gorilla, and macaque (Macaca fuscata). High resolution G-band- ing and replication banding were done as  reported before (Small et al., 1985; Stanyon et al., 1990). DistamycidDAPI staining was according to Wienberg and Stanyon (1988). CISS hybridization to primate chromo- somes was as  described previously (Wien- berg et al., 1990). Briefly, chromosome spe- cific human DNA libraries established from flow sorted chromosomes 5 and 17 and cloned to phages (ATCC Nr. LA05NSO1, LL17NS02) or plasmids (chromosome 17 li- brary kindly provided by J. Gray, Liver- more, CA, U.S.A.) were used as  probes. Slides were mounted with 18 mm x 18 mm cover slips and standard hybridization mix- ture with 2 kg labelled phage library DNA or 300 microgram labelled plasmid library DNA. Labelling was by standard nick-trans- location protocol. Probes were labelled with biotin and detected with fluorescein (FITC) coupled to avidin (Vector Laboratories), di- luted 1 : 1000 in TE. Unlabelled human competitor DNA and salmon sperm carrier DNA was as  described (Lichter et al., 1988). Double hybridization experiments were performed with digoxigenin labelled chro- mosome 17 probe, which was detected with FITC labelled mouse anti-digoxigenin anti- bodies (Boehringer Mannheim) diluted 1 : 1000 in TE, together with biotin labelled human 5 probe that was detected with avi- din coupled to AMCA (Vector Laboratories), diluted 1 : 200 in TE. AMCA signals were amplified once as  described by Pinkel et al. (1986) and analysed with the same filter combination as for DAF'I. Slides were ana- lysed with a Zeiss photomicroscope I11 equipped for FITC and DAPI fluorescence. A B Fig. 1. From left to right: a high resolution G-band comparison of human chromosomes 5 and gorilla 19, and gorilla 4 and human 17.  Human 17 has been in- verted to better demonstrate the banding homology Note the apomorphic teleomeric heterochromatin in the gorilla chromosomes. Photographs were taken on AGFA-CHROM 1000 RS or Kodak T-MAX black and white (400 ASA) films. RESULTS High resolution G-banding clearly showed a striking correspondence of banding be- tween the short arm and a part of the long arm of gorilla 4 and the long arm of human 17. There was an equally good match of banding between the short arm and part of the long arm of human 5 and most of gorilla 19 (Fig. 1). Additional, terminal G-bands were noted in gorilla 4 and gorilla 19. The replication sequence demonstrated that the similarity in banding pattern ex- tended to the DNA replication timing of the G-bands (Fig. 2). In particular, gorilla 19 has a large band on the short arm which is late replicating not only within the chromo- some, but also one of the latest-replicating regions within the entire karyotype. This replication timing is identical to that of band 5p14 in humans. The replication order of gorilla 4p corresponds to the replication or- der of human 17q. However, the replication order of the terminal half of gorilla 19q had no obvious counterpart in the human karyo- type. The gorilla chromosomes had late tele- omeric replicating heterochromatin not present in humans. DNDAPI staining showed that the distri- bution of brilliant fluorescent bands in all species was as previously reported (Schmid RECIPROCAL TRANSLOCATION IN GORILLA A C 247 D Fig. 2. A and B: The replication pattern of human 5 and gorilla 19. A, complete replication; B, late replication pattern; C and D: The replication pattern of gorilla 4 and human 17 (inverted to better demonstrate the replication homology). C, complete replication; D, late replication pattern. Note the additional late replicating teleomeric heterochromatin in the gorilla. hybridized to the entire short arm and a part of the long arm next to the centromere of gorilla 4 and to about half of the long arm of gorilla 19 (Fig. 4c). The human 5 library hy- bridized to the short arm, to half of the long arm adjacent to the centromere of gorilla 19, and to most of the long arm of gorilla 4 (Fig. 4d). In gorilla chromosomes, late replicating teleomeric C-bands were not hybridized with either chromosome 5 or 17 libraries. Fig. 3. Shows the DNDAPI fluorescent band on go- rilla 4. This DA/DAPI band was most probably translo- cated from an ancestral chromosome homologous to hu- man 17 (see text). et al., 1986; Wienberg and Stanyon, 1987, 1988). Relevant to our analysis, a pericen- tromeric bright band was found on the go- rilla 4p, and chimpanzee 19, but not on hu- man 5, chimpanzee 4, or gorilla 19 (Fig. 3). The macaque chromosome had no DNDAPI brilliant bands. The DNA libraries of human 5 and 17 each hybridized to  one chromosome pair in humans, chimpanzees, and macaques. In the gorilla each probe hybridized to four chromosomes (Fig. 4). Human 17 libraries DISCUSSION The hybridization data alone demonstrate that at the molecular level there can be no doubt that a reciprocal translocation oc- curred in the gorilla lineage between ho- mologs to human 5 and 17 (Fig. 5). The high- resolution replication sequence and DA/ DAPI banding also clearly show that a reciprocal translocation has indeed oc- curred. The comparison with outgroup chro- mosomes, from M. fuscata, indicates that this reciprocal translocation in the gorilla is apomorphic. According to Wienberg and Stanyon (1988), the last hominoid common ancestor had a DNDAPI band on the homolog equiv- alent to human 17. The gorilla lacks a DN DAPI band on this chromosome and is the only hominoid to have such a bright band on the homolog to human 5. It is most likely that this DA/DAPI band was derived from an ancestral homolog to human 17 and is additional evidence for the reciprocal trans- 248 R. STANYON ET AL. Fig. 4. CISS hybridization patterns of human 5 and 17 libraries to primate chromosomes. Hybridization was detected either with fluorescein (FITC) (a, b, and c) or AMCA (d). Chromosomes were counterstained with pro- pidiumiodine. a: Chromosome phage 17 library hybrid- ized to chimpanzee chromosomes. One pair of chromo- somes is hybridized. Note the slight cross hybridization to the heterochromatin in some centromeres due to se- quence homology. b: Chromosome plasmid 17 library on M .  