Abstract We have established a series of highly specificpainting probes for human acrocentric chromosomes. Thesechromosomes are involved in the formation of the nucleolarorganizer region (NOR) and show DNA sequence homolo-gies within their pericentric heterochromatin. To date, thesechromosomes have shown considerable cross hybridizationin chromosome painting experiments. Our probe set hasbeen established from primate homologues that are not in-volved in the NOR in that particular species or fromspecies in which highly repetitive sequences have under-gone rapid sequence divergence. The new painting probesshould be of particular value for automated microscopy,for which highly specific signals are required as they arerecorded at low magnification, e.g. when scoring chromo-some 21 domains in interphase nuclei.

Introduction

A chromosome paint is a mixture of DNA sequences thathybridize to the entire length of a specific chromosome.Chromosome painting on metaphase chromosomes andinterphase nuclei has become an important tool for the cy-togenetic analysis of human chromosomes (Carter 1994;Ferguson-Smith 1997). Most of the painting probes avail-able show very little non-specific hybridization to otherchromosomes of the human karyotype (Collins et al.1991). However, chromosomes 13, 14, 15, 21 and 22 areinvolved in the formation of the nucleolar organizer region(NOR) and share sequences coding for ribosomal RNA(Henderson et al. 1972). These chromosomes also showhighly repeated DNA sequence similarities in pericentricheterochromatin (Warburton and Willard 1996) and, so

far, no chromosome-specific alphoid sequences have beenreported. Painting probes from any of these chromosomesresult in cross hybridization to most of the centromeres ofall acrocentric chromosomes and this cannot be suppress-ed by the addition of unlabelled competitor DNA (Cot-1DNA). Therefore, the respective painting is of limited usein molecular cytogenetics. There is, however, demand forspecific painting probes for these chromosomes in clinicalcytogenetics, especially for human chromosomes 13 and21.

Our previous work has shown that paints specific forhuman chromosomes can be used to delineate homolo-gous sequences on chromosomes of non-human primates(Wienberg et al. 1990, 1992; Jauch et al. 1992; Stanyon etal. 1992). Cross hybridization of human highly repetitivesequences is less evident in great ape chromosomes butnearly absent in lesser apes and monkeys. Nevertheless,painting of euchromatic regions shows no intensity differ-ences between the species. This reflects the observationthat, during evolution, repetitive sequences show muchhigher divergence than low copy number sequences(Archidiacono et al. 1995; Warburton and Willard 1996).Therefore, probes from closely related species should pro-vide a highly specific source for chromosome paintingwith reduced cross hybridization to heterochromatin.

Bivariate fluorescence activated chromosome flowsorting (FACS) has been widely used to generate chromo-some painting probes from various non-human mammals(Yerle et al. 1993; Hoebee et al. 1994; Shepel et al. 1994;Rabbitts et al. 1995; Yang et al. 1995; Burkin et al. 1997).Here, we have used FACS of great ape and gibbon chro-mosomes to establish a set of specific painting probes forhuman chromosomes 13, 14, 15 and 21.

Materials and methods

Cell lines for great apes, viz. lines EB 178, EB (JC) and EB 185,were obtained from the European Collection of Animal Cell Cul-tures (ECACC), Porton Down, UK. The siamang gibbon lym-phoblastoid line was established from an individual housed at theMunich Zoo, Germany (Koehler et al. 1995a) and the lymphoblas-

Stefan Müller · Patricia C. M. O’Brien ·Malcolm A. Ferguson-Smith · Johannes Wienberg

A novel source of highly specific chromosome painting probes for human karyotype analysis derived from primate homologues

Hum Genet (1997) 101 :149–153 © Springer-Verlag 1997

Received: 22 May 1997 / Accepted: 16 June 1997

ORIGINAL INVESTIGATION

S. Müller · P. C. M. O’Brien · M. A. Ferguson-Smith ·J. Wienberg (Y)Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QP, UK Tel.: +44-1223-333691; Fax: +44-1223-333346 e-mail: jw@mole.bio.cam.ac.uk

toid cell line for the concolor gibbon was kindly provided by theKunming Cell Bank of the Chinese Academy of Sciences. Humanand primate chromosome preparations were obtained by standardprotocols.

