Somalic cell genetics

Replication studies on prometaphase chromosomes of man, the chimpanzee, the pygmy chimpanzee, the gorilla, and the orangutan reveal great interspeeifie homologies between the autosomes. The early replicating X chromosomes clearly show a high degree of conservation of both the pattern and the time course of replication. An early replicating segment on the short arm of the X chromosomes of man (Xp22.3) which escapes inactivation can be found on the X chromosomes of the great apes as well. Furthermore, the most early replicating segment on the Y chromosomes of all species tested appears tö be homologous to this segment on the X chromosomes. Therefore, these early replicating segments in the great apes may correspond to the pseudoautosomal segment proposed to exist in man. From further cytogenetic characterization of the Y chromosomes it is evident that structural alterations have resulted in an extreme divergence in both the euchromatic and heterochromatic parts. It is assumed, therefore, that, in contrast to the X chromosomes, the Y chromosomes have undergone a rapid evolution within the higher primates. Despite numerous comparative cytogenetic studies in man and the great apes (see, e.g., Turleau et al., 1972; Dutrillaux, 1975; Egozcue, 1975; Schnedl et al., 1975; Miller, 1977; Seuänez, 1979; Schmidetal . , 1981; Yunis and Prakash, 1982), the Y and the late-replicating X chromosomes are only poorly characterized in the great apes. Replication studies on prometaphase human sex chromosomes have revealed a distinet early replicating segment on the distal ends of both Xp and Yp (Müller and Schempp, 1982; Schempp and Müller, 1982), corresponding to the pairing segment in which a synaptonemal complex is formed during male meiosis (Moses et al., 1975; Solari, 1980; Chandley et al., 1984). Furthermore, this early replicating segment on Xp appears to escape inactivation (Race and Sanger, 1975; Mohandas et al., 1979; Müller et al., 1980; Wolf, 1981); therefore, it was suggested that the segSupported by the Deutsche Forschungsgemeinschaft (Schempp 214/3-1). Request reprints f r o m : Dr. W. Schempp, Institut für Humangenetik und Anthropologie, Universität Freiburg, Albertstrassc 11, D-7800 Freiburg i. Br. (Fedcral Republic of Germany). ments on the distal portions of Xp and Yp are a remnant left unchanged during the differentiation of heteromorphic sex chromosomes from originally homomorphic autosomes (Schempp and Meer, 1983). The question arises whether the sex chromosomes of the great apes demonstrate a similar replication behavior. Therefore, we have performed a comparative replication study on prometaphase chromosomes, paying special attention to the sex chromosomes of man, the chimpanzee, the pygmy chimpanzee, the gorilla, and the orangutan. In order to draw a comprehensive comparison of the Y chromosomes of man and the great apes, additional staining procedures for the characterization of heterochromatin were applied. Materials and methods C h r o m o s o m e p r e p a r a t i o n s Chromosome preparations from five normal human males and fcmalcs ( H o m o sapiens [HSA]), five male and three female chimpanzees ( P a n t r o g l o d y t e s [PTR], T N O Primate Center, Rijswijk, The Nethcrlands; Tierpark Hellabrunn, München, FRG; Zoologischer Scx-chromosomc replication in man and the great apes 73 Garten, Frankfurt, FRG; Zoologischer Garten, Berlin, FRG) , two male pygmy chimpanzees ( P a n paniscus [PPA], Zoologischer Garten, Köln, FRG), three male and two female gorillas ( G o r i l l a g o r i l l a [GGO], Zoologischer Garten, Köln, FRG; Zoologischer Garten, Berlin, FRG), and four male and three female Bornean orangutans ( P o n g o pygmaeus [PPY], Zoologischer Garten, Köln, FRG; Tierpark Hellabrunn, München, FRG) were made from peripheral lymphocytes aecording to the method of Pfeiffer (1974), with minor modifications (Schempp and Meer, 1983). Briefly, the cells were cultivated in RPMI 1640 (Gibco), supplemcnted with 15% fetal calf serum and phytohemagglutinin. After 65 h of ineubation, bromodeoxyuridine (BrdU, 10 ug/ml) and fluorodeoxyuridine (FdU, 0.5 ug/ml) were added; 6 h later thecultures were treated with Colcemid (0.05 ug/ml) for 30 min. The cells were then harvested by centrifugation, resuspended in hypotonic Solution (0.4% KCl) for 30 min at 37 C, and fixed in 3:1 methanol:acetic acid. Cells were washed three times with fixative and kept at 4°C overnight. Chromosomes were spread on cold slides by flaming. Slides were air-dried for al least 24 h. S t a i n i n g methods B r d V r e p l i c a t i o n p a t t e r n s . Prometaphase chromosome preparations of the BrdU-lreated cultures were differentially stained with acridine orange (50 ug/ml), resulting in RBA patterns (ISCN, 1985). By adding BrdU to the cultures during the last 6.5 h, thymidine is incorporated inlo early replicating chromosome sites, resulting in bright fluorescence (light bands), while the incorporation of BrdU into late-replicating chromosome material produces dull-red staining (dark bands). Q b a n d i n g . Quinacrine mustard was used aecording to the technique of Caspersson et al. (1970). D i s t a m y c i n A / D A P I b a n d i n g . A slight modification of the technique originally described by Schweizer et al. (1978) was applied. In brief, the slides were ineubated in distamycin A (50 ug/ml in Mcllvainc's buffer, pH 7.0) for 20 min at room lemperature. The slides were then lightly washed in buffer and stained with DAPI (1 ug/ml in Mcllvaine's buffer, pH 7.0) for a furlher 20 min. After a rinse in buffer, the preparations were mounted in equal parls of glycerol and Mcllvaine's. For fluorescence microscopy, a BP 365/FT 395/LP397 (Zeiss) filter combination was used. C b a n d i n g . Constitutive heterochromatin was stained aecording to the method of Sumner (1972). C h r o m o s o m e length measurements Centromere indices of the Y chromosomes of man and the great apes were determined by length measurements of 30 Q-banded metaphase plates of each species. For interspeeifie comparison of the Y chromosomes, the X-chromosome length was used as an