View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Universität München: Elektronischen Publikationen Chromosome Research 1994, 2, 405-410 The origin of human chromosome 2 analyzed by comparative chromosome mapping with a DNA microlibrary J. Wienberg, A. Jauch, H.-J. L0decke, G. Senger, B. Horsthemke, U. Claussen, T. Cremer, N. Arnold & C. Lengauer Received 7 March 1994; received in revised form 5 May 1994; Accepted for publication by M. Schmid 5 May 1994 Fluorescence in situ hybridization (FISH) of that human chromosome 2 was derived by a fusion of microlibraries established from distinct chromo- two primate homologs (Dutrillaux 1979, Yunis & some subregions can test the evolutionary conserva- Prakash 1982). This has been confirmed by fluores- tion of chromosome bands as well as chromosomal cence in situ hybridization (FISH) of a human chromo- rearrangements that occurred during primate evolu- some 2-specific DNA library to primate chromosomes tion and will help to clarify phylogenetic relation- ships. We used a DNA library established by (Wienberg et al. 1990,1992, Jauch et al. 1992). However, microdissection and microcloning from the entire the exact phylogenetic interpretation of the chromo- long arm of human chromosome 2 for fluorescence in some rearrangements noted between these species is situ hybridization and comparative mapping of the still not clear. Owing to the presence of chromosomes of human, great apes (Pan troglo- pericentromeric heterochromatin in great ape chro- dytes, Pan paniscus, Gorilla gorilla, Pongo mosomes, it is difficult to match the homologous pygmaeus) and Old World monkeys (Macaca fuscata bands close to the centromeres by classical banding and Cercopithecus aethiops). Inversions were found studies. From high-resolution chromosome banding, in the pericentric region of the primate chromosome Yunis & Prakash (1982) proposed a common derived 2p homologs in great apes, and the hybridization (synapomorphic) pericentric inversion in the homolog pattern demonstrates the known phylogenetically derived telomere fusion in the line that leads to hu- to human chromosome 2p in a suggested chimpan- man chromosome 2. The hybridization of the 2q zee/human phylogenetic lineage, which was followed microlibrary to chromosomes of Old World monkeys by a fusion event in the human karyotype. gave a different pattern from that in the gorilla and A more secure demonstration of the homology be- the orang-utan, but a pattern similar to that of chim- tween individual bands is provided by fluorescence in panzees. This suggests convergence of chromo- situ hybridization of chromosome band-specific DNA somal rearrangements in different phylogenetic libraries (Lengauer et al. 1991a, b; Trautmann et al. lines. 1991). The construction of such libraries is possible by microdissection and microcloning (Lfidecke et al. Key words: evolution, fluorescent in situ hybridization, microdissection, phylogeny, primates 1989). We have used a microlibrary established from the long arm of human chromosome 2 for comparative chromosome mapping of human and primate species. Introduction The chromosomes of Old World monkeys were used as an 'outgroup' to analyze the direction of Comparative banding analysis between human chro- chromosome changes that occurred during hominoid mosomes and the karyotype of great apes suggests evolution. J. Wienberg (corresponding author) and N. Arnold are at the Institut far Anthropologie und Humangenetik, Universitdt Miinchen, Richard-Wagner Strasse lOft, D-80333 Munich, Germany. Fax: (+49) 89 5203 286. A. Jauch is at the Institut far Humangenetik und Anthropologie, Universit~t Heidelberg, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany. H.-J. Uidecke and B. Horsthemke are at the Institut far Humangenetik, Universit~tsklinikum Essen, Hufelandstr. 55, D-45122 Essen, Germany. G. Senger and U. Claussen are at the Institut far Humangenetik und Anthropologie, Universit4t Jena, Kollegiengasse 10, D-07740 Jena, Germany. C. Lengauer is at the Institut far Humangenetik und Anthropologie, Universit~t Heidelberg, Im Neuenheimer Feld 328, D-69120, Germany, and is currently at the Research Institute of Molecular Pathology, Dr. Bohr Gasse 7, A-1030 Vienna, Austria. ~) 1994 Rapid Communications of Oxford Ltd Chromosome Research Vol 2 1994 405 J. Wienberg et al. Material and methods Fluorescence in situ hybridization Chromosome preparations About 200 ng of PCR amplified biotin-labeled probe was co-precipitated with 10 ~tg of salmon sperm DNA The present study includes chromosome preparation and 30~tg of Cot-1 DNA and the pellet was from human (HSA), Pan troglodytes (PTR), P. paniscus redissolved in 50% formamide, lxSSC and 10% (PPA), Gorilla gorilla (GGO), Pongo pygmaeus dextran sulfate. The