Human chromosome painting probes were the same as thosedistributed by CAMBIO (Cambridge, UK). Bivariate chromosomeflow sorting of primate chromosomes was performed as reportedpreviously for other species (Rabbitts et al. 1995; Ferguson-Smith1997). The flow karyotypes of the single primates will be dis-cussed elsewhere (S. Müller et al. in preparation). Painting probeswere generated by degenerate oligonucleotide-primed polymerasechain reaction protocols as described (Telenius et al. 1992). Peaksin the flow karyotypes containing the homologues to human acro-

centric chromosomes were identified by in situ hybridization tonormal human metaphase preparations and 4,6-diamidino-2-phenyl-indole (DAPI) banding. Fluorescence in situ hybridization (FISH)of primate paints to human chromosomes followed standard proto-cols without changing hybridization stringency.

In the primate multi-colour probe set, the probe specific to hu-man chromosome 13 was derived from the homologue of the sia-mang gibbon (Hylobates syndactylus, chromosome 15, labelledwith biotin-dUTP and Cy3-dUTP; biotin was detected with avidin-Cy5), the probes specific to human chromosomes 14 and 15 werederived from the gorilla and chimpanzee, respectively (Gorilla go-rilla, chromosome 18, labelled with biotin-dUTP and detected withavidin-Cy5; Pan troglodytes, chromosome 16, labelled with fluo-rescein-isothiocyanate-dUTP) and the probe specific to humanchromosome 21 was from the concolor gibbon (H. concolor, chro-mosome 25, labelled with Cy3-dUTP).

Fluorescence intensities were measured by a modified versionof the IPLab SmartCapture extensions software kindly provided byDigitalScientific (Cambridge, UK). The program allows the mea-surement of the mean fluorescence intensities along the chromo-somes perpendicular to the chromosome axis. Ten chromosomeswere scored for each experiment.

Results and discussion

A multi-colour FISH experiment was performed in whichhuman-derived and primate-derived multiplex probescontaining paints specific for human chromosomes 13,14, 15 and 21 were compared. Representative metaphasespreads are shown in Fig. 1. The painting probes isolatedfrom great apes showed markedly reduced cross hybridi-zation with human NOR chromosomes compared with

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Fig. 1 Painting human NOR-bearing chromosomes (chromosomes13–15 and 21) with probes derived from human (a) and from homologous primate (b) chromosomes in a multi-colour format,after merging the 8 bit gray scale images obtained separately forthe green, red and infrared fluorescence with a cooled charge-cou-pled device camera (Photometrics). The merged image is dis-played in false colours (red for Cy3, green for fluorescein isothio-cyanate, blue for Cy5). The human probes show considerable peri-centromeric cross hybridization (pink) on all NOR-bearing chro-mosomes, including chromosome 22, which was not present in theprobe set. This was not evident when hybridizing the primateprobe set comprised of a human chromosome-13-specific probederived from the homologue of the siamang (Hylobates syndacty-lus chromosome 15), the human chromosome-14-specific and chro-mosome-15-specific probes derived from the gorilla and chim-panzee (Gorilla gorilla, chromosome 18; Pan troglodytes, chro-mosome 16), respectively, and the human chromosome-21-spe-cific probe from the concolor gibbon (H. concolor, chromosome25). Differences in the labelling scheme between the human and theprimate probe sets (chromosomes 13 and 21) arise by chance andhave no further meaning

a b

human probes. This was even more evident with probesderived from more distantly related species. In order toverify this observation, the hybridization signals on thetargeted chromosomes and on other chromosomes in-volved in the formation of the NOR were quantified bycomputer densitometry. The hybridization background onhuman chromosome 1 served as a negative control. Anexample for probes specific for human chromosome 13derived from human, chimpanzee and siamang homo-logues is given in Fig. 2. When using a probe specific for

human chromosome 13, significant signal is evident onthe profile for the heterochromatin of chromosome 15.This cross hybridization is less evident with the probe de-rived from the chimpanzee homologue. However, nocross hybridization was found with the probes derivedfrom the gibbon. For euchromatin, signal intensities andsignal to background ratios of all primate derived probeswere the same as for human probes hybridized to humanchromosomes.

The primate-derived probes were used to analyse vari-ous patients with chromosome abnormalities. Two exam-ples showing a trisomy 21 detected with a painting probederived from the concolor gibbon and a reciprocal translo-cation t(6;13)(q25.3;q21.3) detected with a probe specificfor human chromosome 13 and derived from the siamanggibbon are shown in Fig. 3. In both cases, the chromo-some abnormality was also readily detectable in inter-phase nuclei (Fig. 3e, f).