fuscata chromosomes, as in the chimpanzee one chro- mosome pair is hybridized. c and d: The same gorilla metaphase hybridized with human 17 plasmid library (c) which was labelled with digoxigenin and detected with FITC labelled mouse anti-bodies, and human 5 ph- age library (d) labelled with biotin and detected with avidin coupled to AMCA. This procedure allows two dif- ferent probes to be visualized on the same metaphase in different colors by just changing the filter combination of the microscope. With both probes, four chromosomes were labelled, demonstrating the reciprocal translocation. location, even if the molecular basis and sig- nificance of DA/DAPI is not yet known. It is clear that the reciprocal translocation was not a simple exchange of arms, because the hybridization signals of each probe are found on both the short and the long arm of different chromosomes. Consequently, the short arm, the centromere, and a small prox- imal part of the long arm of gorilla 4 is ho- mologous to human 17, while the part of go- 249 RECIPROCAL TRANSLOCATION IN GORILLA 5 Fig. 5. Idiograms of, from left to right, human chro- mosome 5, gorilla 19, human 17, and gorilla 4. Human chromosomes are numbered below. The gorilla chromo- somes are numbered on the left t o  summarize the hy- bridization pattern obtained with human probes. Go- rilla chromosome 4 (extreme right) is inverted to better show the homology to human 17. The D to the right of gorilla chromosome 4 indicates the location of the DA/ DAPI bright band. rilla 19 homologous to human 5 includes the short arm, the centromere, and the proximal half of the long arm. It appears that the cen- tromere of gorilla 4 comes from an ancestral chromosome homologous to human 17, while the centromere of gorilla 19 derives from an  ancestral chromosome homologous to human 5. There are several possibilities to explain the inconsistency between chromosomal ho- mology and gene mapping. TK1, gene as- signment in the gorilla, could be erroneous. Chromosomal rearrangements in vitro in hybrid cell lines are probably more common than previously thought, and the analysis of hybrid cell chromosomes by banding can be difficult. On the other hand, there may be a small, undetected rearrangement which has altered the chromosomal location of TK1 in the gorilla. Notably, there is no good corre- spondence between either the high-resolu- tion banding or replication sequence of the terminal half of gorilla 19 and any part of human 17. CISS hybridization shows that this chromosome section derives from a ho- molog to human 17, but the lack of banding correspondence suggests that it has been subject to further, as  yet not clear, alter- ations. Further study with molecular cyto- genetic techniques could clarify whether or not such rearrangements have involved the TK1 gene. CISS hybridization, in contrast to single gene mapping, signals the presence of all parts of the homologous chromosome in the target species, regardless of where they are. Single genes, however, are localized to spe- cific chromosomal regions, and chromo- somal homologies between mapped genes are interpolations. There is no guarantee that chromosomal regions between markers are actually homologous. A limitation of using whole chromosome probes is that they do not allow the mapping of intrachromosomal rearrangements. Mi- crodissection and microcloning of chromo- some bands, YAC (yeast artificial chromo- somes) and cosmid clones, and pools of single copy sequences can provide probes for chromosome subregions from any species (Ajioka et al., 1990; Wienberg et al., 1990; Arnold et al., 1991; Lengauer et al., 1991). CISS hybridization was recently used to map more complex DNA probes on a single human chromosome (Lichter et al., 19901, then the maximum number of genes (44) that have been mapped to all the chromo- somes of the gorilla (O'Brien and Graves, 1990). CISS hybridization, combined with traditional chromosome techniques, will permit a new level of confidence and utility for reconstructing and clarifying primate ev- olution and human origins. ACKNOWLEDGMENTS We would like to thank A. Wiegenstein and J. Hollatz for photographic work. T.C. was the recipient of a Heisenberg-Stipen- dium from the Deutsche Forschungsgemein- schaft. This work was supported in part by the Deutsche Forschungsgemeinschaft and the Italian Ministry of Universities and Sci- entific Research, 60% and 40% grants, and CNR grant number 90.2476.CT04. LITERATURE CITED Arnold N, Wienberg J, Zachau H-G (1991) Evolution of V-kappa immunoglobulin genes in human and pri- 250 R. STANYON ET AL. mates analysed by molecular cytogenetics (abstract). Am. J. Hum. Genet. (in press). Ajioka JW, Garza D, Johnson D, Carulli JP,  Jones RW, and Hart1 DL (1990) Genome evolution analyzed by cloning large fragments of Drosophila DNA in yeast artificial chromosomes. In MT Cleg and SJ OBrien (eds.): Molecular Evolution. New York: Wiley-Liss, pp. 253-263. Dutrillux B (1975) Sur le nature et l'origine des chromo- somes humanines. Paris:L'expansion Scientifique. 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A new era in mammalian gene mapping: Somatic cell genetics and recombinant DNA methodologies.

