Chromosoma (Bert) (1984) 90: 389-393 CHROMOSOMA 9 Springer-Verlag 1984 Description of a chromosome replication unit in individual prematurely condensed human S-phase chromosomes* H. Hameister 1 and K. Sperling 2 1 Abteilung Klinische Genetik, FrauenstraBe 29, D-7900 Ulm (Donau), Federal Republic of Germany; 2 Institut ffir Humangenetik, Heubnerweg 6, D-1000 Berlin, Federal Republic of Germany Abstract. Mammalian chromosome replication was studied by the aid of premature chromosome condensation (PCC). After induction of PCC the sites of DNA replication appear as "gaps" between condensed chromosomal regions. These condensed particles are unineme before and bineme after DNA replication. The two phases are due mainly to the unineme or bineme nature of the particles. During early S-phase almost all particles are unineme, during late S- phase they are bineme and there is only one transitory stage between these two main stages. Premature chromosome condensation was studied in detail on a specific human chromosome 22 which is marked by its heterochromatin constitution. This led to easy identification of these ele- ments in S-phase PCC (S-PCC) preparations. For each stage of the S-phase there was a reproducible pattern of condensed chromosomal particles making up the whole chromosome. The number of these particles was rather lim- ited and a complementary pattern was found in early versus late S-phase. The pattern of early S-PCC corresponded to the banding pattern of G-banded prometaphase chromo- somes; the pattern of late S-PCC, to R-banded prometa- phase chromosomes. Thus, "gaps" and condensed particles as observed after PCC induction are obviously homologous to chromosome replication units. Replication of constitu- tive heterochromatin occurred during the very late S-phase. During this stage PCC induction led to condensation of the heterochromatin into several small, highly fluorescent particles. negative regions (R-band regions) double in the first half of the S-phase (Dutrillaux et al. 1976). Moreover, a com- plete correspondence of the structural and replicational banding pattern of prometaphase chromosomes was found (Meer et al. 1981 ; Camargo and Cervenka 1982). A struc- tural band was therefore believed to behave as an indepen- dent unit during replication. However, one cannot exclude that more units would be observable if the resolution ob- tained by chromosomal preparation techniques were in- creased (Yunis 1981). We have therefore analyzed prematurely condensed S- phase chromosomes (S-PCC), which are significantly longer than even prophase chromosomes (for review see Sperling 1982). Due to incomplete condensation at the sites of DNA replication the replicons can be directly visualized by light microscopy (Sperling and Rao 1974; R6hme 1975; Lau and Arrighi 1981). However, as the S-PCC appear "pulver- ized", it is normally impossible to identify individual chro- mosomes. We have overcome this difficulty by the study of a human cell line containing a chromosome 22 with a highly fluorescent marker, which allowed identification of this element even in S-PCC. The following questions were investigated: (1) Is the marker chromosome composed of a recognizable and reproducible pattern of condensed parti- cles attached to the heterochromatin? (2) How many differ- ent patterns of condensed particles can be found through- out the whole S-phase? (3) Are any of these patterns compa- rable to the well-known banding pattern of prometaphase chromosomes? Introduction Cytogenetic DNA replication analysis is usually done on mitotic chromosomes. The DNA is labeled during part of the preceding DNA synthetic phase with 3H-thymidine or bromodeoxyuridine. Several hours later the distribution of label is recorded on chromosomes that have entered mitosis. This procedure was introduced by Taylor (1960). When he applied this technique to mammalian chromosomes, he observed a fixed spatial order of chromosomal subsections engaged in DNA replication. A combination of this labeling technique with chromosomal banding showed that G-band- positive chromosomal regions replicate late (Ganner and Evans 1971; Calderon and Schnedl 1973) whereas G-band- * Dedicated to Prof. Marlies Tolksdorf on the occasion of her 60th birthday Materials and methods A diploid cell line derived from the skin biopsy of a normal female containing a chromosome 22 with a brilliant fluores- cent short arm after quinacrine staining was used (Fig. 1). The cells were grown in Eagle's minimal essential medium supplemented with 10% fetal calf serum. Logarithmically growing fibroblasts were fused to mitotic HeLa cells for the induction of premature chromosome condensation (PCC). The procedure for PCC induction has been described earlier (Sperling and Rao 1974). In brief, mitotic HeLa cells and logarithmically growing fibroblasts were mixed at a ratio of 2:1 and incubated with 200 HAU of UV-inacti- vated Sendal virus at 4 ~ C for 15 min. 3H-thymidine (spec. act. 44 Ci/mM) was added at a final concentration of 20 gCi/ml and the cell mixture was then transferred to a