biotin-labeled probe was hybrid- (PPY), Macaca fuscata (MFU) and Cercopithecus ized under suppression conditions (Cremer et al. 1988, aethiops (CAE). Metaphase chromosomes were made Lichter et al. 1988, Pinkel et al. 1988) and detected with from standard preparations of phytohemagglutinin fluorescein-conjugated avidin. Analysis was per- (PHA)-stimulated peripheral lymphocytes or from formed using a Zeiss Axiophot microscope equipped lymphoblastoid cell lines established from blood with epifluorescence and the filter combination for samples kindly provided by the Munich Zoo fluorescein, rhodamine and DAPI (4",6-diaminidino-2- 'Tiergarten Hellabrunn' or from Drs W. Schempp, phenyfindole), Chromosomes were photographed Freiburg, Germany, M. Schmid, W/irzburg, Germany, with Agfachrome (1000 ASA) color slide films or and R. Stanyon, Genoa, Italy. Microscopic slides Kodak T-MAX black and white (400 ASA) films. were prepared by standard techniques. Before The photographed hybridization signals were using the lymphoblastoid cell lines in this study, superimposed over the previous photographed G- chromosomes were checked for aberrations and banded chromosomes to identify the marked chro- were found to be normal diploid, except for one mosomes and the position of signals along the of the gorilla cell lines, which showed a translocation chromosomes. in the chromosome homologous to human chromosome 2q. Results DNA probe Fluorescence in situ hybridization (FISH) of the estab- The microlibrary was established from the entire fished 2q microfibrary to human lymphocyte long arm of the human chromosome 2. metaphase spreads resulted as expected in a specific Microdissection was performed essentially as delineation of the long arm of human chromosome 2 described by Lfidecke et al. (1989) except that the (Figure la). In metaphase spreads from great apes, chromosomes were not GTG banded. Twenty chromo- gibbons and Old World monkeys different hybridiza- some fragments were pooled into the collection tion patterns were observed. drop and microreactions were performed as previ- In both chimpanzees (P. troglodytes and P. paniscus), ously described (L~decke et al. 1989). An aliquot the entire chromosomes PTR 13 and PPA 13 were (100 ng) of the primary amplification products was stained, except for a small part of the short arm that biotin labeled in a 100-~tl assay containing 1 ~tM remained unlabeled. On the chromosomes PTR 12 and universal M13/pUC sequencing and reverse PPA 12 hybridization signals were restricted to the sequencing primer, 10mM Tris-HCL (pH 8.4), short arm also, leaving a small unlabeled band (Figure 50 mM KC1, 1.5 ~tM MgC12, 0.001% gelatin, 200 ~tM lc), which may represent telomeric heterochromatin. dATP, dGTP and dCTP, 100~tM dTTP, 150~tM The hybridization pattern of the gorilla chromo- bio-11-dUTP and 2.5 units of Thermus aquaticus some GGO 11 was similar to that found on the chim- DNA polymerase. After initial denaturation at 96°C panzee chromosomes PTR 13 and PPA 13 except that for 3 min, 25 cycles of polymerase chain reaction the unlabeled telomeric band was more pronounced. (PCR) were carried out with denaturation at 96°C for The labeling of the gorilla chromosome GGO 12 was I min, annealing at 47°C for i min and extension at different from that of both chimpanzee chromosomes 72°C for 2 min, 10 s. PTR 12 and PPA 12, which are supposed to be homologs. Hybridization signals were found on the long arm of gorilla chromosome GGO 12 close to the Chromosome banding centromere, whereas the short arm was not labeled (Figure le). A bright band with the counterstain DAPI Chromosome banding prior to FISH was indicates that the short arm of GGO 12 may be totally achieved according to Klever et al. (1991). Briefly, heterochromatic (not shown). The two orang-utan after routine GTG banding, metaphases were homologs (PPY 12 and PPY 11) were labeled as in the photographed (Agfa-Ortho 25), destained with gorilla, except for the telomeric heterochromatin, fixative (methanol-acetic acid 3:1) and post-fixed which is not present in this species (Figure lg). with 4% formaldehyde in phosphate-buffered saline Hybridization of the 2q microlibrary to Old World (PBS) for 15 min. Post-fixation was essential to pre- monkey chromosomes gave a similar pattern as in the serve chromosome morphology in subsequent FISH two chimpanzees. In both species analyzed one experiments. submetacentric chromosome pair (MFU15, CAE16) 406 Chromosome Research Vol 2 1994 Analysis of human chromosome 2 with a DNA microlibrary was entirely labeled including the short arms (Figure recent in situ hybridization experiments with lh and i). On another submetacentric chromosome centromere and telomere DNA sequences of human pair (MFU9, CAE13) labeling was