An evolutionarily conserved early replicating segment on the sex chromosomes of man and the great apes.

Analyse de la structure fine de chromosomes du Gorille (Gorilla gorilla).

Comparative chromosome band mapping in primates by in situ suppression bybridization of band specific DNA microlibraries.

Cytogenetic analysis by in situ hybridization with fluorescently labelled nucleic acid probes. Cold Spring Harb.

Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries.

Evolution of V-kappa immunoglobulin genes in human and pri- 250 R. STANYON ET AL. mates analysed by molecular cytogenetics (abstract).

Fluorescence in situ hybridization with human chromosome specific libraries: Detection of trisomy 21 and translocations of chromosome 4.

Fluorescent heterochromatin staining in primate chromosomes. Human Wienberg

Genome evolution analyzed by cloning large fragments of Drosophila DNA in yeast artificial chromosomes.

Heterochromatin in the chromosomes of the gorilla:

High resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones.

Hominoid cytogenetics and Evolution. Year-book Phys.

Mammalian genome organization: an evolutionary view.

Mid-prophase human chromosomes.

Molecular cytotaxonomy of primates by chromosomal in situ suppression hybridization.

Report of the committee on comparative gene mapping (HGM10.5).

Sequence of DNA replication in Macaca fuscata chromosomes: An outgroup for phylogenetic comparison between man and apes.

Sur le nature et l'origine des chromosomes humanines. Paris:L'expansion Scientifique.

The attainment of 2000 bands.

The origin of man: A chromosomal pictorial legacy.

Molecular and classical cytogenetic analyses demonstrate an apomorphic reciprocal chromosomal translocation in Gorilla gorilla

Molecular and classical cytogenetic analyses demonstrate an apomorphic reciprocal chromosomal translocation in Gorilla gorilla

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