DNA CHANGES INVOLVED IN THE FORMATION OF METAPHASE CHROMOSOMES, AS OBSERVED IN MOUSE DUODENAL CRYPT CELLS STAINED BY OSMIUM-AMMINE .1. NEW STRUCTURES ARISE DURING THE S-PHASE AND CONDENSE AT PROPHASE INTO CHROMOMERES, WHICH FUSE AT PROMETAPHASE INTO MITOTIC CHROMOSOMES

Background: In the hope of understanding how chromosomes condense at m itosis, we took advantage of a subdivision of the cell cycle into 11 s tages to examine the changes in DNA taking place during the stages pre ceding the emergence of metaphase chromosomes. Methods: To identify DN A changes, pieces of mouse duodenum were fixed in formaldehyde, and se ctions of the rapidly dividing cells of the crypts were stained by the osmium-ammine method, which is specific for the detection of DNA in t he electron microscope. Results: Throughout the cell cycle, DNA is pre sent in nucleofilaments composed of rows of 11-nm-wide nucleosomes. At stage I, during which the DNA-synthesizing or S phase of the cell cyc le begins, some of the nucleofilaments are compacted in the heterochro matin accumulations associated with nuclear envelope and nucleoli, whi le the others are scattered in the nucleoplasm where they appear eithe r ''free'' or ''attached'' to the heterochromatin. This DNA distributi on is similar to that observed in the noncycling cells examined. After the beginning of the S phase, ''free'' nucleofilaments are seen to as semble into structures composed of compacted nucleofilaments and refer red to as ''aggregates''; these make their appearance at stage II and increase in size through stage III up to the end of S during stage IV. Meanwhile, the heterochromatin associated with nuclear envelope and n ucleoli expands toward the nucleoplasm in the form of protrusions refe rred to as ''bulges,'' which gradually enlarge during stages III and I V, while the heterochromatin shrinks and eventually vanishes. On avera ge, a total of 1,171 aggregates and bulges are formed in the nucleus d uring the S phase. At the apparition of stage V, which corresponds app roximately to prophase, aggregates and bulges are rapidly gathered int o an average of 288 spheroidal bodies referred to as ''chromomeres.'' These are connected to one another by nucleofilamentous bridges in suc h a way as to be lined up in rows. The formation of rows of chromomere s represents in the electron microscope the prophasic condensation obs erved in the light microscope, Finally, during stage VIa, which corres ponds to prometaphase, the chromomeres approach one another within eac h row, make contact, and coalesce to become the 40 chromosomes of the mouse, which during stage VIb are organized in the equatorial plate of metaphase. Conclusions: The condensation of metaphase chromosomes occ urs in three main steps. The first and longest takes place during the S phase, as nucleofilaments are assembled into aggregates, while the h eterochromatin gives rise to bulges. The brief second step occurs towa rd the beginning of prophase, when the numerous aggregates and bulges are congregated into a limited number of chromomeres, which are lined up in rows. The third step takes place during the brief prometaphase, when the chromomeres of a row coalesce into a mitotic chromosome. (C) 1995 Wiley-Liss, Inc.