Chromosoma (Berl) (1983) 88:333-342 CHROMOSOMA 9 Springer-Verlag 1983 Cell cycle-specific changes in the ultrastructural organization of prematurely condensed chromosomes Steven K. Hanks 1, 2, 4, Susanne M. Gollin a, s, 6, Potu N. Rao t, 2, Wayne Wray 2, 3 and Walter N. Hittelman 1, 2 1 Department of Developmental Therapeutics, University of Texas, M.D. Anderson Hospital and Tumor Institute at Houston, Houston, TX 77030 USA; 2 University of Texas Health Science Center at Houston, Graduate School of Biomedical Sciences; 3 Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030 USA; 4 present address: Division of Cellular Biology, Department of Basic and Clinical Research, Scripps Clinic and Research Foundation, La Jolla, CA 92037 USA; 5 present address : Kleberg Cytogenetics Laboratory, Department of Medicine, Baylor College of Medicine, Houston, TX 77030 USA; 6 To whom correspondence should be addressed Abstract. Prematurely condensed chromosomes (PCC) of HeLa cells synchronized in different phases of the cell cycle were analyzed by high-resolution scanning electron micros- copy. The purpose of this study was to examine changes in the arrangement of the basic 30-nm chromatin fiber with- in interphase chromosomes associated with progression through the cell cycle. These studies revealed that highly condensed metaphase chromosomes and early G1-PCC consisted of tightly packed looping fibers. Early to mid GI-PCC were more extended and exhibited gyres suggestive of a despiralized chromonema. Further attenuation of PCC during progression through G1 was associated with a grad- ual transition from packed looping fibers to single extended longitudinal fibers. This process occurs prior to the initia- tion of DNA synthesis which appears to be localized within single longitudinal fibers. Following replication of a chro- mosome segment, extended longitudinal fibers were rapidly reorganized into packed looping fiber clusters concomitant with the formation of a multifibered chromosome axis. This results in the characteristic ~ pulverized" appearance of S- PCC when viewed by light microscopy. Subsequently, adja- cent looping fiber domains coalesce, resulting in the uni- formly packed, looping fiber arrangement observed in G2- PCC. Spiralization of the chromonema during the G2-mi- totic transition results in the formation of highly compact metaphase chromosomes. Introduction The idea of a chromosome condensation cycle originated with a model proposed by Mazia (1963). According to this model, chromosomes decondense gradually throughout G1 phase, reach a maximum level of dispersion at the time of replication during S phase, and then begin a recondensa= tion process that culminates in the formation of the maxi- mally condensed metaphase chromosome. The model has received support from a number of investigations using a variety of experimental methods (Ringertz et al. 1969; Pe- derson and Robbins 1972; Zetterberg and Auer 1970; A1- varez 1974; Moser et al. 1975 ; Hildebrand and Tobey 1975; Nicolini et al. 1975). Direct visual evidence for cell cycle- specific changes in the higher-order arrangement of chro- matin fibers within interphase chromosomes is provided by the phenomenon of premature chromosome condensa- tion. Fusion between mitotic and interphase cells induces a rapid breakdown of the interphase nuclear framework and reorganization of interphase chromatin into premature- ly condensed chromosomes (PCC) (Johnson and Rao 1970). In nearly every respect, the processes of premature chromo- some condensation and the normal entry of a nucleus into mitotic prophase appear identical (Johnson and Rao 1970; Matsui et al. 1972; Obara et al. 1974). The only apparent difference is the manner in which the chromosomes are organized following nuclear dissolution. The PCC condense differentially, depending on what stage of the cell cycle a cell is in at the time of fusion (Johnson and Rao 1970; Stenman and Saksela 1971). Under the light microscope, Gl-phase PCC appear as single chromatids. A closer analysis of G1-PCC morpholo- gy reveals a relationship between chromatid length and thickness and the degree of advancement of the cell toward S phase at the time of fusion (Schor et al. 1975; Hittelman and Rao 1976, 1978; Rao et al. 1977; Rao and Hanks 1980). As a cell progresses through GI, its PCC become more extended. The early stages of this decondensation pro- cess appear to involve an uncoiling of the chromonema, resulting in gyred chromatids characteristic of Ohnuki-spi- rals (Ohnuki 1968). Immediately prior to entry into S phase, the PCC have a highly extended morphology and individual chromosomes are often no longer easily discernable. This decondensation process appears to be necessary, although not sufficient, for the initiation of DNA synthesis (Hanks and Rao 1980). The PCC from S phase cells exhibit a characteristic "pulverized" morphology. This appearance results from variable levels of condensation achieved by different chro- mosome segments and is related to their replicative state prior to fusion. Segments of S-PCC that have not yet ini- tiated replication condense into extended single chromatids or remain largely diffuse in appearance. Autoradiographic analyses of PCC spreads from S-phase cells pulse-labeled with 3H-thymidine immediately prior to fusion clearly re- veal a heavy localization of silver grains within gap regions of the PCC (Sperling and Rao 1974; Lau and Arrighi 1981). Thus, segments that were actively engaged in DNA replica- tion at the time of fusion appear under the light microscope as gaps in the continuity of the chromosome. Regions in S- and G2-PCC that have completed replication are highly