The cross hybridization of probes derived from humanacrocentric chromosomes is the result of homologies inboth highly repetitive sequences and sequences specificfor ribosomal DNA. Although alphoid and other repetitivesequences may show rapid divergence in primate evolutionand are therefore unlikely to contribute to cross hybridiza-tion, ribosomal DNA is certainly conserved between thespecies. However, unlike most genomic DNA sequences,

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Fig. 2 Hybridization signals obtained by DNA probes specific forhuman chromosome 13 and derived from human, chimpanzee andsiamang were quantified by computer densitometry: mean fluores-cence intensities were scored on the targeted chromosome and onother chromosomes involved in the formation of the NOR. Humanchromosome 15 served as an example of a non-targeted acrocen-tric and chromosome 1 as a negative control in order to estimatethe signal to background ratio caused by non-specific binding ofthe DNA probes. Ten chromosomes were scored for each experi-ment. The chimpanzee-derived probe showed about 70% reductionof the mean fluorescence intensities in the pericentric region ofnon-targeted human NOR-bearing chromosomes when comparedwith the DNA probe of human origin. The siamang-derived DNAprobe showed no signal above background on other than the tar-geted chromosome. The level of non-specific binding of the DNAprobes detected on chromosome 1 was similar with all DNAprobes

NOR regions in primates show different distributions overvarious non-homologous chromosomes (Seuánez 1979;Stanyon et al. 1995). For example, in the gorilla, chromo-somes homologous to human chromosomes 13, 14 and 15are not involved in NOR formation. The same is true forthe siamang gibbon, where the NOR is found on a seg-ment homologous to human chromosome 3 and on the Ychromosome (Koehler et al. 1995a; Stanyon et al. 1995).Hybridization of the siamang chromosome 3 homologoussegment to human chromosomes results in strong crosshybridization patterns with all NOR-bearing human chro-mosomes (not shown). On the other hand, the copy num-ber of ribosomal DNA sequences on individual chromo-somes may also differ greatly between species. Althoughthe homologue of human chromosome 21 in the concolorgibbon has been shown to contain ribosomal DNA se-

quences (Stanyon et al. 1995), a probe derived from thischromosome results in no cross hybridization when usedon human chromosomes, presumably because of the lowcopy number of ribosomal DNA sequences. A probe forhuman chromosome 22 is not yet available in comparablequality, since the homologue in all species analysed todate is either NOR-bearing or involved in a translocationwith other chromosomes (Wienberg et al. 1992; Koehleret al. 1995a, b; Stanyon et al. 1995; Bigoni et al. 1997).

The results show that chromosome paints derived fromhominoid primates can be used readily in human cytoge-netics to avoid problems encountered with non-specifichybridization signals on non-targeted chromosomes. Thesenew paints should fill the gap in the cytogenetic probe set,which has not yet included specific tools for the delin-eation of NOR-bearing chromosomes. They will also very

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Fig. 3a–f Application of pri-mate chromosome-specificpainting probes to delineatechromosome abnormalities inclinical diagnosis. a, b, e Aconcolor-gibbon-derived probespecific for human chromo-some 21 was used to delineatea trisomy 21. b Three hy-bridization signals are evidenton the targeted chromosome21, whereas no signal is evi-dent on non-targeted chromo-somes. a The same metaphasecounter-stained with DAPI. eThe same probe detected on in-terphase nuclei delineatingthree distinct chromosome do-mains. c, d, f A probe specificfor human chromosome 13 de-rived from the siamang homo-logue delineates a translocationt(6;13)(q25.3;q21.3) with nocross hybridization to chromo-somes not involved in the re-arrangement. d The hybridiza-tion signal detected on meta-phase chromosomes. c Thesame metaphase showing DAPIcounter-staining. f The chro-mosome 13 probe on inter-phase nuclei of the transloca-tion case

much simplify interphase cytogenetics, especially sincehighly specific hybridization is a prerequisite for auto-mated microscopy when signals are recorded at low mag-nification.

Acknowledgements We thank the Kunming Cell Bank of theChinese Academy of Sciences and the Munich Zoo for providingthe gibbon samples. This work was supported by the DeutscheForschungsgemeinschaft (J.W.) and the Medical Research Council(M.A.F.).

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